key: cord-279863-5kxgu4t9 authors: oem, jae-ku; an, dong-jun title: phylogenetic analysis of bovine astrovirus in korean cattle date: 2013-11-23 journal: virus genes doi: 10.1007/s11262-013-1013-0 sha: doc_id: 279863 cord_uid: 5kxgu4t9 bovine astrovirus (bastv) belongs to a genetically divergent lineage within the genus mamastrovirus. the present study showed that bastv was associated with the gastroenteric tracts of cattle in nine positive fecal samples from 115 cattle, whereas no positive samples were found in the brain tissues of 14 downer cattle. interestingly, the positive diarrheal samples were obtained mainly from calves aged 14 days–3 months. bayesian inference tree analysis of the partial orf1ab and capsid (orf2) gene sequences of bastvs identified four divergent groups. eleven bastvs, four porcine astroviruses, and two deer astroviruses (dastvs; ccastv-1 and -2) belonged to group 1; group 2 contained two bastvs (bastk08–51 and bastk10–96) with another two in group 3 (bastk08–2 and bastk08–53); and group 4 comprised the bastv-neuros1 strain derived from a cattle brain tissue sample and an ovine astrovirus. the same divergent groups were obtained when the pairwise alignments were produced using both amino acid and nucleotide sequences. the korean bastvs isolated from infected cattle had a nationwide distribution and they belonged to groups 1, 2, and 3. electronic supplementary material: the online version of this article (doi:10.1007/s11262-013-1013-0) contains supplementary material, which is available to authorized users. astroviruses are single-stranded positive-sense rna viruses that measure approximately 6.4-7.3 kb in length. the family astroviridae comprises two genera: mamastrovirus infects mammals and avastrovirus infects birds [1] . human astrovirus was first reported in children with diarrhea in 1975 [2] and mamastroviruses were found subsequently in a variety of wild hosts, including sheep, cow, pig, dog, cat, red deer, mouse, mink, bat, cheetah, brown rat, roe deer, sea lion, dolphin, and rabbit [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] . bovine astrovirus (bastv) was one of the first astroviruses [17] to be discovered and it has been isolated in the usa and the uk. two bastv serotypes were established based on the results of a virus neutralization assay [18] . the genomic characterization and sequence analysis of astroviruses in bovine fecal specimens collected in hong kong provides evidence of potential recombination in orf2 [19] . recently, the complete genome of a novel bastv associated with neurological disease in cattle was sequenced, i.e., boastv-neuros1, which was phylogenetically related to an ovine astrovirus (oastv). a previous study suggested that boastv-neruos1 infection was a potential cause of neurological disease in cattle [20] . however, genetically diverse lineages of bastvs have not been identified in many countries because there have been few studies of astroviruses derived from cattle. thus, the present study investigated the genetic groupings of korean bastvs and examined their relationships with the age of cattle infected with bastvs. in total, 115 fecal samples were collected from cattle with certain or suspected diarrheal disease at cattle farms throughout korea between january 2008 and december 2010. the cattle comprised 64 calves aged \30 days and 51 cattle aged [30 days. based on the fecal condition, 91 samples were from animals with diarrhea and 24 from nondiarrheic animals. the cattle comprised 84 korean cattle and 31 holstein cattle. nonambulatory cattle which are commonly referred to as ''downer'' cattle are unable to stand or walk. cattle brain tissue samples with a histopathological diagnosis of encephalitis were also collected from 14 downer cattle between 2010 and 2012. out of 14 brain samples collected from downer cattle, 7 were found to be positive for akabane virus and two were bovine viral diarrhea virus (bvdv) whereas no pathogenic agent for encephalitis was detected from the other. viral rna was extracted from the feces using trizol ls b , according to the manufacturer's instructions. bastv was detected in fecal specimens by rt-pcr using a specific primer set for the orf1ab and orf2 regions of bastv (bastv-f, 5 0 -gtgtttggcatgtgggtyaarcc-3 0 and bastv-r: 5 0 -rtcvyybktggtggt-3 0 ), which were designed based on known strains deposited in genbank (accession no. hq916313-hq916317). the rt-pcr process amplified a 965-nt long fragment at 42°c for 30 min, 94°c for 5 min, 94°c for 40 s, 51°c for 40 s, and 72°c for 1 min, followed by 40 cycles using virus-specific conditions. the bastv associated with neurological disease in cattle was also detected in brain tissue specimens by rt-pcr, as described previously [20] . products with the expected size were cloned using the pgem-t vector system ii tm (promega, cat. no. a3610, usa). the cloned gene was sequenced with t7 and sp6 sequencing primers using an abi prism ò 3730xi dna sequencer at the macrogen institute (macrogen co. ltd). the sequences of all the bastv-positive samples were submitted to genbank under accession numbers kf668444-kf668452. nine of 115 fecal samples from korean cattle were positive for bastv and all of the bastvs were related to diarrhea. however, bastv was not detected in 14 cattle brain tissue samples. although bastv was first reported in england in 1987 [17] , the association between bovine astroviruses and gastroenteric diseases in cattle is still not clear. a recent study reported that bastv is not associated directly with severe diarrheic disease in calves under natural conditions [17, 19, 21, 22] . in the present study, nine korean bastvs were associated with clinical diarrhea in cattle where calves aged \1 month accounted for 77.8 % of cases (table 1) . a previous study shows that bastvs were excreted by 60-100 % of calves on farms [21] while a recent study of rectal swab samples from asymptomatic adult cattle showed that only 2.4 % (5/209) contained bastv [19] . this must be because bastvs are more frequent in young calves than adult cattle. to investigate the relationships between astroviruses and other bovine viruses that cause diarrhea in cattle, a screening test was conducted using specific primers for the detection of bovine rotavirus (brv) [23] , bovine coronavirus (bcv) [24] , bvdv [25] , and bovine kobuvirus (bkv) [26] , as described previously. co-infections with other viruses were associated with the clinical symptoms of diarrhea in only two cases: the bastk08/1 strain derived from a 20-day-old calf was coinfected with brv and the bastk10/31 strain from a 14-day-old calf was co-infected with both brv and bvdv (table 1) . although the association between bkv and diarrhea or gastroenteritis is unclear, it was co-infected with six korean bastvs, except for bastk08/51, bastk08/53, and bastk10/35 (table 1) . in cattle, two astrovirus serotypes have been recognized based on serological investigations, i.e., boastv-1 and boastv-2 infections [18] , and recent phylogenetic analyses support the classification of bastvs and the newly discovered astroviruses in roe deer (ccastv) under the proposed mamastrovirus genocluster gi [14, 19] . all of the astrovirus sequences were aligned using the clustal x alignment program [27] . the nucleotide sequences were translated and the shared nucleotide and amino acid sequence identities among the astrovirus strains were calculated using bioedit 7.053 [28] . the analysis of the diversity of bastvs in the present study identified four table) . bayesian trees were generated with mrbayes 3.1.2 [29, 30] using best-fit models, which were selected with mrmodeltest 3.7 [31] for nucleotide sequences and prottest 1.4 [32] for amino acid sequences. markov chain monte carlo analyses were run using 2,000,000 generations for each nucleotide and amino acid sequence. the best-fit model of the orf1ab nucleotide sequence selected by mrmodeltest 3.7 software was trnef?g, according to the results of a hierarchical likelihood ratio test. the likelihood parameter was set to nst = 6 and rate = gamma for the datasets, and the gamma distribution shape parameter was 0.5612. the substitution model rmat was 1. and -2 [19] . recently, the boastv-neuros1 strain was detected in the brain tissues of cattle and the analysis of its genetic diversity showed that it was most closely related to the oastv prototype, which was identified in 1977 [3] , whereas it was phylogenetically distant from a recently reported oastv [33] and the hong kong bastvs [20] . this suggests the occurrence of multiple cross-species transmission events among hosts and other animal species. however, it appears that the histopathogenic findings of encephalitis in korean downer cows were not associated with the detection of boastv-neuros1 in brain tissue. the bi analysis of the partial orf1ab and/or orf2 genes also showed that all of the known bastvs could be separated into four groups (fig 1a, b) , in the same way as the diversity analysis. group 1 of the bi tree contained six hong kong bastvs and five korean bastvs, groups 2 and 3 included only korean bastvs, and the boastv-neuros1 strain was the only member of group 4. in conclusion, the present study identified four bastvs groups based on the phylogenetic analysis and their shared pairwise amino acid sequence identities. the bastv detection rate in cattle feces was higher in calves aged \1 month compared with adult cattle. thus, continuous surveillance of novel diversity in bastvs should be conducted on many cattle farms throughout the world because of the risk of emerging astroviruses associated with neurological disease in cattle. pastk-76 pig (jq696855) pastk-120 pig (jq696856) b76/hk cow (hq916316) b170/hk cow (hq916314) ccastv-2 deer (hm447046) ccastv-1 deer (hm447045) poastv12-4 pig (hm756259) poastv14-4 pig (hm756260) pastk-76 pig (jq696855) pastk-120 pig (jq696856) b170/hk cow (hq916314) b161-1 cow (jf796126) cow pastv-2/2007/hun pig (gu562296) wbastv-1 wildboar (jq340310) pastv/1117/mn pig (jf272561) pastk-114 pig (jq696847) pastk-4 pig (jq696831) pastk-5 pig (jq696832) pastv/1116/pa pig (jf272560) human astroviurs 4 human (dq344027) virus taxonomy. eighth report of the international committee on taxonomy of viruses acknowledgments we are grateful to dr. soo-kyung joo for technical assistance. key: cord-339178-d6f6a5ds authors: pensaert, m. b.; de bouck, p. title: a new coronavirus-like particle associated with diarrhea in swine date: 1978 journal: arch virol doi: 10.1007/bf01317606 sha: doc_id: 339178 cord_uid: d6f6a5ds coronavirus-like particles were detected by electron microscopy in the intestinal contents of pigs during a diarrheal outbreak on 4 swine breeding farms. diarrhea was reproduced in experimental pigs with one of the isolates, designated cv777, which was found to be distinct from the 2 known porcine coronaviruses, transmissible gastroenteritis virus and hemagglutinating encephalomyelitis virus. coronavirus-like particles were detected by electron microscopy in the intestinal contents of pigs during a diarrheal outbreak on 4 swine breeding farms. diarrhea was reproduced in experimental pigs with one of the isolates, designated cv777, which was found to be distinct from the 2 known porcine coronaviruses, transmissible gastroenteritis virus and hemagglutinating encephalomyelitis virus. in 1946, do yr.e and hutc~i~gs (2) described a viral diarrhea, in swine and called it transmissible gastroenteritis. until recently, transmissible gastroenteritis virus was the only virus known to be specifically associated with diarrhea in swine of all ages. in 1976, following the discovery of rotaviruses in different, animal species, a porcine rotavirus was detected in the feces of pigs with diarrhea (14) . diarrhea could be reproduced experimentally in piglets with this virus. in a search for rotaviruses on belgian swine breeding farms with diarrheal problems, a new coronavirus-like particle was detected b y electron microscopic examination of intestinal or fecal samples from sick pigs. the present report describes the morphology of this coronavirus-like particle, and shows that it is distinct from the known porcine coronaviruses and causes diarrhea. up to now, the only coronaviruses isolated from swine have been transmissible gastroenteritis virus {tgev) and hemagglutinating encephalomyelitis virus (hev). tgev has been described as a cause of diarrhea in swine in countries all over the world (13) . numerous studies have been performed on the v i r u s --a n i m a l interactions of tgev, which is usually detected either by its isolation from fecal these studies were supported by the institute for the encouragement of l~esearch in industry and agriculture (iwonl), brussels, belgium. material in cell cultures or by immunoflu orescence in the smm1 intestinal epithelium of infected pigs (7, 12) . tgev infections can also be diagnosed serologically. j:iev was first described in canada in 1962 as a cause of centrm nervous disorders in pigs (4). the same virus was later associated with a disease syndrome called vomiting and wasting disease in several european countries (1, 8) . the virus can easily be detected by cultivation in several porcine cell cultures (11). both tgev and hev have been classified as coronaviruses mainly on the basis of their specific morphology (10). in 1977, a sudden outbreak of diarrhea was observed in swine of all ages on 4 belgian swine breeding farms. the morbidity in sows was very variable and the animals recovered after a diarrhea which lasted 3 to 4 days. all the pigs showed a watery diarrhea. death occurred up to the age of 7 days and the overali mortality rate in these piglets was approximately 50 per cent (9) . it decreased with increasing age. tgev was suspected as the cause of this diarrhea. however, the direct immunofluorescence test for the diagnosis of tgev, which is routinely applied on cryostat sections of the small intestine of sick pigs, was negative for these pigs. the absence of seroneutralizing antibodies to tgev in the blood of sows collected 6 to 12 weeks after the outbreak confirmed that tgev was not involved. in an attempt to arrive at an etiologie diagnosis, fecal material and intestinal contents from pigs of each farm were subsequently processed for examination in an electron microscope by negative staining. they were diluted t to 5 (v/v) in phosphate-buffered saline, ph 7.3 and clarified at 3000 × g, at 4 ° c, for 30 minutes. the supernatant was layered on top of a 20 per cent sucrose solution and centrifuged at 150,000 x g, at 4 ° c, for 40 minutes. the resulting pellet was resuspended in a few drops of distilled water, placed on 200 mesh formvar coated grids, and stained with 2 per cent k-phosphotungstate, ph 6.1. grids were examined using a zeiss em 9 s-2 electron microscope at an acceleration voltage of 60 kv. micrographs used for particle size measurement were taken at an instrumental magnification of 28,000 x, which were then photographically-enlarged to 84,000 x or 168,000 ×. gotavirus particles were not detected. however, eoronavirus-like particles were observed in specimens of pigs from each of the 4 breeding farms. one of the fecal samples containing these coronavirus-like particles was designated cv777 and was used for further studies. the etiologic relationship between the corona virus-like particles, cv777, and the occurrence of diarrhea was established by oral inoculation of a 20 per cent suspension of the fecal material contmning cv777 into a one day old colostrumdeprived pig. the experimental pig was killed 30 hours later, at the height of diarrhea, and a virus stock was prepared from an homogenate of its small intestine and contents. a bacteria free filtrate of the supernatant of a 20 per cent suspension of this material was used for inoculation of 12 colostrum-deprived-hysterectomy-derived piglets, kept i n isolation. seven control pigs were used. the pigs were inoculated at the age of 3 to 15 days. all the inoculated pigs developed a watery diarrhea within 24 to 36 hours after inoculation whereas the control animals remained normal. coronavirus-like particles were detected by electron microscopic examination in the watery feces or intestinal contents of each of the experimentally inoculated pigs. such particles were not found in the feces of the same pigs prior to inoculation or in the fecal samples of the control animals. the particles, shown in figure 1 , had typicm coronavirus morphology. they were pleomorphic with a range in diameter of 95 to 190 nm, including the projections, which were approximately 18 nm in length. most particles were between t30 and t70 nm in diameter. the projections formed a single fringe radiating from the core. they appeared to be club-shaped. only the dilated distal ends of the projections were seen on the micrographs. the negative stain also appeared to settle on the surface of some particles and an electron opaque central area covered by surface projections was often seen (fig. 1 a --a r r o w s and lc) . no internal structure was observed. it was impossible to distinguish these coronavirus-like particles morphologically from tgev or h e v particles from similar preparations. other particles, different from these coronavirus-like particles, were also observed in the majority of the fecal samples. as seen in figure 2 , they were pleomorphic and very variable in size, ranging in diameter from 95 to 650 nm with an average diameter of 190 to 225 rim. they carried numerous short projections, of approximate length 9 nm, on their surfaces. similar particles of unkno~m identity have been described in human and animal fecal samples (3, 5, 6) . in the present studies such particles have also been found in the solid fecal samples of the control pigs. they appeared, therefore, not to be associated with diarrhea. rotaviruses and other recognizable virus particles were not seen in control or experimentally inoculated pigs. as already mentioned, tgev was eliminated as the cause of the diarrhea on the origina 1 farms. additionmly 9 out of the 12 experimentally inoculated pigs, killed at, the heigth of diarrhea, were negative for tge viral antigens in their small intestinal epithelium by the direct immunofluorescenee test. furthermore, the remaining 3 pigs, inoculated with cv777 at the age of 15 days, were allowed to recover after a diarrhea which lasted 4--5 days. a serum sample, collected from these pigs 3 weeks later, did not contain neutralizing antibodies against the cell culture adapted purdue strain of tgev. fig. 2 . one eoronavirus-like particle cv 777 (arrow) together with pleomorphie particles of unknown identity. bar represents 100 nm the possibility that the cv777 particles consisted of h e v was less likely since the latter virus does not cause diarrhea in pigs. cryostat sections of the sma~ intestine of experimentmly inoculated pigs were negative for fluorescence by the direct test using a conjugate directed against the vw572 isolate of h e v (8) . furthermore, the pigs that had been allowed to recover did not possess hemagglutination-inhibiting or seroneutralizing antibodies against this t t e v isolate. preliminary attempts were made to cultivate the coronavirus-like particle, cv777, in primary pig kidney cell cultures and in secondary porcine thyroid cells. four weekly blind passages were made. the cells were examined for cytopathic effect and hemadsorption with chicken red blood cells, and the cell culture fluids were examined for hemagglutination. no evidence of viral replication in the cell cultures was obtained. it is kno~n that the tiev can easily be isolated in primary pig kidney cell cultures using the same criteria (8) . the present data suggest that, as well as tgev and hev, another previously unrecognized coronavirus-like virus is prevalent in swine. the results indicate that diarrhea can be reproduced in experimental pigs with this virus and that it is associated with certain outbreaks of epizootic diarrhea on belgian swine breeding farms. more details on the clinical disease in the field and on the results of the experim e n t a l infections will be reported later. vomiting and wasting disease of piglets a transmissible gastroenteritis in pigs the hunt for viruses in infections of the alimentary s y s t e m : a n immuno etectronmicroscopicat approach a hemagglutinating virus producing encephalomyelitis in baby pigs pleomorphic virus-like particles in human faeces virus-like particles in calves' faeces diagnosis of transmissible gastroenteritis in pigs by immunofluorescence characteristics of a coronavirus causing vomition and wasting in pigs a virus isolated from an apparently new epizootic diarrhea in swine. proe. 5th world int the size and morphology of transmissible gastroenteritis and vomiting and wasting disease viruses of pigs titration of two porcine respiratory viruses in mammalian cell cultures by direct fluorescent antibody staining isolation of the virus of transmissible gastroenteritis (tge) from naturally infected piglets in cell culture transmissible gastroenteritis of swine the isolation of reovirus-like agents (rotaviruses) from acute gastroenteritis of piglets key: cord-339924-tsmnkuhw authors: jung, kwonil; wang, qiuhong; scheuer, kelly a.; lu, zhongyan; zhang, yan; saif, linda j. title: pathology of us porcine epidemic diarrhea virus strain pc21a in gnotobiotic pigs date: 2014-04-17 journal: emerg infect dis doi: 10.3201/eid2004.131685 sha: doc_id: 339924 cord_uid: tsmnkuhw to understand the progression of porcine epidemic diarrhea virus infection, we inoculated gnotobiotic pigs with a newly emerged us strain, pc21a, of the virus. at 24–48 hours postinoculation, the pigs exhibited severe diarrhea and vomiting, fecal shedding, viremia, and severe atrophic enteritis. these findings confirm that strain pc21a is highly enteropathogenic. in june 2013, intestinal contents were obtained from a 1-day-old pig with diarrhea on a farm in ohio, usa. pedv strain pc21a was detected in the sample by reverse transcription pcr (rt-pcr) selective for the nucleocapsid gene (229-557 nt). the partial nucleocapsid gene sequence of pc21a was identical to that of 2 us pedv outbreak strains from colorado, usa: usa/colorado/2013 (genbank accession no. kf272920) and 13-019349 (genbank accession no. kf267450). only coronavirus-like particles were observed in the fecal sample by electron microscopy (figure 1 ). the sample was negative for rotavirus groups a and c and for transmissible gastroenteritis virus/porcine respiratory coronavirus by rt-pcr (7, 8) . the sample was bacteriologically sterilized by using 0.22-µm syringe filters and then prepared as inoculum. nearterm gnotobiotic pigs were delivered aseptically by hysterectomy from a specific pathogen-free sow (9) . six 10-to 35-day-old pigs were randomly assigned to a pedv-infected group (pigs 1-5) or a negative control group (pig 6). information about inoculation and inocula pig-passage number is described in table 1 . pigs 1-3 and 5 were inoculated orally and/or intranasally with 6.3-9.0 log 10 genomic equivalents (ge) of pedv strain pc21a; pig 4 was exposed to the virus by indirect contact with inoculated pig 3. for each sample, the quantity of pedv rna ge was ≈10 6 times higher than plaque assay results for a cell-adapted pedv strain, pc22a. clinical signs were monitored hourly. pig 4 was monitored for longer-term clinical signs and virus shedding. pigs were euthanized for pathologic examination at 3 stages of infection: acute, mid, and later stages (<24 h, 24-48 h, and >48 h, respectively, after onset of clinical signs). the ohio state university institutional animal care and use committee approved all animal-related experimental protocols. fecal or rectal swab samples were prepared as described (9) . virus rna was extracted by using the mag-max viral rna isolation kit (applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. titers of virus shed in feces were determined by taqman real-time rt-pcr using the onestep rt-pcr kit (qiagen, valencia, ca, usa) as reported (10) , with modifications in the forward primer and probe to provide a 100% match to the us strains: forward 5′-cg-caaagactgaacccactaac-3′ and probe fam-tgyyaccayyaccacgactcctgc-bhq. a standard curve was generated by using the pcr amplicon (pedn 229/557) of strain pc21a. the detection limit was 10 ge per reaction, corresponding to 4.8 log 10 and 3.8 log 10 ge/ml of fecal and serum samples, respectively. small and large intestine tissues, lung, liver, heart, kidney, spleen, and mesenteric lymph node were examined grossly and histologically. mean jejunal vh:cd was measured by using pax-it software (paxcam, villa park, il, usa) as described (11) . the frozen tissues were prepared and tested by immunofluorescence staining, as described (12) , for the detection of pedv antigen, using monoclonal antibody 6c8-1 against the spike protein of pedv strain dr13 (provided by daesub song, korea research institute of bioscience and biotechnology, daejeon, korea). acute, severe watery diarrhea and vomiting developed in all inoculated pigs. clinical signs developed 24-48 h after inoculation, regardless of the inoculum dose or number of inoculum pig passages (table 1 ). pig 4, which was followed longer, also exhibited dehydration, loss of bodyweight, and lethargy, but it consumed most of the milk that was offered. however, ≈120 h after onset of clinical signs, pig 4 collapsed after showing signs of disorientation and emaciation. immune electron microscopy, using a gnotobiotic pig hyperimmune serum to pedv, showed only pedv particles in the intestinal contents. for the pig-passaged pc21a strain, rt-pcr/pcr results were negative for transmissible gastroenteritis virus/porcine respiratory coronavirus (7), rotavirus groups a-c (8), caliciviruses (13, 14) , astroviruses (15) , circoviruses, enterovirus, kobuvirus, and bocavirus. for pigs 1 and 2, the detection of fecal virus shedding 24-48 h after inoculation coincided with the onset of clinical signs; for pigs 3 and 4, fecal shedding occurred before the onset of clinical signs (table 1) . by macroscopic examination, all infected pigs exhibited typical pedv-like lesions, characterized by thin and transparent intestinal walls (duodenum to colon) and accumulation of large amounts of yellowish fluid in the intestinal lumen (figure 2, panel a) . the stomach was filled with curdled milk, possibly due to reduced intestinal peristalsis. the other internal organs appeared normal. histologic lesions included acute diffuse, severe atrophic jejunitis (figure 2 , panel b) and mild vacuolation of superficial epithelial cells and subepithelial edema in cecum and colon ( figure 2 , panel c). these findings were similar to those in conventional pigs naturally infected with asian or us strains of pedv and in caesarean-derived, colostrum-deprived pigs experimentally infected with cv777 (2,3,5,6). the mean jejunal vh:cd of the 5 infected pigs ranged from 1.2 to 3.4, probably depending on the stage of infection (table 1) , and that of the negative control pig was 6.3 (±0.2). vh:cd for pig 4, which was euthanized at a later stage of infection, was 1.5 (±0.2), a ratio indicative of continued cellular necrosis. neither clinical signs nor lesions developed in the negative control pig during the experiment. immunofluorescence-stained cells were observed mainly in the epithelium of atrophied villi of small (duodenum to ileum) and large intestines (table 2; figure 2 , panels d-f), as reported in other studies (2, 3, 5) . the immunofluorescence was confined to the villous epithelial cells (figure 2 , panels d-f). a few immunofluorescence-stained cells were detected infrequently in the peyer patches of pig 4. lung tissues of the infected pigs did not show immunofluorescence staining, indicating that pedv does not infect figure 1 . electron micrograph of a us porcine epidemic diarrhea virus (pedv) particle detected in a field fecal sample collected during a 2013 outbreak of ped on a farm in ohio, usa; the fecal sample from which pedv strain pc21a in this study was obtained was from a pig on the same farm during the same outbreak. the sample was negatively stained with 3% phosphotungstic acid. scale bar = 50 nm. lung tissues under the conditions tested. although pc21a strain replicated in cecum and colon epithelial cells, cellular necrosis and villous atrophy were not evident. whether pedv infection of the large intestine contributes to the severity of ped is unclear. all infected pigs tested at acute or later stages of infection had viral rna titers of 4.8-7.6 log 10 ge/ml in serum samples (table 1) . these titers were similar to those for field samples tested by real-time rt-pcr; 11 (55%) of 20 acutephase serum samples collected from 13-to 20-week-old pigs with diarrhea from ohio had viral rna titers of 4.0-6.3 ge/ml. the early, severe diarrhea and vomiting and the pedv fecal shedding at high titers may be accompanied by viremia. no infected pigs had detectable viral rna in serum samples obtained before inoculation, and no negative control pig had detectable viral rna during the experiment. (4); -, no cells showed staining. ‡at 24 days of age, noninoculated pig 4 was exposed by indirect contact to pig 3 (at pih 0) through small holes drilled into the stainless steel divider panel located between the 2 pigs in the shared pig tub isolator unit. clinical signs and virus shedding were monitored after indirect contact. diarrhea and vomiting developed in pig 4 approximately 2-4 h after clinical signs developed in pig 3 (i.e., in pig 4, signs developed 46-50 h after indirect contact with inoculated pig 3); pig 4 was euthanized 120 h after the onset of clinical signs. in 2013, the first us outbreaks of the rapidly spreading porcine virus, pedv, caused a high number of pig deaths and substantial economic losses (1,2); however, little was known about progression of the disease. our data confirm that us pedv pc21a is highly enteropathogenic and acutely infects the entire intestine, but the jejunum and ileum are the primary sites of infection. pc21a infection causes severe atrophic enteritis accompanied by viremia that leads to severe diarrhea and vomiting. fighting a deadly pig disease: industry, veterinarians trying to contain ped virus, new to the us emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences the pathogenesis of an enteric infection in pigs, experimentally induced by the coronaviruslike agent, cv-777 pathology of experimental cv777 coronavirus enteritis in piglets. i. histological and histochemical study an immunohistochemical investigation of porcine epidemic diarrhoea in situ hybridization for the detection and localization of porcine epidemic diarrhea virus in the intestinal tissues from naturally infected piglets development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs detection and genetic diversity of porcine group a rotaviruses in historic (2004) and recent (2011 and 2012) swine fecal samples in ohio: predominance of the g9p[13] genotype in nursing piglets the effects of simvastatin or interferon-alpha on infectivity of human norovirus using a gnotobiotic pig model for the study of antivirals multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus the effects of transplacental porcine circovirus type 2 infection on porcine epidemic diarrhoea virus-induced enteritis in preweaning piglets porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections prevalence and molecular characterization of porcine enteric caliciviruses and first detection of porcine kobuviruses in us swine characterization and prevalence of a new porcine calicivirus in swine, united states novel astroviruses in insectivorous bats we thank james e. collins and doug marthaler for kindly providing us pedv sequences for primer design and testing for enterovirus, kobuvirus, and bocavirus; j. hanson, g. meyers, and r. mccomick for assistance with animal care; x. wang, m. lee, s.s. wagner, a. veeramani, and chun-ming lin for technical assistance; andrea kaszas for assistance with electron microscopy; and v. anastasia for helpful discussion.salaries and research support were provided by state and federal funds appropriated to the ohio agricultural research and development center, the ohio state university. this work was supported in part by a grant from the national pork board (no. 13-222 to q.w. and l.j.s.).dr jung is a veterinary pathologist at the ohio state university. his major research interests include diagnostic molecular pathology, pathogenesis, and immune responses to enteric viral infections, using germ-free animal models. key: cord-285585-tigj7fhc authors: cleveland, christopher a.; denicola, anthony; dubey, j.p.; hill, dolores e.; berghaus, roy d.; yabsley, michael j. title: survey for selected pathogens in wild pigs (sus scrofa) from guam, marianna islands, usa date: 2017-05-03 journal: vet microbiol doi: 10.1016/j.vetmic.2017.05.001 sha: doc_id: 285585 cord_uid: tigj7fhc pigs (sus scrofa) were introduced to guam in the 1600’s and are now present in high densities throughout the island. wild pigs are reservoirs for pathogens of concern to domestic animals and humans. exposure to porcine parvovirus, transmissible gastroenteritis, and leptospira interrogans has been documented in domestic swine but data from wild pigs are lacking. the close proximity of humans, domestic animals, and wild pigs, combined with the liberal hunting of wild pigs, results in frequent opportunities for pathogen transmission. from february–march 2015, blood, tissue and ectoparasite samples were collected from 47 wild pigs. serologic testing found exposure to brucella spp. (2%), toxoplasma gondii (11%), porcine reproductive and respiratory syndrome (prrs) virus (13%), porcine circovirus type 2 (36%), pseudorabies virus (64%), actinobacillus pleuropneumoniae (93%), lawsonia intracellularis (93%), and porcine parvovirus (94%). eleven (24%) samples had low titers (1:100) to leptospira interrogans serovars bratislava (n = 6), icterohaemorrhagiae (n = 6), pomona (n = 2), and hardjo (n = 1). kidney samples from nine pigs with leptospira antibodies were negative for leptospira antigens. numerous pigs had metastrongylus lungworms and three had stephanurus dentatus. lice (hematopinus suis) and ticks (amblyomma breviscutatum) were also detected. no antibodies to influenza a viruses were detected. in contrast to the previous domestic swine survey, we found evidence of numerous pathogens in wild pigs including new reports of pseudorabies virus, prrs virus, brucella, and leptospira in pigs on guam. these findings highlight that domestic swine-wild pig interactions should be prevented and precautions are needed when handling wild pigs to minimize the risk of pathogen transmission. wild pigs are nearly globally distributed and are hosts for many parasites and bacterial and viral pathogens, some of which are transmissible to agricultural animals, wildlife, and humans (barrios-garcia and ballari, 2012; bevins et al., 2014) . numerous studies have investigated the prevalence and distribution of various pathogens in wild pigs and the risk they pose for pathogen transmission in the united states and europe, but there are relatively few reports from southeast asia (baroch et al., 2015; hill et al., 2014; pedersen et al., 2014 pedersen et al., , 2015 pepin et al., 2016) . wild pig populations are increasing dramatically on several pacific islands, yet data on pathogen exposure are limited or absent. wild pigs were introduced to guam, an unincorporated territory of the united states located in the marianna island chain, in the late 1600's and despite current liberal hunting regulations, populations continue to increase (conry, 1988) . this population increase has led to severe ecological damage and agricultural losses. the only report of swine pathogens on guam was limited to domestic pigs (dugies et al., 2000) . the lack of data on pathogens in wild pigs is a concern as increased swine populations have led to increased pig-human interactions. recent reports of leptospirosis in residents and tourists is one concern and although rodents tend be considered the most common reservoir of leptospira, there is evidence of leptospira in domestic swine on guam (dugies et al., 2000) , and wild pigs in numerous countries have antibodies to leptospires (jansen et al., 2007; corn et al., 1986) . in 2015, there was an effort to decrease wild pig populations within fenced areas of two military bases on guam to minimize the ecological damage caused by the pigs. samples collected from these animals were used to conduct a comprehensive surveillance project on pathogen exposure of wild pigs on guam. andersen air force base (aafb) is a 4135 ha military installation in northern guam (13.5875°n, 144.9244°e) . forested portions of the base contain high quality native habitat, some of which is included in the guam national wildlife refuge. the naval base guam naval munitions site (nbg nms) is centrally located on the island and covers approximately 5723 acres (13.44000°n 144.65250°e). removal of wild pigs was conducted via strategic sharp-shooting techniques conducted by well-trained shooters using suppressed 0.223 caliber rifles mounted with scopes. during removal, shooters only fired if the situation met the following criteria: 1) there was certainty that the animal would be dispatched and not escape, 2) if other animals were nearby, every animal had a high probability of being dispatched, and 3) it was safe to dispatch the animal. the shooting methods followed the american veterinary medical association's guidelines for humane euthanasia of animals (avma, 2013) . pigs were aged based on tooth eruption patterns and wear. animal and sample collection procedures were reviewed and approved by uga's institutional animal care and use committee (a2014 09-021). immediately after euthanasia, blood samples were collected via cardiocentesis and placed into ethylenediaminetetraacetic acid (edta) and plain tubes (greiner bio-one, monroe, nc). clotted blood was centrifuged at 1250g for 15 min and serum was removed and frozen at −20°c until diagnostic testing. whole blood was also frozen at −20°c until testing. representative ectoparasites were collected and preserved in 95% ethanol. information on pathogens we screened pigs for as well as diagnostic assays and diagnostic laboratories used are listed in table 1 . most pigs were serologically tested for all of the pathogens listed; however, due to limited sample volume, some pigs were only tested for selected pathogens. small sections of lung were fixed in formalin and processed for routine histology. small sections of kidneys were also fixed in formalin and if antibodies to leptospira were detected, they were tested for leptospira antigens by immunohistochemistry (ihc). if any gross lesions were noted during necropsy, they were also collected in formalin for histologic examination. pigs were not systematically examined for internal parasites, but if any were seen they were collected in formalin for identification. body weights were compared between male and female swine using a two-sample t-test. prevalence of pathogen exposures was compared between males and females and between adult and juvenile swine using fisher's exact test. all testing assumed a two-sided alternative hypothesis and p < 0.05 was considered statistically significant. analyses were performed using commercially available statistical software (stata version 13.1, statacorp lp, college station, tx). from the two sites, a total of 47 wild pigs were sampled including six (12.8%) juveniles and 41 (87.2%) adults; 18 (38.3%) were males and 29 (61.7%) were females. weight (kilograms) was recorded for 32 individuals: 5 juveniles and 27 adults. all juveniles weighed < 11.3 kgs and the mean ± sd weight of the 12 adult males (47.1 ± 16.5) was significantly greater than that of the 15 adult females (25.4 ± 4.1; p < 0.001). all pigs were positive for at least one of the pathogens included in the study. exposure to or infection with influenza a virus (iav), brucella, porcine epidemic diarrhea virus, babesia spp., transmissible gastroenteritis/porcine respiratory coronavirus and trichinella spp. was rare or absent (table 1 ). based on antibody testing, a high percentage of pigs were exposed to actinobacillus pleuropneumonia (app; 93%), lawsonia intracellularis (93%), and porcine parvovirus (94%). exposure to multiple serotypes of app was also common ( table 2 ). based on mat testing, antibodies to four leptospira serovars were detected in 11 pigs (tables 1 and 3) , with some individuals having antibodies to more than one serovar. kidney samples from nine pigs with leptospira antibodies were negative for leptospira antigens by ihc. juvenile pigs had a significantly higher prevalence of antibodies to porcine circovirus type 2 (6/6 [100%]) compared with adults (10/ ; p = 0.017, respectively). there was no difference in prevalence between males and females for any pathogen. only 38 pigs were examined for ectoparasites; lice (hematopinus suis) and ticks (amblyomma breviscutatum) were found on 12 (32%) and seven (18%) pigs, respectively. lung samples from 32 pigs were examined histologically and eight (25%) were positive for metastrongylus lungworms. stephanurus dentatus were found in abdominal lesions from three pigs and one pig had a liver abscess with intralesional nematode larvae which could not be identified. our results indicate that wild pigs on guam are exposed to multiple pathogens of zoonotic and agricultural importance. guam has many free-range pig operations which have an increased risk of domestic pigwild pig interactions. previous work on guam was restricted to domestic pigs and in contrast to our data, domestic pigs were exposed to relatively few pathogens (duguies et al., 2000) . clinically, several diseases were noted, but the only pathogen detected by serologic testing was parvovirus (7/14, 50%). although domestic pigs were not serologically tested for app or l. intracellularis, porcine pleuropneumonia and proliferative enteritis were noted. domestic pigs tested negative for antibodies to pseudorabies virus (n = 65), porcine reproductive respiratory syndrome virus (n = 16), brucella spp. (n = 66), leptospira spp. (n = 52), swine influenza virus (n = 61), transmissible gastroenteritis virus (n = 27), mycoplasma hyopneumoniae (n = 70), and trichinella spp. (n = 53) while we found serologic evidence of the first four pathogens in wild pigs (duguies et al., 2000) . porcine pleuropneumonia, caused by app, is a highly infectious disease that is economically important for domestic swine. although known to occur on guam among domestic pigs, prevalence was unknown (duguies et al., 2000; bossé et al., 2002) . clinical disease and mortality resulting from infection with one or more app serotypes can vary geographically; however, serotype 2 has been consistently associated with morbidity and mortality in domestic pigs (baroch et al., 2015) . over half of the wild pigs from guam had antibodies for serotypes 1-2-9-11. reproductive disease in domestic pigs due to porcine parvovirus (ppv) has been reported on guam (duguies et al., 2000; ruiz-fons et al., 2006) . in addition to reproductive failure due to ppv infection, coinfection with porcine circovirus type 2 (pcv2) in domestic pigs can result in mortalities (ellis et al., 2000) . we report in wild pigs the first evidence of pcv2 on guam; surveillance and vaccination for these pathogens may be warranted. l. intracellularis has been documented worldwide in domestic and wild pigs and is the etiologic agent of proliferative enteropathy (chiriboga et al., 1999) . the high prevalence of these three pathogens in wild pigs, indicates that transmission to domestic pigs is a risk. exposure to leptospira interrogans and toxoplasma gondii, two important zoonoses, was common in wild pigs on guam. leptospirosis is a growing concern in guam ( mason et al., 1998) . while exposure to several serotypes was detected, prevalence is likely underestimated because antigens from serotypes readily available for testing in our us laboratory were used rather than those likely present in guam/other pacific nations. regardless, exposure of pigs does not implicate them in human cases due to the diverse reservoir-range, but suggests that proper ppe should be utilized when in contact with pigs. domestic pigs have not been previously tested for t. gondii but evidence of t. gondii was noted in goats (duguies et al., 2000) . this parasite has a wide host range and can pose a risk to native avian fauna that do not have an evolutionary history with the parasite (work et al., 2000) . in addition, t. gondii is zoonotic; due to the popularity of wild pig hunting for sport and population management, hunters in guam should properly butcher and cook wild pig meat (hill et al., 2014; conry 1988) . several pathogens were not detected or were rare in wild pigs. no evidence of exposure to trichinella spp., iav, or coronaviruses associated with enteric and respiratory disease was found; one pig had antibodies to porcine epidemic diarrhea virus. previously, trichinella was absent from domestic pigs, but because of the zoonotic potential of this parasite, surveillance and education campaigns to ensure pork is thoroughly cooked (which would also kill any t. gondii present) should continue. swine influenza, a zoonosis, is relatively common in domestic pigs worldwide, but exposure of wild pigs is not ubiquitous and varies geographically (smith et al., 2009) . while prevalence was high in a study in southern china (luo et al., 2013) , in korea, the us and spain, seroprevalence was generally low (< 20%) and varied seasonally; it is possible that our sample size was insufficient to detect exposure in guam (hall et al., 2008; corn et al., 2009; feng et al., 2014) . enteric and respiratory coronaviruses, which have been reported in several asian countries, are of significant concern due to high morbidity and mortality in naïve pig populations (song and park, 2012) . porcine epidemic disease virus is clinically similar to transmissible gastroenteritis; both are coronaviruses that can lead to acute diarrhea in all age groups of swine, often resulting in poor body condition or mortalities (lee, 2015) . one wild pig had antibodies to brucella; the species could not be distinguished based on serologic testing. serology indicates if brucella is present in a population, but culture and/or advanced molecular assays are required to confirm species (leiser et al., 2013) . brucellosis, a zoonotic disease, has been documented in wild pig populations globally (leiser et al., 2013) . lice (h. suis) were common on guam, similar to studies on domestic and wild pigs worldwide (girişgin et al., 2009) . h. suis can transmit swine poxvirus and classical swine fever virus but there is no evidence that these viruses are present on guam. amblyomma breviscutatum were found on several pigs but little is known about this tick species; it is believed to have been historically present on guam and other islands in greater micronesia (vander velde and vander velde, 2013) . currently, there are no data on pathogens associated with a. breviscutatum. our testing method for helminth parasites was limited due to time and cleveland et al. veterinary microbiology 205 (2017) 22-25 importation restrictions; we only examined small sections of lung tissue (or lesions if noted). confirmed infections with metastrongylus lungworms and s. dentatus were relatively common, and likely underrecognized because of testing method. both metastrongylus lungworms and s. dentatus, previously reported in domestic pigs on guam, can impact domestic pig health (duguies et al., 2000) . our study highlights the importance of surveillance efforts for pathogens transmitted by wild pigs on guam as many of the pathogens circulating in wild pigs can cause disease in domestic swine. we detected serologic evidence for multiple zoonotic pathogens, necessitating simple yet effective preventative measures. wearing appropriate ppe, practicing good hygiene, washing all utensils and surfaces that have come into contact with butchered wild pigs, and thoroughly cooking meat prior to consumption may assist in preventing infection. management of wild pigs is often controversial as they are seen as a food source for hunters or in some cases may be considered native species. the wild pig populations on guam are not native, harbor pathogens of importance to domestic swine and people, and very importantly, cause major damage to the environment leading to economic losses for farmers and extreme habitat destruction for native species of wildlife. guidelines for the euthanasia of animals, edition. avma, schaumburn, il exposure of feral swine (sus scrofa) in the united states to selected pathogens impact of wild boar (sus scrofa) in its introduced and native range: a review consequences associated with the recent range expansion of nonnative feral swine actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection detection of lawsonia intracellularis in faeces of swine from the main producing regions in brazil management of feral and exotic game species on guam survey of selected diseases in wild swine in texas pathogen exposure in feral swine populations geographically associated with high densities of transitional swine premises and commercial swine production animal health survey for guam 1999 coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome influenza a subtype h3 viruses in feral swine occurrence of haematopinus suis linnaeus, 1758 (insecta, anopluridae) on a wild boar (sus scrofa) influenza exposure in united states feral swine populations surveillance of feral swine for trichinella spp. and toxoplasma gondii in the usa and host-related factors associated with infection leptospirosis in urban wild boars porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus feral swine brucellosis in the united states and prospective genomic techniques for disease epidemiology exposure to swine h1 and h3 and avian h5 and h9 influenza a viruses among feral swine in southern china leptospira interrogans antibodies in feral pigs from new south wales identification of brucella suis from feral swine in selected states in the usa widespread detection of antibodies to leptospira in feral swine in the united states contact heterogeneities in feral swine: implications for disease management and future research seroprevalence of six reproductive pathogens in european wild boar (sus scrofa) from spain: the effect on wild boar female reproductive performance diversity of piroplasms detected in blood-fed and questing ticks from several states in the united states origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines known and potential ticks and tick-borne pathogens of micronesia fatal toxoplasmosis in free-ranging endangered 'alala from hawaii primary financial support for this project came from the us department of the navy (n40192-14-r-8000 and n40192-14-r-8005). additional financial support was provided by the wildlife management agencies of the southeastern cooperative wildlife disease study member states through the federal aid to wildlife restoration act (50 stat. 917) and by the us department of the interior cooperative agreement g11ac20003. the authors thank the personnel at the diagnostic laboratories for diagnostic support and r.l. poulson for comments on this manuscript. key: cord-281679-xmbnpawj authors: meekins, david a.; morozov, igor; trujillo, jessie d.; gaudreault, natasha n.; bold, dashzeveg; artiaga, bianca l.; indran, sabarish v.; kwon, taeyong; balaraman, velmurugan; madden, daniel w.; feldmann, heinz; henningson, jamie; ma, wenjun; balasuriya, udeni b. r.; richt, juergen a. title: susceptibility of swine cells and domestic pigs to sars-cov-2 date: 2020-08-16 journal: biorxiv doi: 10.1101/2020.08.15.252395 sha: doc_id: 281679 cord_uid: xmbnpawj the emergence of sars-cov-2 has resulted in an ongoing global pandemic with significant morbidity, mortality, and economic consequences. the susceptibility of different animal species to sars-cov-2 is of concern due to the potential for interspecies transmission, and the requirement for pre-clinical animal models to develop effective countermeasures. in the current study, we determined the ability of sars-cov-2 to (i) replicate in porcine cell lines, (ii) establish infection in domestic pigs via experimental oral/intranasal/intratracheal inoculation, and (iii) transmit to co-housed naive sentinel pigs. sars-cov-2 was able to replicate in two different porcine cell lines with cytopathic effects. interestingly, none of the sars-cov-2-inoculated pigs showed evidence of clinical signs, viral replication or sars-cov-2-specific antibody responses. moreover, none of the sentinel pigs displayed markers of sars-cov-2 infection. these data indicate that although different porcine cell lines are permissive to sars-cov-2, five-week old pigs are not susceptible to infection via oral/intranasal/intratracheal challenge. pigs are therefore unlikely to be significant carriers of sars-cov-2 and are not a suitable pre-clinical animal model to study sars-cov-2 pathogenesis or efficacy of respective vaccines or therapeutics. the emergence of sars-cov-2, the causative agent of covid-19, has resulted in a global pandemic with over 20 million cases and 740,000 deaths as of august 13, 2020 [1, 2] . sars-cov-2 causes a respiratory disease in humans with a broad clinical presentation, ranging from asymptomatic or mild illness to severe fatal disease with multi-organ failure [3] [4] [5] [6] . sars-cov-2 is rapidly transmissible via contact with infected respiratory droplets and can also be transmitted by asymptomatic carriers [6] [7] [8] . to curb viral spread, countries have instituted varying levels of social distancing policies, which have significant negative economic and social impacts [9] . mitigating the effects of this unprecedented pandemic will necessitate the development of effective vaccines and therapeutics, which will require well-characterized and standardized preclinical animal models. sars-cov-2 is a member of the betacoronavirus genus that includes the pathogenic human viruses sars-cov-1 and mers-cov [2, [10] [11] [12] . while details of the origin of sars-cov-2 are unknown, evidence indicates it emerged from a zoonotic spillover event, with bats and perhaps pangolins as probable origin species [2, [13] [14] [15] . the potential for a reverse zoonotic event, i.e. human-to-animal transmission, is possible and of significant concern to animal and public health [16] [17] [18] . instances of natural human-to-animal transmission of sars-cov-2 have been reported with covid-19 patients in domestic settings (dogs and cats), zoos (lions and tigers), and farms (mink) [18] [19] [20] . therefore, investigations into the infectivity of sars-cov-2 in various animal species with human contact are essential to assess and control the risk of a spillover event and to establish the role these animals may play in the ecology of the virus. several studies have determined the susceptibility of different animal species to sars-cov-2 via experimental infection [20, 21] . cats, hamsters, and ferrets are highly susceptible to sars-cov-2 infection, demonstrate varying clinical and pathological disease manifestations, readily transmit the virus to naïve animals, and mount a virusspecific immune response [22] [23] [24] [25] [26] [27] [28] . dogs are mildly susceptible to experimental sars-cov-2 infection, with limited viral replication but with clear evidence of seroconversion in some animals [22] . poultry species seem to be resistant to sars-cov-2 infection [22, 26] . these findings establish the respective utility of different animal species as pre-clinical models to study sars-cov-2. several lines of evidence suggest that pigs could be susceptible to sars-cov-2 infection. pigs are susceptible to both experimental and natural infection with the related betacoronavirus, sars-cov-1, and demonstrate seroconversion [29, 30] . structure-based analyses predict that the sars-cov-2 spike (s) protein receptor binding domain (rbd) binds the pig angiotensin-converting enzyme 2 (ace2) entry receptor with similar efficiency compared to human ace2 [31] . single-cell screening also indicated that pigs co-express ace2 and the tmprss2 activating factor in a variety of different cell lines, and sars-cov-2 replicates in various pig cell lines [2, 26, 32, 33] . despite these preliminary data indicating that pigs could be susceptible to sars-cov-2 infection, two recent studies revealed that intranasal inoculation of three and twelve pigs, respectively, with 10 5 pfu or tcid50 of sars-cov-2 did not lead to any detectable viral replication or seroconversion [22, 26] . however, the single route of intranasal inoculation used in these studies suggests that additional investigations are necessary before definitive conclusions can be made regarding susceptibility of pigs to sars-cov-2. in the present study, we determined the susceptibility of swine cell lines and domestic pigs to sars-cov-2 infection. two different porcine cell lines were found to be permissive to sars-cov-2 infection showing cytopathic effects (cpe). domestic pigs were challenged via simultaneous oral/intranasal/intratracheal inoculation with a 10 6 tcid50 dose of sars-cov-2. sars-cov-2 did not replicate in pigs and none of them seroconverted. furthermore, the virus was not transmitted from sars-cov-2 inoculated animals to sentinels. the present findings, combined with the other studies [22, 26] , confirm that pigs seem resistant to sars-cov-2 infection despite clear susceptibility of porcine cell lines. pigs are therefore unlikely to play an important role in the covid-19 pandemic as a virus reservoir or as a pre-clinical animal model to study sars-cov-2 pathogenesis or develop novel countermeasures. sars-cov-2 usa-wa1/2020 isolate (genbank accession # mn985325) [34] was eighteen pigs (mix of males and females, five weeks of age) were used in the study. pigs were acquired from a source guaranteed free of swine influenza virus (siv), porcine circovirus-2 (pcv-2), and porcine reproductive and respiratory syndrome virus (prrsv) infection. the study outline is illustrated in figure 1 . upon arrival, pigs were acclimated for 3 days prior to sars-cov-2 inoculation. nine pigs were designated as uninfected negative controls and housed in separate bsl-2 facilities. three of these uninfected negative control pigs were humanely euthanized at 3 days post challenge (dpc) to provide negative control clinical and tissue samples. the nine principal infected pigs were housed in the same room in two separate groups (4 or 5 pigs each; gross pathological examinations on major organs were performed and respiratory tissue samples were collected and either stored in 10% neutral-buffered formalin or stored as fresh samples at -80˚c. blood and swab samples were all filtered using a 0.2µm filter prior to storage at -80˚c. rna was isolated from blood, swabs, and tissue samples, using a magnetic bead-based protocol in a bsl-3+ laboratory at the bri at ksu. lung tissue homogenates (200 mg per 1ml dmem; 20% w/v) were prepared by thawing tissue, mincing it into 1mm sections, followed by lysis in a 2 ml sure-lock tube containing 5 mm stainless steel homogenization beads using the tissuelyser lt (qiagen, germantown, md, usa) for 30 seconds at 30 hz followed by 1 min of 30 hz while keeping the sample cold. following clarification via a 3-minute centrifugation (3,000xg; room temperature), supernatants were mixed with an equal volume of rlt lysis buffer. blood and clinical swabs were directly mixed with an equal volume of rlt lysis buffer. 200 µl of each sample lysate was used to extract rna using a magnetic bead-based during post mortem examinations, the upper and lower respiratory tract, central nervous system, lymphatic and cardiovascular systems, gastrointestinal and urogenital systems, and integument were evaluated. lungs were removed in toto and the percentage of the lung surface that was affected by macroscopic lesions was estimated by single veterinarian experienced in evaluating gross porcine lung pathology as previously described [36, 37] . lungs were evaluated for gross pathology such as edema, congestion, discoloration, atelectasis, and consolidation. tissue samples of interest were collected and either fixed in 10% neutral-buffered formalin for histopathological examination or frozen at -80˚c for rt-qpcr testing. tissues were fixed in formalin for 7 days, then transferred to 70% ethanol (thermofisher scientific, waltham, ma, usa) prior to trimming and paraffin embedding following standard automated protocols used in the histology section of the kansas state veterinary diagnostic laboratory. following embedding, tissue sections were cut and stained with hematoxylin and eosin and evaluated by a board-certified veterinary pathologist who was blinded to the treatment groups. to detect sars-cov-2 antibodies in sera, indirect elisas were performed observed. neutralizing sera from sars-cov-2-infected cats from a separate study [38] was used as a positive control. to determine the consensus sequence of the usa-wa/1/2020 virus and to analyze if there were any nucleic acid substitutions in the sars-cov-2 virus after passage in porcine cell lines, rna was extracted from cell culture supernatant as described above. the rna was then subjected to rt-pcr amplification using a tiledprimer approach to amplify the entire sars-cov-2 genome as described previously [39] . briefly, the pcr amplicons were pooled and subjected to library preparation for next generation sequencing using the nextera xt library prep kit (illumina, san diego, ca, usa). the library was normalized and sequenced using a miseq nano v2 2x250 sequencing kit. the sequence was then analyzed by mapping reads to the parent sequence (genbank accession # mn985325) [34] to generate a consensus sequence. the sars-cov-2 usa-wa1/2020 isolate, which was isolated from a human patient in washington state, usa, was used as the parent stock for the study [34] . the to determine the effect of sars-cov-2 infection in domestic pigs, nine sixweek-old sars-cov-2 seronegative piglets were inoculated with a total of 1 x 10 6 tcid50 of the usa-wa1/2020 isolate, which was passaged once in swine st cells ( figure 1 ). the challenge material (total 4 ml) was administered orally (1 ml), intranasally (1 ml; 0.5 ml each nostril) and intratracheally (2 ml) after sedation of the animals. at 1-day post challenge (dpc), six uninoculated sentinel contact pigs were comingled with the principal inoculated animals (3 animals per pen). daily rectal temperatures were recorded for each pig and clinical signs were monitored daily, including observations for signs of lethargy, hyporexia, respiratory distress (coughing, labored breathing, nasal discharge), and digestive issues (diarrhea or vomiting). no significant temperature elevation or change in rectal temperature was observed in the principal inoculated nor sentinel contact pigs throughout the study (figure 3) . moreover, no obvious clinical signs were observed for any of the principal inoculated nor sentinel pigs throughout the 21-day observation period. to detect viral replication in the principal and sentinel pigs, clinical samples were subjected to rt-qpcr to detect the sars-cov-2 n gene ( table 2) table 2 ). the only the exception was a low suspect positive result in a nasal swab at 1 dpc in a principal inoculated pig #161, for which one of two qpcr replicates yielded a low fluorescent amplification curve with a ct of 37 (table 2) . moreover, viral rna was not detected in any lung sample collected at post-mortem examination on 4, 8 and 21 dpc (table 2 ). in addition, gross and histopathological analysis of trachea and lung from the principal challenged pigs did not reveal the presence of any obvious pathological lesions ( table 3 , figure 4 ). these results indicate that sars-cov-2 failed to replicate in the respiratory and digestive tract as well as the blood in orally/intranasally/intratracheally inoculated pigs throughout an observation period of 21 days. this is confirmed by the fact that the principal infected pigs failed to transmit sars-cov-2 to co-mingled sentinel animals. to determine whether the orally/intranasally/intratracheally inoculated pigs sars-cov-2 is a zoonotic agent, and a detailed understanding of the susceptibility of various animal species to sars-cov-2 is central to controlling its spread [16, 17] . in addition, the development of animal models that emulate covid-19 in humans is essential for pre-clinical testing of novel vaccines and therapeutics [20] . in this study, we inoculated nine pigs with a high dose of sars-cov-2 that was passaged once in porcine cells. simultaneous oral/intranasal/intratracheal inoculation did not result in any detectable viral rna in the blood, the oral/nasal/rectal cavities, or the lungs. also, none of the co-mingled, sentinel contact pigs shed viral rna. moreover, a virus-specific immune response characteristic of infection was not observed within the 21-day study period in the principal infected or sentinel pigs. the transient nature of the igm and igg response observed in pig #848 could indicate cross-reactivity of antibodies directed against a porcine coronavirus such as porcine epidemic diarrhea virus [40] . such antibodies could be maternally derived and therefore transient as the lack of sars-cov-2 specific reactivity by the end of the study might suggest. in contrast to previous sars-cov-2 swine studies [22, 26] , the present study used a more stringent inoculation procedure (intratracheal and oral, in addition to intranasal) and 1 log higher titer of virus inoculum (10 6 vs 10 5 ). in addition, the inoculum in the present study was passaged once in porcine st cells. these results, combined with previous intranasal pig inoculation studies [22, 26] , indicate that pigs seem to be resistant to sars-cov-2 infection, are unlikely to be a sars-cov-2 carrier animal species, and are also not suitable as an animal model for research. the results of the present and previous sars-cov-2 inoculation studies in pigs are intriguing in light of the findings that the porcine ace2 receptor seems highly compatible with the sars-cov-2 rbd, suggesting that pigs could be susceptible to sars-cov-2 infection [2, 31] . pigs are susceptible to both experimental and natural infection with sars-cov-1 [29, 30] . however, the experimental sars-cov-1 infection was via simultaneous intranasal/oral/intraocular/intravenous inoculation [29] , thus the actual route(s) of sars-cov infection cannot be determined. recently, several porcine cell lines have been shown to be permissive to sars-cov-2 infection [26, 33] ; in addition, single-cell screening studies showed that porcine ace2/tmprss2 expression are compatible with infection [32] . in contrast to previous reports that some porcine cell lines are susceptible to sars-cov-2 infection, but show no cpe [26, 33] , we found that both st and pk-15 cell lines are susceptible to infection and observed cpe after two or four passages, respectively. the absence of sars-cov-2 replication and transmission in the present and two previous pigs studies [22, 26] seems to lessen the need to monitor pig populations for sars-cov-2 during the ongoing pandemic. however, the evidence described above suggests pig susceptibility should not be disregarded, because all pig studies to date have used rather young pigs and commercially available pig breeds/genetics. we also have to be aware that unforeseen genetic changes in the sars-cov-2 genome may result in a better compatibility of the virus for pigs in the future. pigs are considered to be an excellent model for studying human infectious diseases based on their relatedness to humans in terms of anatomy and immune responses and they have been found to be much more predictive for the efficacy of therapeutics when compared to rodent models [41] . however, the results presented here indicate that pigs are not a suitable preclinical model for sars-cov-2 pathogenesis studies and the development and efficacy testing of therapeutics and/or vaccines. a recently available article indicates that while pigs are not susceptible to sars-cov-2 infection, neutralizing antibody responses were detected in pigs infected via intramuscular or intravenous inoculation routes [42] ; this indicates that pigs could be used for immunogenicity studies related to sars-cov-2. however, the use of pigs to monitor sars-cov-2 immune responses must be careful to screen for cross-reactive maternal antibodies derived from other coronaviruses [43] . alternate pre-clinical animal models, namely non-human primates, syrian hamsters, transgenic or transduced mice expressing human ace2, ferrets, or even cats need to be considered to gain additional insights into sars-cov-2 pathogenesis and virulence. comprehensive characterization of sars-cov-2 pathogenesis in preclinical animal models and the establishment of standardized infection and testing protocols will be crucial for the development of much-need countermeasures to combat covid-19. 1 swabs/blood were tested from samples on 0, 3, 5, 7, 10, and 14 dpc. lung tissue was collected 4, 8, and 21 dpc. 2 swabs/blood were tested from samples on 0, 3, 5, and 10 dpc. lung tissue was collected on 21 dpc 3 swabs/blood were tested on 0 dpc. lung tissue was collected on 3 dpc for these uninfected controls. *one pig (#161) had a ct signal of 37.72 (3.82x10 4 copy number/ml) for 1/2 of rt-qpcr wells on 1 dpc. magnification is 10x for main images and 20x for inserts. coronavirus disease (covid-19) situation report -206 a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical presentation of covid-19: case series and review of the literature epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of coronavirus disease 2019 in china transmission, diagnosis, and treatment of coronavirus disease 2019 (covid-19): a review early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia temporal dynamics in viral shedding and transmissibility of covid-19 the socio-economic implications of the coronavirus pandemic (covid-19): a review coronaviridae study group of the international committee on taxonomy of v. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2. nat microbiol severe acute respiratory syndrome (sars): a review middle east respiratory syndrome coronavirus (mers-cov): a review the proximal origin of sars-cov-2 probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak possible bat origin of severe acute respiratory syndrome coronavirus 2. emerg infect dis is covid-19 the first pandemic that evolves into a panzootic? vet ital covid-19 and veterinarians for one health, zoonotic-and reverse-zoonotic transmissions a critical needs assessment for research in companion animals and livestock following the pandemic of covid-19 in humans. vector borne zoonotic dis are animals a neglected transmission route of sars-cov-2? pathogens evidence for sars-cov-2 infection of animal hosts. pathogens infectivity, virulence, pathogenicity, host-pathogen interactions of sars and sars-cov-2 in experimental animals: a systematic review susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2. science transmission of sars-cov-2 in domestic cats infection and rapid transmission of sars-cov-2 in ferrets sars-cov-2 is transmitted via contact and via the air between ferrets simulation of the clinical and pathological manifestations of coronavirus disease 2019 (covid-19) in golden syrian hamster model: implications for disease pathogenesis and transmissibility pathogenesis and transmission of sars-cov-2 in golden hamsters susceptibility of pigs and chickens to sars coronavirus sars-associated coronavirus transmitted from human to pig. emerg infect dis receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus single-cell screening of sars-cov-2 comparative tropism, replication kinetics, and cell damage profiling of sars-cov-2 and sars-cov with implications for clinical manifestations, transmissibility, and laboratory studies of covid-19: an observational study. lancet microbe severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, united states. emerg infect dis division of viral diseases. real-time rt-pcr panel for detection -2019-novel coronavirus pathogenic and antigenic properties of phylogenetically distinct reassortant h3n2 swine influenza viruses cocirculating in the united states comparison of pathogenicity and transmissibility of influenza b and d viruses in pigs. viruses sars-cov-2 infection, disease and transmission in domestic cats ncov-2019 sequencing protocol v2 v.2 lactogenic immunity and vaccines for porcine epidemic diarrhea virus (pedv): historical and current concepts the pig: a model for human infectious diseases pigs are not susceptible to sars-cov-2 infection but are a model for viral immunogenicity studies emerging and re-emerging we gratefully thank the staff of ksu biosecurity research institute, the the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. key: cord-009820-fi54s0x7 authors: andries, k.; pensaert, m.; callebaut, p. title: pathogenicity of hemagglutinating encephalomyelitis (vomiting and wasting disease) virus of pigs, using different routes of inoculation date: 2010-05-13 journal: j vet med b infect dis vet public health doi: 10.1111/j.1439-0450.1978.tb00754.x sha: doc_id: 9820 cord_uid: fi54s0x7 summary: forty‐eight pigs were inoculated by different routes with the vw 572 isolate of the hemagglutinating encephalomyelitis (vomiting and wasting disease) virus. all piglets inoculated by the combined oral — nasal route (16) or into the infraorbital nerve (3) became sick after an incubation period of 5 days. six of the 7 pigs inoculated into the stomach wall, 6 of the 8 pigs inoculated intramuscularly and 3 of the 5 pigs inoculated intracerebrally became ill after an incubation period of 3–3.5 days. none of the pigs inoculated either intravenously or into the abdominal cavity or into the stomach lumen became sick. all diseased pigs showed the vomiting and wasting syndrome. in oronasally inoculated pigs, killed during the early stages of disease, the virus was reisolated consistently from the tonsils and respiratory tract but irregularly from the pons + medulla and the stomach wall. pigs inoculated by other routes were positive for virus when sick. all except one of the pigs which remained healthy had seroconverted. the site of virus replication which gives rise to the vomition could not be determined. it was concluded from the present studies that virus spread within the body occurs along nerve pathways. zusammenfassung: die pathogenität von hämagglutinierendem enzephalomyelitis‐virus (kümmern und erbrechen) bei shweinen nach infektion über vershiedene inokulationswege achtundvierzig schweine wurden übegr verschiedene inokulationswege mit dem vw 572‐isolat des hämagglutinierendtn enzephalomyelitis‐virus (kümmern und erbrechen) infiziert. alle schweine, die entweder kombiniert oro‐nasal (16) oder über den infraorbitalnerv (3) infiziert wurden, erkrankten nach einer inkubationszeit von 5 tagen. sechs der sieben über die magenwand inokulierten, 6 oder 8 intramuskulär und 3 der 5 intrazerebral infizierten tiere wurden nach einer inkubationszeit von 3–3,5 tagen krank. bei den schweinen, die intravenös oder in die bauchhöhle bzw. direkt in den magen inokuliert wurden, kamen erkrankungsfälle nicht vor. alle erkrankten schweine zeigten das syndrom des kümmerns und erbrechens. von oro‐nasal infizierten schweinen, die während des frühstadiums der erkrankung getötet wurden, konnte das virus regelmäßig von den tonsillen und dem respirationstrakt und gelegentlich vom gewebe des pons‐medulla‐bereiches sowie aus der magenwand reisoliert werden. von schweinen, die nach infektion über andere routen erkrankten, ließ sich immer virus isolieren. alle tiere, die nicht erkrankten (mit ausnahme eines ferkels) bildeten jedoch antikörper. der ort der virusvermehrung, mit dem das erbrechen zusammenhängt, ließ sich nicht ermitteln. die ergebnisse der vorgelegten untersuchungen lassen den schluß zu, daß die virusausbreitung im körper über die nervenbahnen erfolgt. résumé: pathogénicité du virus hémagglutinant de l'encéphalomyélite du porc (dépérissement et vomissement) après infection par différents modes d'inoculation 48 porcs ont été infectés selon différents procédés d'inoculation avec l'isolement vw 572 du virus hémagglutinant de l'encéphalomyélite (dépérissement et vomissement). tous les porcs infectés soit par la voie combinée oronasale (16) soit par le nerf infraorbital (3) tombèrent malades après une incubation de 5 jours. 6 des 7 animaux infectés par la paroi stomacale, 6 des 8 par voie intramusculaire et 3 des 5 intracérébralement tombèrent malades après un temps d'incubation de 3–3,5 jours. il n'y a pas eu de maladie chez les porcs inoculés par voie intraveineuse, dans l'abdomen ou directement dans l'estomac. tous les porcs malades ont présenté le syndrome de dépérissement et de vomissement. chez les animaux infectés par voie oro‐nasale et sacrifiés au début de la maladie, on a pu régulièrement réisoler le virus à partir des amygdales et de l'appareil respiratoire, parfois du tissu de la région «pons‐medulla» et de la paroi stomacale. le virus a toujours été isolé chez les porcs tombés malades après un mode d'infection différent. tous les animaux qui ne furent pas malades (à l'exception d'un porcelet) formèrent des anticorps. l'endroit de multiplication du virus lié au syndrome de vomissement n'a pas été déterminé. les résultats de ces essais permettent de conclure que la propagation du virus dans le corps se fait par voie nerveuse. resumen: la patogeneidad del virus hemoaglutinante de la encéfalomielitis (hipotrepsia y vómitos) en el cerdo tras infección a través de vías diversas de inoculación se infectaron cuarenta y ocho cerdos a través de diferentes vías de inoculación con el aislamiento vw 572 del virus hemoaglutinante de la encéfalomielitis (hipotrepsia y vómitos). todos los cerdos infectados bien con arreglo al procedimiento combinado buco‐nasal (16) o bien a través del nervio infraorbitario (3) enfermaron tras un tiempo de incubación de 5 días. seis de siete animales inoculados a través de la pared gástrica, 6 de 8 por vía intramuscular y 3 de 5 por vía intracerebral enfermaron tras un tiempo de incubación de 3–3,5 días. no se registraron casos de enfermedad en los cerdos inoculados por vía intravenosa o en la cavidad abdominal resp. directamente en el estómago. todos los cerdos que enfermaron presentaban el síndrome de hipotrepsia y vómitos. de los cerdos infectados por vía buco‐nasal, que se sacrificaron durante el estadio precoz de la enfermedad, se pudo reaislar el virus con regularidad de las tonsilas y del tracto respiratorio y, en ocasiones, del tejido correspondiente al ámbito puente de varolio‐medula, así como de la pared gástrica. se logró siempre aislar virus de cerdos que enfermaron tras infección por otras vías. sin embargo, todos los animales que no enfermaron (excepción hecha por un lechón) produjeron anticuerpos. no se logró descubrir el lugar en donde se multiplicaba el virus, hecho relacionado con los vómitos. los resultados de los estudios presentados permiten llegar a la conclusión de que la propagación del virus en el organismo acontece a través de las vías nerviosas. in 1961, an infectious encephalomyelitis of viral origin was observed in pigs in canada (7) . the clinical illness was characterised by a prodromal stage with anorexia, depression, retching and vomiting, followed by motor disorders (1) . the virus responsible for this disease was later found to be a porcine coronavirus (8; 13) which, because of its hemagglutinating properties, was called hemagglutinating encephalomyelitis virus or hev (7) . an infectious disease of suckling pigs, characterised by vomiting and wasting, was described in england in 1969 (2) and later observed in other european countries (4; 6; 11; 15; 16) . the disease, called vomiting and wasting disease, was shown to be caused by a virus antigenically and structurally similar if not identical to the hev from canada (3; 12; 13). a similar coronavirus, which caused mainly vomiting and wasting symptoms in hysterectomy-derived colostrum-deprived pigs was also reported from the u.s.a. (9). using the same field isolates mengeling was able to reproduce experimentally the two clinical entities, the encephalitic form and the vomiting and wasting form (10) . it was, therefore, concluded that both types of the disease, are caused by the same virus. few studies have been performed on the pathogenesis of this viral infection. transmission experiments have proved that reproducible results could be obtained only if colostrum-deprived pigs were used (7; 14). clinical disease has been succesfully reproduced after inoculation of the virus by oral, intranasal, intramuscular and intracerebral routes (7) . in other studies, the virus was reisolated with regularity from brain, respiratory tract and oronasal swabs, but only if the pigs were examined during the early stages of the disease (3; 12) . hemagglutination inhibiting and seroneutralizing antibodies were demonstrable starting at 6 and 9 days after inoculation respectively (12). these antibodies are suspected to preclude virus isolation at later stages of the disease (5; 9; 12). the present studies were primarily designed to determine whether a virus isolate, obtained from pigs with the vomiting and wasting syndrome only, could produce clinical signs after inoculation by different routes. at the same time, some information was obtained on the optimal conditions for virus reisolation and on the distribution of the virus in different organs particularly after oronasal inoculation. 2.1. virus the virus strain, designated vw 572, was isolated in belgium in 1972 as previously described (12) . the virus stock used represented the loth passage in primary pig kidney (ppk) cells and contained 10"' tcid,, per ml. the studies were performed in hysterectomy-derived-colostrum-deprived (hdcd) pigs which were individually housed in horsfall type units. they were inoculated at the age of 3 to 5 days using different routes of inoculation. a blood sample was withdrawn prior to inoculation to assure the absence of specific viral antibodies. sixteen piglets were inoculated by combined oral and nasal (on) route, using 2 ml. of stock virus. in seven piglets, 0.7 to 1 ml. of stock virus was inoculated directly into the stomach wall after laparotomy around the pyloric sphincter at four inoculation sites. five pigs were inoculated with 1 ml. of stock virus directly into the gastric lumen after laparotomy by inserting a needle through a canula which perforated the stomach wall. in two pigs, 1 ml. of stock virus was brought into the abdominal cavity after laparatomy. two pigs were inoculated with 1 ml. of stock virus intravenously into the ear vein. five pigs were inoculated in the cerebral cortex, using 1 ml. of stock virus, by perforating the skull with a needle. four pigs were inoculated into the infraorbital nerve with 0.5 ml. of stock virus by insertion of the needle into the infraorbital canal. eight pigs were inoculated with 1 ml. of stock virus intramuscularly either in the neck muscles or in the foreleg muscles or in the hindleg muscles. all pigs were observed three times a day for clinical signs. when sick, pigs were killed at time intervals varying from one to five days after the appearance of clinical signs and different tissues were collected for virus isolation. from the on inoculated pigs, serum was collected at the time of killing for detection of hemagglutination inhibiting (hi) and seroneutralizing (sn) antibodies. the pigs which did not become sick during the present study were followed clinically until 3 weeks after the inoculation. during the last week of the observation period, a serum sample was collected and examined for the presence of hi-antibodies. from the pigs killed at different time intervals after inoculation, the following tissues were collected for virus isolation : nasal mucosa, tonsils, lungs (apical and cardiac lobes), pyloric region of the stomach, pons and medulla combined, cerebrum, cerebellum and blood clot. from each organ, a 20 per cent suspension (w/v) was prepared in phosphate buffered saline by homogenisation. after centrifugation at 7500 x g., the supernatant fluid was collected and 0.1 ml. inoculated in each of 10 tubes with fully sheeted primary pig kidney (ppk) cells. after four days, hemadsorption with chicken erythrocytes was attempted on 5 tubes. the 5 remaining tubes were used for making a second passage. the cells and culture medium were subjected to two cycles of freezing and thawing and, after centrifugation a t 700 x g., the supernatant was inoculated in 5 tubes with fresh monolayers of ppk-cells. hemadsorption was performed on all tubes 4 days later. the procedures used for the hemagglutination inhibition and seroneutralisation test were previously described (12) . in the presentation of the results, differentiation will be made between the on inoculated pigs and the pigs inoculated by other routes. the number of pigs in the on inoculated group was sufficiently high to allow a more complete study since animals could be killed at several time intervals after the appearance of clinical signs. for the other routes of inoculation, it was the main purpose to see whether clinical illness could be obtained or not. the overall results obtained after on inoculation of the 16 pigs are presented in table i . the individual results of these pigs are presented in table 2 . all pigs were free of hiand sn-antibodies at the time of inoculation. it can be seen from the tables that all on inoculated pigs became ill. the incubation period varied from 3 to 7 days with an average of 5 days. the earliest clinical signs were characterised by anorexia and vomition which suddenly appeared. the vomition usually started after the uptake of milk. the vomit first consisted of milk curds and changed, after a few hours, to a yellow foamy fluid. as soon as the piglets took up more milk, curds were seen again. vomition and retching movements were very regularly present during the first 24 hours. afterwards, the frequency of vomition decreased but the appetite remained markedly reduced. the smaller pigs became emaciated and moribund after 3 to 4 days. the course of the disease was somewhat slower in the stronger pigs. virus could be isolated from 11 of the 16 pigs. as shown in table 2 , the virus was isolated most consistently from the lungs and tonsils. it was sometimes isolated from the pons + medulla and nasal mucosa, and only in 2 pigs from the stomach wall. virus was not isolated from the cerebrum, the cerebellum and the blood clot. all pigs which were killed 6 days or later after inoculation had hi-antibodies, whereas sn-antibodies were detected starting 7 days after inoculation. the pons + medul li a of these pigs was positive by immunofluorescence. the overall results obtained in the pigs inoculated by other routes are presented in table 1 . none of the pigs inoculated into the stomach lumen, the abdominal cavity or the ear vein had become sick at 3 weeks after inoculation. all these animals, except one of the pigs inoculated into the gastric lumen, had hi-antibodies at that time. a variable number of pigs became sick after inoculation by other routes. the clinical disease was not different from that observed after on inoculation. in table 2 , the sick pigs were considered to be positive for virus when it was isolated from one or more of the collected organs, the inoculated organ not included. six of the seven pigs inoculated in the pyloric region of the stomach became sick after an average incubation period of 3 days. from two of the six pigs, the virus was reisolated from the stomach wall only whereas, in the remaining four, the virus could be detected in other tissues also. hi-antibodies (titer 64) were present in the serum of the pig without clinical signs at the end of the observation period. of the intracerebrally and intramuscularly inoculated pigs, three of the five and six of the eight respectively became sick after an average incubation period of 3 * / 2 days, all the diseased pigs were positive for virus. the pigs which remained healthy had built up hi-antibodies at titers varying from 6 to 128 at the end of the experiment. all three pigs inoculated into the infraorbital nerve became sick after an average incubation period of 5 days and were positive for virus. the clinical disease and the results of virus isolation obtained upon combined oral and nasal (on) inoculation were similar to those previously described (12) . the virus was most consistently reisolated from the respiratory tract which is probably the natural way of infection. seroneutralizing antibodies appear soon after the beginning of the clinical symptoms and may be a limiting factor for the further succesful isolation of the virus from organs. for the diagnosis of hemagglutinating encephalomyelitis by virus isolation, pigs should therefore be examined preferably within two days after the start of clinical signs. the negative results obtained in pigs 220 and 21.1, killed within this period of time, are probably due to the fact that tonsils and pons + medulla were not collected for virus isolation. the pons + medulla of these pigs was later found to be positive by immunofluorescence. the irregularity with which virus was isolated from the stomach wall and pons + medulla does not allow any conclusions to be drawn about the exact target organ, if it is supposed that virus replication in one of these organs is responsible for clinical signs. this irregularity may be due to the lack of sensitivity of the virus isolation technique. recently, it has been found that centrifugation of tissue suspensions at 7500 x g. instead of 2000 x g. can remove up to 1 log,, tcid,, of hemagglutinating encephalomyelitis virus. in this way, virus isolation from a tissue suspension with little infectious virus could give a false negative result. it is known from electron microscopic studies that hev during intracellular replication is mainly present in cytoplasmic vesicles with a large amount of membranous material (8). the removal of virus by centrifugation may, therefore, be due to sedimentation of particles associated with subcellular fragments. upon inoculation into the stomach lumen, clinical signs were not observed. this finding indicates that virus reaching the gastric lumen after swallowing does not initiate clinical disease. however, the seroconversion observed in 4 of the 5 pigs indicates that a subclinical infection has occurred. the ability to reproduce typical disease after a very short incubation period in 6 of the 7 pigs inoculated in the stomach wall may be significant in the light of the pathogenesis. in two of these piglets, virus was isolated from the stomach wall only. this points to the stomach wall as a possible target organ for the virus. it is, however, not excluded that the isolated virus is a residue of the inoculum rather than a result of virus replication. the first possibility is rather improbable since these pigs were killed 3 and 4 days after inoculation respectively. in the present experiments, not all the pigs became sick after intracerebral inoculation and the disease consisted of the vomiting and wasting syndrome. these results are somewhat different from those described by greig, who obtained clinical signs consisting of motor central nervous disorders in all inoculated pigs (7) . this may be related to the observation that, with the present virus isolate, only the vomiting and wasting syndrome has been observed, not only in the original outbreak but also in experimental pigs. it should be mentioned, however, that mengeling was able to reproduce both clinical entities with the same virus isolate (10) . the inability to find the virus in the cerebral cortex of on inoculated pigs may indicate that the present isolate has little or no affinity for this part of the central nervous system. it is not impossible that, in intracerebrally inoculated pigs, the appear-ance of clinical signs is determined by the ability of the virus to reach the pons + medulla, another possible target organ, from the deposition site. the findings that none of the pigs became sick after intravenous inoculation and that the virus was never isolated from the blood clot of pigs killed at different time intervals after on inoculation indicate that the target organ is not reached through viraemia. on the other hand, the ability to obtain typical disease after inoculation into the infraorbital nerve suggests that virus spread within the body occurs via nerve pathways. whether clinical signs are obtained in pigs after intramuscular inoculation may be dependent on the ability of the virus to enter into nerves. from the present studies, it was impossible to determine the exact target organ of the vw 572 virus. the results indicate that virus replication, either in the stomach wall or in the pons + medulla or possibly in both, may be responsible for causing the vomiting and wasting syndrome in pigs. forty-eight pigs were inoculated by different routes with the vw 572 isolate of the hemagglutinating encephalomyelitis (vomiting and wasting disease) virus. all piglets inoculated by the combined oralnasal route (16) or into the infraorbital nerve (3) became sick after an incubation period of 5 days. six of the 7 pigs inoculated into the stomach wall, 6 of the 8 pigs inoculated intramuscularly and 3 of the 5 pigs inoculated intracerebrally became ill after an incubation period of 3-3.5 days. none of the pigs inoculated either intravenously or into the abdominal cavity or into the stomach lumen became sick. all diseased pigs showed the vomiting and wasting syndrome. in oronasally inoculated pigs, killed during the early stages of disease, the virus was reisolated consistently from the tonsils and respiratory tract but irregularly from the pons + medulla and the stomach wall. pigs inoculated by other routes were positive for virus when sick. all except one of the pigs which remained healthy had seroconverted. the site of virus replication which gives rise to the vomition could not be determined. it was concluded from the present studies that virus spread within the body occurs along nerve pathways. the study was supported by the belgian research institute for industry and agriculture (instituut tot aanmoediging van het wetenschappelijk onderzoek in nijverheid en landbouw), brussels, belgium. the technical assistance of gerry van (3) la patogeneidad del virus hemoaglutinante de la enckfalomielitis (hipotrepsia y vbmitos) en el cerdo tras infeccibn a travds de vias diversas de inoculacibn se infectaron cuarenta y ocho cerdos a travks de diferentes vias de inoculacibn con el aislamiento vw 572 del virus hemoaglutinante de la enckfalomielitis (hipotrepsia y v6mitos). todos 10s cerdos infectados bien con arreglo a1 procedimiento combinado buco-nasal (1 6 ) o bien a travks del nervio infraorbitario (3) enfermaron tras un tiempo de incubacidn de 5 dias. seis de siete animales inoculados a travks de la pared glstrica, 6 de 8 por via intramuscular y 3 de 5 por via intracerebral enfermaron tras un tiempo de incuba-ci6n de 3-3,5 dias. no se registraron casos de enfermedad en 10s cerdos inoculados por via intravenosa o en la cavidad abdominal resp. directamente en el estbmago. todos 10s cerdos que enfermaron presentaban el sindrome de hipotrepsia y v6mitos. de 10s cerdos infectados por via buco-nasal, que se sacrificaron durante el estadio precoz de la enfermedad, se pudo reaislar el virus con regularidad de las tonsilas y del tract0 respiratorio y, en ocasiones, del tejido correspondiente a1 lmbito puente de varolio-medula, asi como de la pared gistrica. se logr6 siempre aislar virus de cerdos que enfermaron tras infecci6n por otras vias. sin embargo, todos 10s animales que no enfermaron (excep-ci6n hecha por un lech6n) produjeron anticuerpos. no se logr6 descubrir el lugar en donde se multiplicaba el virus, hecho relacionado con 10s v6mitos. los resultados de 10s estudios presentados permiten llegar a la conclusi6n de que la propagaci6n del virus en el organism0 acontece a travds de las vias nerviosas. vomiting and wasting disease in piglets isolement en france et identification du virus de la maladie du vomissement et du dcpcrissement des porcelets (coronalike virus) lesions induced by hemagglutinating enccphalomyelitis virus strain 67 n in pigs vomiting and wasting disease in piglets a hemagglutinating virus producing encephalomyelitis in baby pigs characteristics of a coronavirus (strain 67 n) of pigs experimentally induced infection of newborn pigs with hemagglutinating encephalomyelitis virus strain 67 n pathogenicity of field isolants of hemagglutinating encephalomyelitis virus from neonatal pigs characteristics of a coronavirus causing vomition and wasting in pigs the size and morphology of tge and vomiting and wasting disease viruses of pifs a viral encepha omyelitis of pigs. further studies on the transmissibility of the disease in ontario vomiting and wasting disease of piglets key: cord-348522-r7ev9br6 authors: englund, stina; hård af segerstad, carl; arnlund, frida; westergren, eva; jacobson, magdalena title: the occurrence of chlamydia spp. in pigs with and without clinical disease date: 2012-01-26 journal: bmc vet res doi: 10.1186/1746-6148-8-9 sha: doc_id: 348522 cord_uid: r7ev9br6 background: within the genera chlamydia, the development of refined diagnostic techniques has allowed the identification of four species that are capable of infecting pigs. the epidemiology, clinical, and zoonotic impacts of these species are however largely unknown. the study aimed to investigate the presence of chlamydia spp. in the intestines of growing pigs and in conjunctival swabs from finisher pigs, and relate the findings to clinical signs. results: by histology, 20 of 48 pigs had intestinal lesions that may be consistent with chlamydial infection. by pcr, forty-six of the pigs were positive whereas two samples were inhibited. sequencing of 19 dna extracts identified these as chlamydia suis. by immunohistochemistry, 32 of 44 samples were positive and a significant relationship was detected between macroscopically visible intestinal lesions and a high degree of infection. by real-time pcr, a significant difference was detected between pigs with and without conjunctivitis when a ct value of 36 was employed but not when a ct value of 38 was employed. conclusions: chlamydia suis was demonstrated in most samples and overall, no correlation to clinical signs was detected. however, a correlation was noted between samples with a high degree of infection and the presence of clinical signs. it is possible, that the intensive pig production systems studied might predispose for the transmission and maintenance of the infection thus increasing the infectious load and the risk for disease in the pig. the chlamydiaceae family including the genera chlamydia is a well-recognised cause of disease in many animal species including cats, ruminants, birds and humans [1, 2] . within the genera, nine distinct species have been identified. four of these have been described in pigs (chlamydia (c.) abortus, c. psittaci, c. pecorum, and c. suis) and related to reproductive disorders, conjunctivitis, enteritis, pneumonia, polyarthritis, pleuritis and polyserositis [1, [3] [4] [5] . the epidemiology, clinical, and zoonotic importance of these species are however largely unknown [6] [7] [8] . however, c. suis seems to be both common and widespread, often occurring in mixed infections with c. abortus and c. pecorum [2] . in gnotobiotic pig challenge studies from usa, c. suis caused dose-dependent diarrhoea in young piglets. histologically, villi atrophy, tip erosions, necrosis, inflammatory changes and lymphangitis were noted in the distal small intestine. chlamydial antigens were demonstrated in enterocytes and chlamydiae were reisolated from tissue specimens and faecal swabs [9, 10] . however, weaned pigs developed microscopical lesions but remained clinically healthy [11] . similar lesions have also been demonstrated in clinical cases of diarrhoea in weaned pigs [12] . however, other studies have not been able to confirm a causal relationship [3, 8] . a few studies have addressed conjunctivitis [13] . chlamydiae were seen in eight pigs by ultrastructural examination and were isolated in two pigs [4] , and subclinical conjunctivitis was experimentally induced in 3-day-old gnotobiotic piglets [14] . isolation is the gold standard for diagnosis but many strains are difficult to cultivate [2, 5] . other methods includes serology, immunohistochemistry, histology, and pcr on faeces, mucosal swabs or tissue specimens [2] . because of cross-reactivity, some tests may lack sensitivity and specificity [5, 12, 15] . in a study comparing four diagnostic methods, c. suis were demonstrated in 42% of the samples by pcr and in 33% by culture. the sensitivity was 94.4% and specificity 81.0%, whereas the two elisas performed considerably weaker. pcr was concluded to be a reliable, highly sensitive and specific tool for detection of c. suis [16] . the present study aimed to investigate the presence of chlamydia spp in the intestines of growing pigs and in conjunctival swabs from finisher pigs, and relate those findings to the occurrence of clinical signs. the study was approved by the ethical committee for animal experiments, uppsala, sweden. all herd owners had given an informed consent prior to the study. dna from enteric specimens originated from a previous study on growing pigs with diarrhoea [17] . the samples originated from the major pig producing areas of sweden, i.e. in the south-western and eastern parts of the country, and included 36 pigs from six herds with poor performance and diarrhoea in growing pigs, and 12 pigs from four herds with good performance and no diarrhoea. from each of the poor performance herds, three case and three control pigs were chosen, and from each of the four good performance herds, three control pigs were chosen. the case pigs had diarrhoea that had commenced within 2 days and no other diseases were evident. the control pigs were matched to the case pigs for age and sex, but showed no signs of clinical disease. the control pigs had a mean age of 72 days and a mean weight of 18.7 kg, and the case pigs had a mean weight of 12.1 kg. the pigs submitted from the good performance herds had a mean age of 67 days and a mean weight of 23.1 kg. all animals were weaned at approximately 5 weeks of age and none of the pigs had been treated with antibiotics. the pigs were transported to the laboratory and euthanized within 15 min prior to necropsy. further, animals from three finisher herds were sampled based on the present occurrence of conjunctivitis. the pigs originated from several piglet-producing herds and were introduced to the finisher herds at 25-30 kg. b.w., i.e. 4-14 weeks prior to sampling. they were kept 10 pigs per pen in units of 300 pigs each. in two herds, 7 case and 7 control pigs were selected from each of 3 units, and in one herd, 20 case and 20 control pigs from one unit were selected. the case pigs were selected based on clinical signs of moderate to severe conjunctivitis, defined as hyperaemia and chemosis with epiphora and/or muco-purulent secretion. the control pigs were selected from the same pens as the case pigs but showed no or only mild conjunctivitis, i.e. none to slight hyperaemia in the conjunctiva and no chemosis or epiphora. the necropsies were carried out at the department of pathology at the national veterinary institute, uppsala, or at analycen, skara. the animals were stunned with electricity, weighed and exsanguinated, and necropsy was immediately performed. all gross lesions were noted, and specimens for histological examination were taken from ileum and from all macroscopically visible lesions. the samples were fixed in 10% buffered formalin, embedded in paraffin blocks, sectioned and stained with haematoxylin and eosin according to standard protocols. the finisher pigs were snared and sterile cotton swabs were rubbed against the conjunctiva in the conjunctival sac in the left and right eye, respectively. the swabs were placed in sterile tubes and transported to the laboratory. intestinal samples were prepared from a 0.5 cm 3 piece of frozen mucosa from the distal ileum. dna was extracted by phenol/chloroform and precipitated by ethanol [18, 19] . dna from the conjunctival swabs was extracted according to k. sachse [20] and all samples were analysed by real-time pcr according to everett and others [21] . the primers tqf and tqr targeted the 23 s ribosomal dna and detected all members of the family chlamydiaceae. multiple negative controls were included and dna from cp. abortus and c. suis (kindly provided by k. sachse, friedrich-loeffler-institute, jena, germany) were used as positive controls. to detect false negative pcr results due to inhibiting agents, an internal amplification control (mimic) was constructed and used as previously described [22] . the primers used in the mimic producing pcr were tqfactin (5'gaaaagaacccttgttaagggagc-catgtaccctggcattg-3') and tqractin (5'-cttaactccctggctcatcatggatccacacg-gagtacttgc-3'). the sequence of the rox-labelled mimic probe used in real-time pcr was 5'-ccgacag-gatgcagaaggagatca-3'. the conjunctival swabs were analysed both undiluted and diluted 1:100 in accordance with the standard protocol applied at the nvi, and a positive reaction in at least one of these dilutions was regarded as positive. in the statistical analyses, threshold values below ct 36 were regarded as positive whereas values between ct 36 and ct 38 were regarded as doubtful. the results were calculated separately for the respective thresholds. values above ct 38 were regarded as negative or were excluded in the calculations. the difference in detection rate of chlamydia spp. between pigs with moderate to severe conjunctivitis and the control pigs were analysed by chi-square test. sequence analysis was performed on 19 samples from the intestinal specimens giving a strong positive reaction in the pcr. pcr was performed with primers 16sf2 and 23r according to everett and andersen [23] on dna samples from seven case pigs and six control pigs from poor performance herds and six pigs from good performance herds. the predicted pcr product of 600 bp as well as one pcr product of larger and one of smaller size was purified prior to sequencing by using the gfx pcr dna and gel band purification kit (amersham bioscience europe, gmbh germany). the purified products were sequenced with the same primers used in the pcr and by using the bigdye terminator v3.1 cycle sequencing kit (applied biosystems, foster city, ca, usa) in combination with ethanol/edta/ sodium acetate precipitation, according to the protocol supplied. thermocycling was performed in a geneamp 2700 thermocycler (applied biosystems). the sequencing products were subjected to electrophoretic separation and on-line detection on an abi prism 3100 genetic analyzer (applied biosystems), followed by blast search. paraffin-embedded intestinal specimens were available from 44 of the 48 growing pigs. 4-μm thick sections of the formalin-fixed, paraffin-embedded blocks where cut and placed onto positively charged slides (polysine™, menzel-gläser, braunschweig, germany). prior to immunostaining, deparaffinisation and hydration where done in xylene and graded ethanol to distilled water. after hydration, a blocking for endogenous peroxidase where done in 0.03% h 2 o 2 in tris-buffered saline (tbs) and nonspecific binding sites where blocked with 2% bovine serum albumin (bsa). antigen retrieval was performed by heat induced epitope retrieval (hier) in retrieval buffer ph = 9 (tbs-edta), using microwave as heat source. hier was performed by heating the polysine™-slides immersed in retrieval buffer for 7 min at 750 w followed by 14 min at 340 w. after completed heating procedure, the slides remained in the retrieval buffer at room temperature for 20 min. the slides where incubated with a mouse monoclonal antibody against chlamydia, clone aci (progen biotechnik gmbh, germany) diluted 1:100. visualisation of the bound primary antibody was achieved by using dako envision™ + system utilising an hcp-labelled antimouse monoclonal antibody (glostrup, denmark), and the presence of the relevant antigen was detected with 3,3'-diaminobenzidine (dab, nvi, sweden). slides were weakly counterstained with haematoxylin. the staining included at least one positive and one negative control section. the positive control originated from previously confirmed routine cases. the results were graded as negative (-), sparse (+), moderate (++) or abundant (++ +) occurrence. no gross lesions were observed in the pigs from the good performance herds. in the pigs from the poor performance herds, five control pigs and 20 pigs with diarrhoea had macroscopic lesions consistent with lawsonia (l.) intracellularis infection and one had a parasitic colitis. by histology, lesions that may be consistent with chlamydial infection [9] were noted in 20 pigs: villus atrophy was noted in six pigs with diarrhoea and in ten control pigs from the poor performance herds. epithelial exocytosis or necroses were noted in five pigs with diarrhoea, in one control pig from the poor performance herds, and in one pig from the good performance herds (table 1) . in the previous study [17] , brachyspira pilosicoli were demonstrated in eight case pigs and five control pigs, campylobacter jejuni; in one case pig and four control pigs, yersinia enterocolitica in three case and five control pigs, haemolytic escherichia (e.) coli in two case and two control pigs, clostridium perfringens in two case pigs, and l. intracellularis were previously demonstrated in 11 case and eight control pigs from the poor performance herds. in addition, haemolytic e. coli had been demonstrated in two pigs from the good performance herds. rotavirus had been demonstrated in one case pig. coronavirus was not included in the study, since sweden has previously been shown to be free from table 1 the findings in intestinal specimens from growing pigs with diarrhoea (case), clinically healthy control pigs from the same poor performance herds (casecontrol), and from healthy pigs originating from good performance herds (control), examined by immunohistochemistry (ihc), necropsy, and pcr. transmissible gastroenteritis and porcine epidemic diarrhoea. balantidium coli were demonstrated in one case and three control pigs from the poor performance herds, and in one pig from the good performance herds. of the 48 enteric samples analysed, 46 were positive for chlamydiaceae (table 1) . two samples could not be evaluated due to amplification inhibition. the major band of 600 b.p. found in all samples was confirmed as c. suis in the 19 samples subjected to sequence analysis. the larger pcr product described in the methods' section was 98-100% identical to escherichia coli whereas the smaller pcr product did not match any sequence in the blast search. in the pcr analyses on the conjunctival swabs, one pig with conjunctivitis and three control animals together with their matched counterparts were excluded from the statistical calculations, since the pcr was partially or totally inhibited. thus, in total, 116 conjunctival swabs were included in the statistical analyses. of these, three samples from pigs with conjunctivitis gave weakly positive reactions (ct > 38 in undiluted samples) and were judged as negative. employing a cut-off value of ct 36, 45 samples from pigs with conjunctivitis and 35 control pigs were determined as positive by pcr. using a cut-off value of ct 38, 48 samples from pigs with conjunctivitis and 42 control pigs were determined as positive. a statistically significant difference (p = 0.03) was detected between pigs with and without conjunctivitis when a ct value of 36 was employed. the difference was mainly related to one of the herds (18 positive pigs with conjunctivitis and 12 positive control pigs). when a ct of 38 was employed, no significant differences were noted. the results are shown in table 1 . among the case pigs, 6 (40%) were negative or were carrying a low-grade infection. among the control pigs from good performance herds, 9 (75%) were negative or sparsely (+) infected. the two control specimens in which the pcr analyses had been inhibited were graded as + and ++, respectively, by immunohistochemistry. a significant (p < 0.05) relationship was detected between the presence of clinical signs and a high degree (++ or +++) of infection. no relationship was detected between the occurrence of histological lesions and the demonstration of chlamydial antigen by immunohistochemistry. the pig intestine is considered as the natural reservoir for c. suis, and the microbe seems generally to be well adapted to its host [1] . in the present study, chlamydia spp. were demonstrated in high prevalence in all herds investigated. this is consistent with the few studies that have previously addressed the occurrence of chlamydia spp. in pig herds. overall, it was not possible to relate the demonstration of the microbe to the presence of diarrhoea. however, based on the immunohistochemistry, pigs with diarrhoea might have been more heavily infected than the healthy pigs. this is in consistency with the results from another study on 447 pigs submitted for necropsy, where it was not possible to relate the presence of chlamydia spp. to the occurrence of diarrhoea, but in 12 cases, it was the only pathogen found [6] . similar experiences have also been noted in calves and sheep [24] [25] [26] . in the study by becker et al. [13] , c. suis was significantly (p < 0.0001) related to conjunctivitis in extensive pig production systems, whereas in intensive farming systems, high prevalences (88-90%) were found in both pigs with conjunctivitis and in clinically asymptomatic pigs. this is in accordance with the findings in the present study. becker et al. discussed, that environmental factors might predispose to infection. however, in one of the herds in the present study, factors such as emission of ammonium and carbon dioxide gases, air movements and overcrowding were investigated, but it was not possible to relate any environmental factor to the occurrence of conjunctivitis (data not shown). however, in one stable a high relative humidity (83%) was noted that might facilitate microbial survival. in the present study, the pigs originated from several piglet producing herds and it should be emphasized that intensive farming systems with the mixing of pigs from several sources also imply increased opportunities for microbial spread among animals of different immunological status. in fact, in one herd a significant relationship was noted between conjunctivitis and the presence of chlamydia spp. when the lower threshold value was applied in the real-time pcr. this might further indicate that the infectious load is important in the development of disease. although chlamydia spp. was demonstrated in the ileal sections by immunohistochemistry in the present study, lesions compatible to those described in gnotobiotic pigs [9] were only noted in 50% of the pigs. several authors speculate that the development of lesions may depend on different factors such as the virulence of the strain, the infectious dose, the route of infection, or the age and the immunological status of the host [1, 6, 8, 10, 11, 27] . since young, naive pigs seem to develop lesions in response to an experimental infection [9] , host immunity might be induced at an early age in the field [11, 28] . it is also possible that co-infections with other presumptive pathogens might act synergistically to exacerbate the lesions or that lesions induced by one microbe might increase the susceptibility to other infections. several pathogens have been discussed in this respect [3, 6, 11, 13, 29, 30] . most of the intestinal lesions described in the present study were shown to be related to infections with l. intracellularis or b. pilosicoli [17] . some studies also report the occurrence of mixed infection with several chlamydial species [3, [31] [32] [33] . in wild boars, c. psittaci was the dominating species but c. suis and c. abortus was also demonstrated by sequencing [31] . in the present study, preliminary data obtained by pcr-rflp indicated the co-infection by other species [34] . however, sequencing of the amplicons revealed the involvement of c. suis only, whereas other bands detected by pcr originated from e. coli and un-identified bacterial species. the results underline the difficulties involved in the diagnosis and a large variation in reported prevalences between various detection methods exist [3, 15, 16, 35] . in the present study, the problem was circumvented by the use of an internal probe and a high melting temperature in the real-time pcr, combined with sequencing of the amplicons that assured a high specificity. chlamydia suis was found in high prevalences in growing pigs with or without diarrhoea, and in finisher pigs with or without conjunctivitis. overall, no correlation to clinical signs was detected. however, a correlation was noted between samples with a high degree of infection and the presence of clinical signs. it is possible, that the intensive pig producing systems investigated in the present study might predispose for the transmission and survival of the microbe, thus increasing the infectious load and the risk for disease in the pig. interestingly, no other species of the chlamydiaceae family were detected, as also supported by the findings in other animal species in swedish surveys [36] . chlamydial infection in animals: a review chlamydiaceae infections in pig pcr-based detection of chlamydial infection in swine and subsequent pcr-coupled genotyping of chlamydial omp--gene amplicons by dna-hybridization, rflpanalysis, and nucleotide sequence analysis conjunctivitis and keratoconjunctivitis associated with chlamydiae in swine chlamydial infections of domestic ruminants and swine: new nomenclature and new knowledge prevalence of intestinal chlamydial infection in pigs in the midwest, as determined by immunoperoxidase staining intestinal chlamydia in finishing pigs immunohistologischer nachweis von chlamydia psittaci/pecorum und c. trachomatis im ferkel-darm intestinal lesions caused by two swine chlamydial isolates in gnotobiotic pigs experimental enteric infection of gnotobiotic piglets with chlamydia suis strain s45 intestinal lesions caused by a strain of chlamydia suis in weanling pigs infected at 21 days of age small intestinal chlamydia infection in piglets intensively kept pigs pre-disposed to chlamydial associated conjunctivitis conjunctivitis caused by a swine chlamydia trachomatis-like organism in gnotobiotic pigs chlamydial zoonoses. dtsch ärtzebl int detection of chlamydia suis from clinical specimens: comparison of pcr, antigen elisa, and culture diarrhoea in the growing pig-a comparison of clinical, morphological and microbial findings between animals from good and poor performance herds routine diagnostics of lawsonia intracellularis performed by pcr, serological and post mortem examination, with special emphasis on sample preparation methods for pcr diagnosis of contagious caprine and contagious bovine pleuropneumonia by pcr and restriction enzyme analysis. int symp diagn contr livestock dis nucl tech int atomic energy agency detection and differentiation of chlamydiae by nested pcr rapid detection of the chlamydiaceae and other families in the order chlamydiales: three pcr tests detection of mycobacterium avium subsp. paratuberculosis in tissue samples by single, fluorescent and nested pcr based on the is90 gene identification of nine species of the chlamydiaceae using pcr-rflp behavior of different bovine chlamydial agents in newborn calves pathologic changes in intestinal chlamydial infection of newborn calves high frequency of chlamydial co-infections in clinically healthy sheep flocks an experimentally induced chlamydia suis infection in pigs results in severe lung function disorders and pulmonary inflammation experimental chlamydia psittaci serotype 1 enteric infection in gnotobiotic piglets: histopathological, immunohistochemical and microbiological findings intestinal chlamydia in pigs concurrent infection of enterocytes with eimeria scabra and other enteropathogens in swine occurrence of chlamydiaceae spp. in a wild boar (sus scrofa l.) population in thuringia (germany) mixed infections with porcine chlamydia trachomatis/ pecorum and infections with ruminant chlamydia psittaci serovar 1 associated with abortions in swine detection of all chlamydophila and chlamydia spp. of veterinary interest using speciesspecific real-time pcr assays the occurrence of chlamydia species in intestinal specimens from swedish grower pigs. the 20th international pig veterinary society congress prevalence of chlamydial infection in breeding sows investigation of chlamydophila spp. in dairy cows with reproductive disorders the occurrence of chlamydia spp. in pigs with and without clinical disease we wish to thank marianne persson for skilful technical assistance. we also wish to thank the herd owners who kindly supplied the pigs and allowed us to enter the herds. authors' contributions se was responsible for the pcr investigation and sequencing of dna from the intestinal specimens, analysis and interpretation of these data, and revised the manuscript. chs made an intellectual contribution by drafting the manuscript and revising it critically. fa was responsible for collecting of the conjunctival swabs, pcr analysis and interpretation of these data, and revised the manuscript. ew was responsible for the immunohistochemical analyses, interpretations of these data, and revised the manuscript. mj was planning the studies, collected and prepared the intestinal specimens, and was responsible for drafting and writing the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-302155-hksmt48i authors: mclean, rebecca k.; graham, simon p. title: vaccine development for nipah virus infection in pigs date: 2019-02-04 journal: front vet sci doi: 10.3389/fvets.2019.00016 sha: doc_id: 302155 cord_uid: hksmt48i nipah virus (niv) causes a severe and often fatal neurological disease in humans. whilst fruit bats are considered the natural reservoir, niv also infects pigs and may cause an unapparent or mild disease. direct pig-to-human transmission was responsible for the first and still most devastating niv outbreaks in malaysia and singapore in 1998–99, with nearly 300 human cases and over 100 fatalities. pigs can therefore play a key role in the epidemiology of niv by acting as an “amplifying” host. the outbreak in singapore ended with the prohibition of pig imports from malaysia and the malaysian outbreak was ended by culling 45% of the country's pig population with costs exceeding us$500 million. despite the importance of niv as an emerging disease with the potential for pandemic, no vaccines, or therapeutics are currently approved for human or livestock use. in this mini-review, we will discuss current knowledge of niv infection in pigs; our ongoing work to develop a niv vaccine for use in pigs; and the pig as a model to support human vaccine development. nipah virus (niv) is an enveloped, single stranded, negative sense rna paramyxovirus, genus henipavirus. the natural hosts and wildlife reservoirs of niv are old world fruit bats of the genus pteropus (1). both nipah and the related hendra virus possess a number of features that distinguish them from other paramyxoviruses. of particular note is their broad host range which is facilitated by the use of the evolutionary conserved ephrin-b2 and -b3 as cellular receptors (2) . the niv attachment glycoprotein (g) is responsible for binding to ephrin-b2/-b3 (3). following receptor binding, the g protein dissociates from the fusion (f) protein. subsequently, the f protein undergoes a series of conformational changes which in turn initiates fusion of the viral and host membrane allowing entry (4) . during viral replication, the f protein is synthesized and cleaved into fusion active f1 and f2 subunits. these subunits are subsequently transported back to the cell surface to be incorporated into budding virions, or facilitate fusion between infected and adjacent uninfected cells (5) . this cell-to-cell fusion results in the formation of multinucleated cells called syncytia, and greatly influences the cyopathogenicity of niv as it allows spread of the virus, even in the absence of viral budding (5, 6) . niv infection is currently classed as a stage iii zoonotic disease, meaning it can spill over to humans and cause limited outbreaks of person-to-person transmission (7, 8) . niv outbreaks have been recognized yearly in bangladesh since 2001 as well as occasional outbreaks in neighboring india (figure 1 ). these outbreaks have been characterized by person-to-person transmission figure 1 | previous locations of henipavirus infection outbreaks. nipah and hendra virus distribution map highlighting the range of the natural wildlife reservoir, pteropus spp. bats [adapted from (9) ]. and the death of over 70% of infected people (10, 11) . in may 2018, the first ever outbreak in southern india was reported. a total of 19 niv cases, of which 17 resulted in death, were reported in the state of kerala. pteropus giganteus bats from areas around the index case in kozhikode, kerala, were tested at the national high security animal diseases laboratory at bhopal. of these, 19% were found to be niv positive by rt-pcr (12) . characteristics of niv that increase the risk of it becoming a global pandemic include: humans are already susceptible; many niv strains are capable of person-to-person transmission; and as an rna virus, niv has a high mutation rate (13) . niv has been found to survive for up to 4 days when subjected to various environmental conditions, including fruit bat urine and mango flesh (14) . whilst survival time was influenced by fluctuations in both temperature and ph, the ability for niv to be spread by fomites could play a role in outbreak situations. the first and still most devastating niv outbreak occurred in peninsular malaysia from september 1998 to may 1999 (15, 16) . the link to pigs in this outbreak was obvious as 93% of the infected patients had contact with pigs (17) . if a niv strain were to become human-adapted and infect communities in southeast asia where there are high human and pig densities and pigs are a primary export commodity, infection could rapidly spread and humanity could face its most devastating pandemic (8, 11, 18) . in september 1998, there was an outbreak of severe febrile encephalitis among pig farmers in the state of perak, malaysia, that was associated with a high mortality rate. a total of 265 cases of encephalitis, of which 105 resulted in death, were confirmed. these deaths were initially thought to be due to japanese encephalitis (je), an endemic disease in malaysia. however, with most cases occurring in men who worked with pigs, the epidemiological characteristics of this disease were distinct from those of je, where ∼75% of cases occur in children aged 0-14 years (19) (20) (21) . the epidemiological link was from fruit bats infecting pigs that then served as amplifier hosts, resulting in transmission to humans through close contact (22) . as a result of movement of infected pigs and humans to other states in malaysia, by february 1999 similar diseases were recognized in both pigs and humans in new outbreak areas (23). in the following month, there were 11 cases of respiratory illness and encephalitis amongst singapore abattoir workers who had handled pigs imported from the outbreak areas in malaysia (15) . due to this, the importation of pigs from malaysia ceased which in turn ended the outbreak in singapore. the outbreak in malaysia ended when 1.1 million pigs (45% of the country's pig population) were culled from outbreak and surrounding areas (17, 24) . the niv outbreak incurred significant economic costs and long-term damage to the malaysian pig industry: us$582 million in direct costs and lost market revenue, including us$97 million in compensation to farmers for the 1.1 million pigs slaughtered and 36,000 jobs lost (25) . to this date, malaysian pig farming is only permitted in "identified pig farming areas." pigs also suffered during the 1998/99 malaysian outbreak, but this was only diagnosed as part of the investigation following the human cases. the severity of symptoms of niv infection in pigs varied with age. in suckling pigs (<4 weeks old), mortality could be high (up to 40%) and labored breathing and muscle tremors were evident. in growing pigs (1 to 6 months), an acute febrile (>39.9 • c) illness was observed with respiratory signs ranging from increased or forced respiration to a harsh, loud non-productive cough, open mouth breathing, and epistaxis (26) . in some cases these respiratory signs were accompanied by one or more of the following neurological signs: trembles, neuralgic twitches, muscle fasciculation, tetanic spasms, incoordination, rear leg weakness, or partial paralysis. pigs of this age had high morbidity and low mortality (<5%) (26) (27) (28) . some animals over 6 months of age died rapidly (within 24 h) without signs of clinical disease. respiratory signs were reported in adult pigs, as with younger animals, although these were less obvious (labored breathing, bloody nasal discharge, increased salivation) and neurological signs included head pressing, bar biting, tetanic spasms and convulsions. first trimester abortions were also reported (26) (27) (28) . in an experimental infection study, pigs were inoculated subcutaneously with a niv isolate from the central nervous system of a fatally infected human patient. infection elicited respiratory and neurological symptoms consistent with those observed in naturally infected malaysian pigs, which included febrile illness, incoordination, mucosal nasal discharge, and persistent cough (29) . pigs inoculated orally with the same dose did not show clinical signs although they still shed virus. in a second study, piglets were inoculated oronasally with a human niv isolate (30) . all infected animals showed a transient increase in body temperature between 4 and 12 days post-infection. two of these animals developed transient respiratory signs, mild depression and a hunched stance. both these studies concluded that niv infection in pigs had no pathognomonic features i.e., the clinical signs observed were non-specific. this can make field diagnosis of niv infection in pigs difficult, as observed in the outbreak in malaysia (16, 28) . the name proposed for the disease caused by niv infection of pigs was "porcine respiratory and neurological syndrome" (also known as "porcine respiratory and encephalitis syndrome"), or, in peninsular malaysia, "barking pig syndrome" (28) . niv infection was included as the sixth pig disease notifiable to the oie world organization for animal health (31) . the oie approve diagnostics and recommends preventative and control measures for a range of transboundary livestock diseases. despite the importance of niv as an emerging disease with the potential for pandemic, no therapeutics or vaccines are approved for use in humans or livestock species. due to the lethal nature of niv infection, producing a safe, live attenuated vaccine with no potential for reversion is difficult. however, recombinant niv mutants, attenuated in hamster and ferret models, have been shown to generate strong neutralizing antibody responses (32, 33) . more commonly, niv vaccine approaches have focused individual candidate antigens delivered as subunit vaccines or using viral vectors. the most studied vaccine candidate is the soluble form of the g protein (sg) from the related hendra virus (hev). hev and the niv malaysia strain share between 68 and 92% amino acid homology between their proteins; with f and g proteins sharing 88 and 83% homology, respectively (34) . both f and g envelope glycoproteins are regarded as vaccine candidate antigens since they are the targets of niv neutralizing antibodies (35) . an adjuvanted hev sg protein subunit-based vaccine (equivac r hev, zoetis) has been licensed in australia to protect horses against hev and to reduce the zoonotic risk to humans (36) . equivac r hev protects ferrets and african green monkeys (agms) after experimental challenge with niv, as well as hev (37, 38) . surprisingly, this vaccine failed to protect pigs from experimental niv challenge (39) . since the vaccine induced cross-neutralizing antibodies but not measurable t cell responses, the authors concluded that both arms of the adaptive immune response may be required for protection against niv and hev. these studies also potentially highlight that adjuvants can have species specific effects and tailoring of adjuvants to the target species may be required or considered in the context of preclinical models. the experimental viral vectored vaccine candidates for niv include vesicular stomatitis virus, rabies virus, canarypox virus (alvac strain), adeno-associated virus (aav), measles virus, newcastle disease virus (ndv) and venezuelan equine encephalitis virus (40) . alvac expressing niv g or f (alvac-g and alvac-f) was found to protect pigs against niv challenge 2 weeks after the second immunization (41) . high titres of niv neutralizing antibodies were induced with the alvac-g vaccine, while despite the low levels of neutralizing antibodies induced by the alvac-f; all vaccinated pigs were protected against virulent niv challenge. recombinant attenuated ndv expressing niv glycoproteins have been shown to induce long lasting niv-specific nabs in pigs, with the vector expressing niv g performing better than niv f (42). however, no challenge was performed in this study and it remains to be determined whether these paramyxovirus-based vaccine candidates are efficacious. compared to canarypox vectors, ndv-based vectors have a number of advantages including their high titer propagation in chicken eggs removing the requirement for cell culture (41, 42) . despite these encouraging results and the continued threat posed by niv, no vaccine candidate has progressed toward market for either pigs or humans. the promising performance of experimental niv and hev vaccines in animal models and the licensure of equivac r hev, as a "one health" vaccine to safeguard animal and human health, strongly support the proposition that a safe and effective niv vaccine may be developed for pigs to reduce the severe economic consequences of niv outbreaks and the threat to public health. with partners, we have initiated a project that aims to develop such a vaccine. we are systematically analyzing the immunogenicity and protective efficacy of three niv vaccine candidates in pigs: (1) an adjuvanted niv sg protein (orthologous to the equivac r hev vaccine), (2) niv g protein delivered by a replication-deficient simian adenoviral vector (chadox1 niv g), and an adjuvanted, molecular clamp stabilized niv f (mcsf) protein. chadox1 is a multispecies vector with an established human and livestock safety profile (43) . chadox1 offers the potential for both single dose efficacy and thermostabilization (44, 45) . the molecular clamp is a proprietary stabilization domain that preserves the f protein in its native "pre-fusion" form, which should enhance immunogenicity and thermostability. in depth analyses of t cell and antibody responses are being conducted to identify correlates of vaccine-induced protection. we will examine the durability of niv-neutralizing antibodies and other immune responses associated with protection, including a comparison of a singleshot vs. homologous prime-boost immunization regimes. incontact animals will be introduced to assess transmission of challenge virus from vaccinates or unvaccinated control animals. the sporadic nature of niv outbreaks means that the commercial development of niv vaccines for use in pigs (other livestock or humans) is limited and animal health companies are of the opinion that niv vaccines will have limited marketability. our ongoing studies should help facilitate this by developing a safe and efficacious prototype niv vaccine that is amenable to "surge production" and discrimination of infection in vaccinated animals (diva) capability. subsequent development and licensure of this vaccine will require engagement with international, regional, and national agencies and the creation of dependable markets via the establishment of niv vaccine banks. the oie world fund manages vaccine banks and the delivery of vaccines for avian influenza, rabies, foot-andmouth disease, and peste de petit ruminants (46) . vaccine banks ensure the procurement and delivery of high quality vaccines mass-produced in line with oie intergovernmental standards. critically these vaccine banks can be rapidly deployed when required and this model appears most appropriate in the context of reactive emergency vaccination programmes to aid niv outbreak control. vaccines can play a major component in an emergency response against emerging infectious disease, with the main aim to reduce virus spread between susceptible hosts (47) . the precise decisions on control strategies will be complex and vary for different regions. factors such as: herd density, production systems, the presence of susceptible wildlife, the impact on export trade and current opinions on economic vs. ethical factors will likely play a role. one strategy to halt a niv outbreak would be to deploy a stockpiled vaccine for ring vaccination around the niv affected area. this approach was utilized in the 2016 ebola outbreak in guinea and showed great promise in terms of disease containment and elimination (48) . for such a strategy, a vaccine with single-dose efficacy and a rapid onset of immunity preventing virus transmission would be preferential. this is likely to be best achieved with a viral-vectored (45) or mrna vectored vaccine (49) . the highly unpredictable nature of niv outbreaks means that it is highly unlikely that niv vaccines would be used routinely by pig producers. one strategy that could help ensure that immunity to niv is maintained in pig herds could involve the engineering of niv g into a live attenuated viral vaccine, such as pseudorabies, which are widely used in countries at-risk. the recent ebola and zika epidemics highlighted how poorly prepared we were to deal with these new and emerging diseases. there has therefore been a global drive to develop vaccines against these diseases and improve preparedness. the coalition for epidemic preparedness innovation's (cepi's) was established in 2016 with a mandate of financing and coordinating the development of new human vaccines to prevent and contain infectious disease epidemics. cepi selected niv, lassa virus and middle east respiratory syndrome-coronavirus, three pathogens from the who's list of priority diseases needing urgent r&d attention as its initial focus (50, 51) . the who's list of priority diseases is part of the r&d blueprint, which identifies priority diseases and addresses gaps in the global scientific community to increase preparedness for future outbreaks. the main aim of the blueprint is to fast-track the availability of effective tests, vaccines, and medicines that can be used to save lives and avert large scale crises (51) . in 2002, the us food and drug administration (fda) established the "animal rule" for regulatory approval of vaccines and therapeutics for which efficacy testing in humans is impossible, therefore requiring relevant animal models that represent a disease model similar to that of the human disease (52) . vaccine efficacy studies in animal models aim to identify specific vaccine-induced correlates of protection including neutralizing antibodies or cell-mediated responses (53) . in 2015, a vaccine to protect against anthrax was the first to be approved through the "animal rule" (54) . the licensing pathway for the "animal rule" requires that immunogenicity results from clinical trials must be consistent with previously identified immune correlates associated with protection (52) . therefore, identifying reliable markers of vaccine-generated immunity becomes critically important for pathogens such as niv. large animal models have been shown to more accurately predict vaccine outcome in humans in comparison to small animal models (55) therefore defining correlates of vaccineinduced protection in pigs, may play an important role in supporting subsequent human vaccine licensure under the "animal rule." animal models can be validated for a particular disease according to a number of different criteria, which include "face" and "predictive" validity. for face validity there must be similarities in the pathology and clinical symptoms between the animal model and the human disease (56). as discussed above, niv infection of pigs causes a similar respiratory and neurological syndrome as seen in human infections. although, disease severity in pigs may be considered lower than in humans. the predictive validity of a model means that clinically effective interventions demonstrate a similar effect in the animal model (56) . no clinical trials of niv vaccine candidates have been reported to compare with vaccine performance in animal models, including the pig. as noted above, the success of the equivac r hev vaccine in horses and other animal models was not replicated in swine (38, 39) , highlighting a potential issue of predicative validity when comparing niv vaccines between animal species, which may extend to humans. on the other hand, pigs have been used successfully as models to study many human infectious diseases (57) (58) (59) (60) (61) (62) (63) , including niv infection (64) . there is also a growing appreciation that pigs provide a superior animal model for influenza a virus infection and immunity and should play a more prominent role as a model for human influenza vaccine development (65) . the success of the pig as an experimental animal model is partly due to their similarities with humans in terms of anatomy, immunology, and physiology, but also due to their manageable behavior and size, and by the general ethical acceptance of using pigs for experimental purposes instead of non-human primates (55, 63, 66) . the niv outbreaks in malaysia and singapore demonstrated that pigs can play a key role in the epidemiology of niv by acting as an amplifier host. the region most at risk of niv infection has some of the highest pig population densities found anywhere in the world, which are rising fast due to the demand of a growing human population. this increases the risk of niv transmission to pigs and humans. the development of a niv vaccine for use in pig populations would decrease the major risk niv poses to the developing pig industries, as well as to the livelihoods of poor livestock keepers in southeast asia. the use of non-human animal models is crucial for vaccine development against diseases such as niv since efficacy testing in humans is impossible. the pig model may therefore contribute to human vaccine development, supporting human vaccine licensure under the animal rule. pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission functional studies of host-specific ephrin-b ligands as henipavirus receptors structural basis of nipah and hendra virus attachment to their cell-surface receptor ephrin-b2 unraveling a three-step spatiotemporal mechanism of triggering of receptorinduced nipah virus fusion and cell entry organ-and endotheliotropism of nipah virus infections in vivo and in vitro role of endocytosis and cathepsinmediated activation in nipah virus entry origins of major human infectious diseases the pandemic potential of nipah virus virus distribution map nipah virus encephalitis reemergence enhancing preparation for large nipah outbreaks beyond bangladesh: preventing a tragedy like ebola in west africa available online at nipah virus edits its p gene at high frequency to express the v and w proteins henipavirus susceptibility to environmental variables outbreak of nipah-virus infection among abattoir workers in singapore nipah virus outbreak in malaysia case-control study of risk factors for human infection with a new zoonotic paramyxovirus, nipah virus, during a 1998-1999 outbreak of severe encephalitis in malaysia pig production in cambodia, laos, philippines and vietnam: a review nipah virus: a recently emergent deadly paramyxovirus nipah virus encephalitis outbreak in malaysia japanese encephalitis and its epidemiology interdisciplinary approaches to understanding disease emergence: the past, present, and future drivers of nipah virus emergence new virus fingered in malaysian epidemic introduction to modern virology available online at the status, public response and challenges in overcoming emerging and exotic diseases -nipah virus disease experience. in: national congress on animal health and production: environmental care in animal production nipah virus infection of pigs in peninsular malaysia experimental nipah virus infection in pigs and cats bacterial infections in pigs experimentally infected with nipah virus infections and infestations in force the nonstructural proteins of nipah virus play a key role in pathogenicity in experimentally infected animals the immunomodulating v and w proteins of nipah virus determine disease course molecular characterization of nipah virus, a newly emergent paramyxovirus a treatment for and vaccine against the deadly hendra and nipah viruses hendra virus vaccine, a one health approach to protecting horse, human, and environmental health a recombinant hendra virus g glycoprotein-based subunit vaccine protects ferrets from lethal hendra virus challenge a recombinant hendra virus g glycoprotein subunit vaccine protects nonhuman primates against hendra virus challenge protection against henipaviruses in swine requires both, cell-mediated and humoral immune response status of vaccine research and development of vaccines for nipah virus recombinant nipah virus vaccines protect pigs against challenge newcastle disease virusvectored nipah encephalitis vaccines induce b and t cell responses in mice and long-lasting neutralizing antibodies in pigs a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity potency of a thermostabilised chimpanzee adenovirus rift valley fever vaccine in cattle chimpanzee adenovirus vaccine provides multispecies protection against rift valley fever available online at emerging and neglected infectious diseases: insights, advances, and challenges efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial zika virus protection by a single low-dose nucleosidemodified mrna vaccination available online at research and development blueprint for action to prevent epidemics new drug and biological drug products; evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible. final rule can ebola virus vaccines have universal immune correlates of protection? first vaccine approval under the fda animal rule large animal models for vaccine development and testing animal models in translational medicine: validation and prediction staphylococcal wound infection in the pig: part i a pig model of acanthamoeba keratitis: transmission via contaminated contact lenses animal models for gastric helicobacter immunology and vaccine studies the benefits of using diverse animal models for studying pertussis animal models of ventilator-associated pneumonia swine influenza h1n1 virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human h1n1 influenza virus the pig: a model for human infectious diseases animal models of henipavirus infection: a review swine as a model for influenza a virus infection and immunity contribution of the swine model in the study of human sexually transmitted infections all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-257922-tbkitz7m authors: sookhoo, jamie r. v.; brown-jordan, arianne; blake, lemar; holder, ridley b.; brookes, sharon m.; essen, stephen; carrington, christine v. f.; brown, ian h.; oura, christopher a. l. title: seroprevalence of economically important viral pathogens in swine populations of trinidad and tobago, west indies date: 2017-05-18 journal: trop anim health prod doi: 10.1007/s11250-017-1299-3 sha: doc_id: 257922 cord_uid: tbkitz7m the objective of this study was to evaluate the seroprevalence and identify the strains of swine influenza virus (swiv), as well as the seroprevalence of porcine parvovirus (ppv), transmissible gastroenteritis virus (tgev), porcine reproductive and respiratory syndrome virus (prrsv), porcine respiratory coronavirus (prcv), porcine circovirus type 2 (pcv-2), and classical swine fever virus (csfv) in pigs in trinidad and tobago (t&t). blood samples (309) were randomly collected from pigs at farms throughout t&t. serum samples were tested for the presence of antibodies to the aforementioned viruses using commercial elisa kits, and the circulating strains of swiv were identified by the hemagglutination inhibition test (hit). antibodies against swiv were detected in 114 out of the 309 samples (37%). out of a total of 26 farms, 14 tested positive for swiv antibodies. hi testing revealed high titers against the a/sw/minnesota/593/99 h3n2 strain and the ph1n1 2009 pandemic strain. antibodies against ppv were detected in 87 out of the 309 samples (28%), with 11 out of 26 farms testing positive for ppv antibodies. antibodies against pcv-2 were detected in 205 out of the 309 samples tested (66%), with 25 out of the 26 farms testing positive for pcv-2 antibodies. no antibodies were detected in any of the tested pigs to prrsv, tgev, prcv, or csfv. monitoring and surveillance for viruses that affect swine are crucial in the prevention and control of diseases that can cause severe economic losses to the swine populations of trinidad and tobago (t&t) and the wider caribbean region. to date, there have been limited or no published studies investigating the prevalence of viruses circulating in t&t's swine populations. in late 2015, according to the world animal health information system (wahis) database, columbia reported to have experienced classical swine fever (csf) limited to one or more zones and porcine reproductive and respiratory syndrome (prrs) was reported to be present in columbia and suriname. viruses such as classical swine fever virus (csfv), porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus type 2 (pcv2), and swine influenza virus (swiv) have been reported to be circulating in venezuelan swine herds (kwiecien 2013) . the close proximity (7 km) of trinidad to venezuela, and the known illegal trade of animals and animal products between the two countries, renders trinidad a high-risk location for the introduction of these viruses. the objectives of this study were to assess the prevalence of selected viruses, namely porcine parvovirus (ppv), transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), csfv, pcv-2, prrsv, and swiv in the pig populations of t&t and to identify the strains of swiv that are circulating in t&t pigs. understanding the background levels of these viruses in swine populations in t&t will enable the development of science-based risk assessments, thus aiding the successful prevention, management, and control of future viral outbreaks within t&t and the caribbean region. this will improve the health status and productivity of swine and pork production in t&t. the sample size (n = 309) was estimated by using an anticipated true herd-level prevalence of 10%, tolerance of 7.5%, a 90% confidence level, and herd-level specificity and sensitivity of 70 and 90% respectively. this sample size was achieved as a modification of the cannon and roe (1982) formula for livestock disease surveys as modified by humphry et al. 2004 . sampling was conducted on pig farms throughout t&t. sampling was carried out from october 2013 to february 2015, and samples were taken from 5 large pig farms and 21 small pig farms. three of the small farms had animals slaughtered at public abattoirs. a map showing the location of the farms and abattoirs sampled is shown in fig. 1 . small farms were classified as operations with 5-50 pigs and large farms as operations with 100-500 pigs. none of the pigs sampled were vaccinated for any of the viruses under investigation. two large farms were sampled on slaughter days. twentyfive pigs were randomly selected and these samples were used. on the 3 large farms where slaughtering was not being carried out, a randomization process was used. each finisher, breeding sow, and boar were designated a number, the numbers were then placed into a bag, and 25 numbers were drawn at random. these 25 pigs were sampled for antibody testing. in total, 125 samples were collected from 5 large farms. ten samples were taken at random from each of the 3 small farms that slaughtered their pigs in public abattoirs. these 10 samples were taken at slaughter. the remaining 18 small farms were visited, and 10 samples were taken at random. on farms with fewer than 10 pigs, all of the pigs on the farm were sampled. in total, 184 samples were collected from 21 small farms. blood was taken from the right anterior vena cava of pigs using 18g 1.5-in. needles for nursery pigs and 3-in. needles for adults. samples were collected in red-topped (no anticoagulant) tubes and centrifuged in a beckman model tj-6 centrifuge at 2500 rpm for 10 min to separate serum. the samples were collected from finisher pigs, and where possible, samples from older animals were taken. -large farms -small farms serum antibodies to influenza were detected by the hi test according to standard methods (organization for animal health 2016). to reduce non-specific reactions, the sera were treated with 100 u/ml of receptor-destroying enzyme at 37°c for 1 h, inactivated at 56°c for 30 min, and adsorbed with 30% (v/v) chicken red blood cells (crbcs) overnight at 4°c. following treatment, the starting dilution of serum was 1/5, and doubling dilutions were prepared, prior to the addition of an equal volume of both four hemagglutinating units of virus and 1% crbcs. following incubation at 4°c for 30 min, the plates were examined for hemagglutination of crbcs. hi titers were recorded as the reciprocal of the highest initial dilution of serum which completely inhibited hemagglutination. lf2 lf3 lf4 lf5 sf1 sf2 sf3 sf4 sf5 sf6 sf7 sf8 sf9 sf10 sf11 tf1 tf2 tf3 tf4 tf5 tf6 tf7 tf8 tf9 lf3 lf4 lf5 sf1 sf2 sf3 sf4 sf5 sf6 sf7 sf8 sf9 sf10 sf11 tf1 tf2 tf3 tf4 tf5 tf6 tf7 tf8 tf9 lf2 lf3 lf4 lf5 sf1 sf2 sf3 sf4 sf5 sf6 sf7 sf8 sf9 sf10 sf11 tf1 tf2 tf3 tf4 tf5 tf6 tf7 tf8 tf9 to 100% (fig. 2a) . the pigs sampled in tobago were all negative for swiv antibodies. hi testing on a selection of 12 of the elisa positive samples revealed high titers against the a/sw/minnesota/593/99 h3n2 strain of swiv as well as the ph1n1 2009 pandemic strain ( table 1) . one of the pigs also showed a high titer against the a/perth/16/09 h3n2 strain. antibodies to ppv were detected in 28% (87 out of 309) of sampled pigs and on 42% (11 out of 26) of the farms sampled, including 3 large farms in central and eastern trinidad. approximately 80% of ppv positives corresponded to an hi titer of 10,240 to 20,480 according to the test kit insert (lsivet™ porcine parvovirosis-serum, lissieu, france), suggesting that field infections of ppv were occurring in these swine. the percentage of pigs testing positive for antibodies to ppv on each farm ranged from 13 to 93% (fig. 2b ). the pigs sampled in tobago were all negative for ppv antibodies. antibodies to pcv-2 were detected in 66% (205 out of 309) of sampled pigs from t&t and on 96% (25 out of 26) of the farms sampled on both islands, including the 5 major farms in trinidad. of the total number of pigs sampled from trinidad, 43% (113 out of 264) tested positive for pcv-2 antibodies which were found on 87.5% (14 out of 16) of trinidad farms. of the total number of pigs sampled from tobago, 43% (19 out of 44) were positive for antibodies to pcv-2. of the 10 farms sampled from tobago, all tested positive for pcv-2 antibodies. approximately 80% of the pcv-2 positives corresponded to an antibody titer of 3000 or higher according to the test kit insert (biochek pcv-2 antibody test kit-holland) suggesting that field infections of pcv-2 were occurring in these swine. the percentage of pigs testing positive for antibodies to pcv-2 on each farm ranged from 30 to 100% (fig. 2c ). ten farms were positive for antibodies to swiv, ppv, and pcv2, and three farms were positive for antibodies to both swiv and pcv2, but not ppv. one small farm (sf11) was positive for antibodies to swiv and ppv but not pcv2, and two small farms (sf1 and sf4) as well as all of the tobago farms were positive for only pcv2. no antibodies were detected in any of the sampled pigs against prrsv, tgev/prcv, or csfv. out of approximately 35,000 domestic pigs in t&t, 309 (~1%) were sampled and tested for the presence of antibodies to the viruses under investigation. the sample size (n = 309) was estimated by using an anticipated true herd-level prevalence of 10%, tolerance of 7.5%, a 90% confidence level, and herd-level specificity and sensitivity of 70 and 90% respectively (cannon and roe 1982) . the amount of pigs sampled in this study was constrained by issues of free access to farms. many farmers were reluctant to allow their pigs to be sampled due to the invasive nature of the blood sampling procedure and the resulting high levels of stress caused to the animals. the more common approach to determining sample size in this type of study uses the formula developed by cannon and roe (1982) where p is the anticipated population proportion which is assumed at 70% and z is 1.96 which is the approximate value of the 97.5 percentile point of a normal distribution curve used to construct approximate 95% confidence intervals. this value is found using a z α/2 table where the confidence level used is 95% and α is 0.05. using the canon and roe formula, the sample size n was calculated to be 322 samples; however, to reduce the number of pigs to be sampled further, the confidence can be relaxed (decreased) and the tolerance, increased (humphry et al. 2004 ) allowing for a sample size of 309. swiv can cause severe respiratory signs in pigs, with morbidity rates sometimes reaching 100%. the primary economic impact is related to retarded weight gain resulting in an increase in the number of days to reach market weight (organization for animal health 2009). in the uk, the financial loss resulting from reduced weight gain in finishing pigs alone due to swiv has been estimated at approximately £7 per pig (morilla et al. 2008) . in this study, antibodies against (rajao et al. 2013) . the 37% seroprevalence found in pigs of t&t is therefore consistent with similar studies carried out in central and north american countries and suggests that swiv is endemic in the trinidadian domestic swine population, but not tobago. in order to detect the likely strains of swiv circulating in the pigs, a selection of 12 elisa positive samples were further tested using hit. a combination of h3n2 strains was selected to try to understand the relative incidence of h3n2 in pigs within the country. due to significant antigenic diversity, strains were selected from both european pigs, which may be representative of global strains but also more recent human strains, given the frequency of transmission from humans to pigs which may result in the establishment of a stable lineage of virus. the pandemic strain ph1n1 2009 was also included, which is known to be present in pigs worldwide at present. the h3n2 serology results (table 1) show different reactivity patterns. a/sw/minnesota/593/99 is most significant since this is the strain of h3n2 virus that has undergone reassortment in north american pigs through multiple generations over several years (anderson et al. 2015) . interestingly, it is well known that many of the pig populations of trinidad originated from minnesota farms indicating a potential introduction pathway. high titers of this strain were observed in the samples tested which would be indicative of quite recent exposure/active circulation of such viruses. one of the samples showed quite a high titer against the a/perth/16/09 h3n2 strain which is a contemporary human virus that does not have a stable lineage in pigs. as expected, reactivity to the european viruses was low as there is very little transfer of swine between europe and trinidad. the pandemic ph1n1 2009 strain showed strong reactivity in all of the samples tested which indicates probable circulation of virus or recent exposure. in the case of the small farms sampled in this study, all of the owners lived either on the farm or within 5 km of their farm and several other houses were present in close proximity to the pig farms. this close proximity of people and pigs could enhance both zoonotic and reverse zoonotic transmission of influenza viruses from humans to pigs, thus maintaining influenza virus circulation in the swine herds (australian pork industry biosecurity programme, apibp 2003). these factors are akin to those which defined traditional influenza epicenters in southeast asia (ma et al. 2009 ). personal hygiene, the granting of sick leave to pig handlers showing flu-like symptoms, as well as biosecurity, should therefore be encouraged on both small-and large-scale farms. such measures should include risk assessment checks for visitors, the use of protective clothing, respiratory protection for people to reduce viral dissemination (zoonotic and reverse zoonotic), handwashing before and after handling animals, restriction on sharing of equipment and tools between farms, and controls relating to the movement of animals and vehicles in and out of the farm. training of workers on pig farms to recognize influenza-like symptoms in humans and pigs should also be carried out (adeola et al. 2015) . porcine parvovirus (ppv) causes severe economically devastating reproductive symptoms in breeding sows. it is known to cause abortion and is associated with stillbirths, mummifications, embryonic death, and infertility (smedi). ppv infection is one of the most common and important causes of infectious infertility in swine. ppv is ubiquitous and worldwide in its distribution; it is therefore an infection which needs to be carefully managed (porcine parvovirus, the pig site 2014). this study identified 87 out of the 309 serum samples (28%) as antibody positive for ppv. interestingly, markedly different levels of seroprevalence for antibodies to ppv were observed across the different swine farms in t&t with seroprevalence levels on farms ranging from 12 to 92% (fig. 2b) . antibodies were not observed in any of the pigs sampled on two of the largest farms in trinidad, suggesting that the biosecurity and husbandry measures practiced on these two farms may be effective at stopping this introduction of ppv onto the farms. interestingly, despite the relatively high seroprevalence for ppv observed in trinidadian domestic pigs, there are limited reports of reproductive problems in the pigs. this could be due to farmers simply not reporting reproductive issues on their farms when they occur or alternatively could be due to a state on endemic stability on the affected farms, meaning that all the breeding sows are being infected with ppv prior to their first pregnancy. on the farms that contained serologically positive pigs, it was found that all the breeding sows that were sampled on these farms had antibodies to the virus prior to their first pregnancy, which would explain the lack of ppvrelated reproductive clinical signs reported to the veterinary services. it is, however, very important to note that some pig farms in trinidad, and all the pigs sampled on tobago, tested negative for ppv antibodies. these ppv-negative farms should be particularly careful when bringing pigs into their farms, ensuring that they avoid the introduction of ppvpositive animals. these farmers should ensure that they test all pigs for ppv antibodies and antigen prior to introduction, or alternatively, they should vaccinate their breeding sows prior to their first pregnancy. all pig farmers from ppvnegative farms should be advised of the importance of maintaining high levels of biosecurity in order to keep their farms free of ppv. all of the samples taken from pigs on the island of tobago (n = 45) tested negative for antibodies to both swiv and ppv. in light of this, tobago pig farmers should be encouraged to maintain a closed system. tobago has approximately 20 small backyard pig farms throughout the island, 10 of which were sampled. these tobago pig farmers usually breed and rear their pigs on their farms and may occasionally buy replacement sows from neighboring farms in tobago to reduce excessive inbreeding. pig farmers from tobago should therefore be advised to avoid importing pigs from trinidad and should maintain their current husbandry practices in order to ensure that their pigs remain seronegative for swiv and ppv. any pigs, including boars, coming into the country should be quarantined and tested for the presence of these viruses. farmers from tobago should be educated on the serological situation of their pigs and be made aware of the risks they face in the event of their naïve pigs becoming exposed to these viruses. the farmers should also be educated on biosecurity measures to be implemented to ensure the prevention of ppv and siv being introduced into their pig population. interestingly, trinidad has at least three pig farms which, on occasions, have imported semen from the usa. this may possibly be the source of ppv infection in the country. pcv-2 is one of the top three most economically important swine pathogens, behind prrsv and mycoplasma pneumoniae (thacker 2013) . if pigs are left unvaccinated for pcv-2, producers could see up to a us$20 loss per pig (thacker 2013) . high seroprevalences (82.4%) of pigs for pcv-2 antibodies have been reported in canada (liu et al. 2002) , the usa (80%) (nawagitgul et al. 2002) , taiwan (83.5%) (wang et al. 2004) , northern ireland (>55%) (walker et al. 2000) , and columbia (83.6%) (monroy et al. 2014 ). in the present study, the overall seroprevalence for pcv-2 antibodies in t&t pigs was found to be 66%. there is, however, little to no evidence for pcv-2-associated diseases in t&t pigs. given the high seroprevalence for pcv-2 observed in the t&t pigs, there is clearly a need to closely monitor pig herds for evidence of pcv-2-associated diseases. routine surveillance for clinical signs and the regular examination of specimens from abattoirs should be adopted. farmers should also be encouraged to adopt sound husbandry practices such as age segregation, good sanitation as well as measures to minimize stress and avoid overcrowding. the application of these measures is essential to avoid the development of diseases in pigs that are associated with pcv-2 infection such as post-weaning multisystemic wasting syndrome (pmws). interestingly, some farms showed very high seroprevalence levels for certain viruses, whereas other farms showed low seroprevalence levels. it is possible that this was due to ongoing infection at the time of sampling. alternatively, this could have been due to the different management practices and stocking densities on the farms. high population densities are known to facilitate the rapid spread of pathogens throughout livestock populations. a study in belgium found that the number of pigs per pen was positively associated with swine influenza h3n2 seropositivity (maes et al. 2000) . ewald et al. (1994) also found that a high pig density was a risk factor for herds to become infected with influenza h1n1 and h3n2 viruses and furthermore that a large number of pigs per pen creates physiological stress, which in turn can alter the immune system and predispose pigs to infection. the reduced seroprevalence levels for swiv observed on the small compared to the large farms may have been associated with the lower stocking densities, as well as the lower overall numbers of pigs, on the small farms. it is known that respiratory viruses are less efficiently maintained on small as opposed to large pig farms (maes et al. 2000; poljak et al. 2008; simon-grife et al. 2011) . the two small farms (sf8 and sf11), on which over 80% of the pigs were seropositive for swiv, were the two largest of the small farms sampled (with close to 30 pigs). the pigs on these farms were kept at a very high stocking density, which may have been the reason for the high seroprevalence for swivobserved on those farms. economic losses resulting from viruses such as siv and pcv2 are usually related to a decrease in average daily gain (adg) and reduced feed conversion efficiency in affected pigs. these viruses may also result in increased carcass condemnation at slaughter and treatment costs for ill pigs (van alstine 2012). for siv, seropositive and virus-positive pigs have been found to have significantly decreased growth performance compared to seronegative pigs, even though feed intake was not decreased. reduced feed conversion efficiency led to lower average daily growth, additional feed requirements, and longer time needed to reach the 100 kg body weight (er et al. 2014 ). pigs vaccinated for pcv2 have been shown to deliver a sizable return on investment of up to approximately us$20 per pig over unvaccinated pigs (gillespie et al. 2009 ). also, in the case of ppv, it has been suggested that the cost of an epidemic could result in losses of up to us$50 per sow (cutler and gardner 1988) . losses of between us$20 and $50 per pig would be devastating to the pig industry in t&t and could result in the closure of affected farms. this study highlights the importance of carrying out regular serological monitoring for economically important viruses of swine that are circulating in the region. although antibodies to csf, prrs, tge, and prcv were not observed in pigs from t&t, it is important to continue monitoring for these viruses, as outbreaks of prrs and csf have recently been reported in south american countries neighboring trinidad, including venezuela, ecuador, suriname, and columbia (world animal health information system, wahis interface 2015). it is well known that domestic species, including pigs, are illegally imported into trinidad from the south american mainland, especially venezuela. although the porcine coronaviruses (tge and prcv) have not previously been reported to be present in trinidad, the presence of these viruses has been suspected for many years, possibly due to previous importation of pigs and pig semen from the usa and canada. this study has revealed that these viruses are not present in domestic pigs in t&t. in conclusion, this study shows that swiv and ppv are present and circulating in trinidadian domestic pig populations; however, these viruses were not observed to be present in pigs sampled from tobago. strains of swiv confirmed as likely to be circulating in pigs on the island of trinidad include a north american h3n2 strain and the ph1n1 2009 pandemic strain. pcv-2, however, was observed to be circulating in domestic pigs from the islands of both trinidad and tobago. pigs on both islands of trinidad and tobago did not have antibodies to csf, prrs, tge, and prcv. detection of pandemic strain of influenza virus (a/h1n1/pdm09) in pigs, west africa: implications and considerations for prevention of future influenza pandemics at the source characterization of co-circulating swine influenza a viruses in north america and the identification of a novel h1 genetic clade with antigenic significance livestock disease surveys-a field manual for veterinarians a blueprint for pig health research. australian pig research council adverse effects of influenza a(h1n1)pdm09 virus on growth performance of norwegian pigs-a longitudinal study at a boar testing station factors associated with the occurrence of influenza a virus infections in fattening swine porcine circovirus type 2 and porcine circovirus-associated disease ecology and molecular epidemiology of avian and swine influenza a viruses in guatemala a practical approach to calculate sample size for herd prevalence surveys. preventative veterinary medicine venezuela's pig industry after 14 years of socialism. pig progress website seroprevalence of porcine circovirus type 2 in swine populations in canada and costa rica serological and molecular prevalence of swine influenza virus on farms in northwestern mexico the pig as a mixing vessel for influenza viruses: human and veterinary implications herd factors associated with the seroprevalence of four major respiratory pathogens in slaughter pigs from farrow-tofinish pig herds detection and molecular characterization of porcine circovirus type 2 from piglets with porcine circovirus associated diseases in columbia trends in emerging viral infections of swine modified indirect porcine circovirus (pcv) type 2-based and recombinant capsid protein (orf-2)-based enzyme-linked immunosorbent assays for detection of antibodies to pcv virologic and serologic surveillance for human, swine and avian influenza virus infections among pigs in the north-central united states swine influenza technical disease card organization for animal health. influenza a virus of swine prevalence of and risk factors for influenza in southern ontario swine herds in porcine parvovirus (ppv) serological evidence of swine influenza in brazil seroprevalence and risk factors of swine influenza in spain merck animal health: technical services bulletin respiratory system in disease of swine development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type 2 characterization of porcine circovirus type 2 in taiwan world animal health information system (wahis interface) -version 2; disease information, disease distribution maps acknowledgements we thank the pig farmers of trinidad and tobago for providing access to their pigs for sampling. we are grateful to the academic and technical staff of the uwi school of veterinary medicine especially mr. roger malcolm and dr. marc driscoll who were particularly instrumental in the sampling collection stages. we owe our gratitude to the director of veterinary public health, dr. saed rahaman, and the livestock and livestock products board of trinidad and tobago. we also thank the uwi trinidad and tobago research and development impact fund (rdi fund) for funding the study. we thank natalie mcginn at the animal and plant health agency-weybridge for technical support. conflict of interest the authors declare that they have no competing interests.ethical approval all procedures performed in studies involving animals were in accordance with the ethical standards of the university of the west indies (uwi) research and development impact fund and the uwi ethics committee. key: cord-261925-nsq837z1 authors: denner, joachim; mueller, nicolas j. title: preventing transfer of infectious agents date: 2015-08-24 journal: int j surg doi: 10.1016/j.ijsu.2015.08.032 sha: doc_id: 261925 cord_uid: nsq837z1 xenotransplantation using pig cells, tissues and organs may be associated with the transfer of porcine infectious agents, which may infect the human recipient and in the worst case induce a disease (zoonosis). to prevent this, a broad screening program of the donor animals for putative zoonotic microorganisms, including bacteria, viruses, fungi and others, using sensitive and specific detection methods has to be performed. as long as it is still unknown, which microorganism represents a real risk for the recipient, experience from allotransplantation should be brought in. due to the fact that pigs can be screened long before the date of transplantation, xenotransplantation will become eventually safer compared with allotransplantation. screening and selection of animals free of potential zoonotic microorganisms, caesarean section, vaccination and/or treatment with chemotherapeutics are the strategies of choice to obtain donor animals not transmitting microorganisms. in the case of porcine endogenous retroviruses (pervs) which are integrated in the genome of all pigs and which cannot be eliminated this way, selection of animals with low virus expression and generation of genetically modified pigs suppressing perv expressions may be performed. xenotransplantation using pig cells, tissues and organs has to overcome three hurdles before being applied in the clinic for the treatment of organ failure: immunological rejection, physiological incompatibility and transfer of infectious agents. the microbiological safety of xenotransplantation is an important issue, however it can be managed easily. the risk of infection is also known in allotransplantation. numerous infectious agents have been transmitted together with human donor transplants, including human cytomegalovirus (hcmv), human immunodeficiency virus-1 (hiv-1) and rabies virus [1] . since xenotransplantation allows screening the donor animals beforehand, most risks can be excluded by careful testing and xenotransplantation finally will be a microbiologically safer technology compared with allotransplantation. like all animals, pigs carry numerous microorganisms in their digestive tract and on their skin, and therefore cells, tissues and organs to be used for transplantation should be removed under aseptic conditions. the number of microorganisms present in the tissues and organs of interest should be zero [2] . in some reviews concerning the microbiological safety of xenotransplantation numerous microorganisms are listed which were thought to induce zoonoses when transmitted to the human recipient [2] . zoonosis means that the microorganisms not only infect the new host, but cause a disease. in general, bacteria, fungi, parasites and viruses may be transmitted. however, at present it is rather difficult to classify most of the porcine microorganisms into pathogenic and non-pathogenic for human recipients. in addition, when a microorganism is pathogenic in the pig it does not mean that it is also pathogenic in humans and vice versa. the risk of transmission is certainly higher when pharmaceutical immunosuppression to prevent immunological rejection of the transplant will be applied. therefore it is still unclear which microorganisms should be monitored. the auckland island pigs which had been used in the first clinical trials performed by the new zealand company lct were screened regularly for 10 bacteria, 15 viruses and toxoplasma (table 1 ) [3] . the g€ ottingen minipigs which are used for numerous biomedical investigations are screened regularly for 27 bacteria, 16 viruses, three fungi and four parasites (http//www.minipigs.dk/). an additional screening of the g€ ottingen minipigs involved perv [4] , hepatitis e and 89 other microorganisms [5, 6] . in general, most microorganisms found in pigs to be used for xenotransplantation may be eliminated by specified pathogen free (spf) or designated pathogen-free (dpf) breeding of the animals. in the case there is a bacterial or fungal infection in the donor pig, treatment with antibiotics or chemotherapeutics may be successful. at the moment hepatitis e virus (hev), porcine cytomegalovirus (pcmv), porcine circoviruses (pcv), porcine lymphotropic herpes viruses (plhv), and porcine endogenous retroviruses (pervs) are thought to pose the main risk for reasons to be discussed below and therefore these microorganisms will be analysed in the next chapters in more details. in most cases hev causes self-limiting hepatitis in humans. whereas hev of the genotype (gt) 1 and gt2 are found in people, are transmitted mainly by contaminated water and are causing a high mortality during pregnancy, hev gt3 and gt4 are swine viruses and do not cause a disease in pigs, however, when they infect humans they may cause in rare cases a zoonotic disease (for review see [5, 7] ). a severe hepatitis after infection with hev gt3 and gt4 was observed only in the case of other underlying liver diseases. importantly, neurological disorders have also been described for hev gt3 and gt1. note, that only hev gt3/4 may pose a risk when xenotransplantation using pig cells, tissues and organs is performed, not gt1 or gt2. usually hev gt3/4 are transmitted by contaminated meat or direct contact with infected pigs. hev gt3 rna was detected in pig liver at grocery stores and infectious virus could be isolated [5, 7] . hev transmission by shellfish and vegetables possibly contaminated by pig manure as well as by blood transfusion and allotransplantion was also reported. a chronic infection was more likely to develop in immunosuppressed patients, including hiv-1 infected individuals [5, 7] . sensitive pcr-based methods have been developed to determine a hev infection and to genotype the virus. detection of hev and its elimination from pigs seems not to be easy. first, the virus is heterogeneous, e.g., 10 subtypes of genotype 3 exist, what makes it difficult to design efficient pcr or real-time pcr. second, the virus load seems to be very low so that even highly sensitive pcrs may be unable to detect the virus. although hev gt3/4 are widely distributed, the prevalence in pigs, especially in multitransgenic pigs generated for usage in xenotransplantation, is not well studied. in contrast, the non-transgenic auckland island pigs, generated by living cell technologies (lct) in new zealand are better table 1 microorganisms tested in auckland island pigs used for islet cell transplantation [3] . [3] . although hev is very common in pigs in new zealand, the auckland island pigs were free of all hev [8] . another well investigated breed is the g€ ottingen minipig produced by ellegaard in denmark. these animals are used worldwide for numerous biomedical investigations. the herd was established by entry of animals obtained by caesarean sectioning and colostrum deprivation. despite this, hev was found in one study in 100% of the animals (7 of 7) [9] , in another, using real-time pcr and western blot analysis detecting antibodies against hev, hev was found in only very few animals [6] . the result suggested a transplacental mother-to-piglet transmission of the virus. this observation may help to explain how the virus entered the pig herd despite caesarean sectioning and other precautions. it remains unclear, whether the absence of the virus in all older g€ ottingen minipigs is due to the elimination of the virus possibly by the immune system or due to the limits of the detection methods. in order to eliminate hev from a herd, a hev elimination program was proposed ( fig. 1 ) [5] . elimination should include selection of hev negative animals using highly sensitive real-time pcr. since it is not clear, whether the animals are truly negative, or carry hev in concentrations below the sensitivity of the detection method, a treatment step should be included using ribavirin, a guanosine analogue used to stop viral rna synthesis. although there are no data on the treatment of hev infection with ribavirin in pigs, ribavirin has been successfully used for the treatment of other virus infections in pigs [10] . another strategy may include a vaccination step, e.g. using a vaccine based on a recombinant orf-2 fragment of hev gt1 that has been approved by the chinese fda [11] . since hev gt1-4 represents only one serotype, the induced antibodies should be protecting from infection with all genotypes [12] . immunisation of pigs with orf-2 of pig hev gt3 resulted in effective protection [12] , indicating that pigs can be immunized and mount an effective antiviral immune response. as human cytomegalovirus (hcmv) causes severe transplant rejection in allotransplantation [13, 14] , considerable concern is warranted on the potential pathogenicity of porcine cytomegalovirus (pcmv) in the setting of xenotransplantation. pcmv is endemic in the world pig population, it is acquired early in life and pcmv infection results in seroconversion and lifelong latent infection [15] . pcmv spreads by both vertical and horizontal transmission [15, 16] . active infection causes fatal systemic failure in piglets less than 3 weeks of age. the clinical symptoms of infected piglets include pneumonia and inclusion body rhinitis with a high mortality rate. pcmv-infected sows are prone to abortion, with pathological changes including edema in the heart and other organs [15] . pcmv can remain latent in adult pigs. the ubiquitous nature of herpesviruses, including pcmv, means that these viruses should be a major focus in the development of xenotransplantation. herpesviruses are able to infect other species. porcine cells can be infected by hcmv [17] , indicating that the pig transplants may be infected when the recipient is hcmv positive, and pcmv can infect human cells [18] . entry of hcmv into porcine endothelial cells depends on both the cellular vascular origin and the viral strain [19] . when pcmv was transmitted by the pig transplant into baboons, the baboon cmv was activated causing invasive disease and consumptive coagulopathy, the pcmv was mainly replicating in the pig transplant causing ureteric necrosis in one transplant [20e22]. when baboons received pig kidneys from pcmv-infected pigs, the survival time was 14.1 days in comparison to 48.3 or 53 days when organs from uninfected animals were transplanted [23] . in a similar experiment with cynomolgus monkeys the difference was 9.2 days versus 28.7 days [24] . alone the presence of the virus had such an important influence on transplant survival. when cd55-transgenic large white pigs were analyzed, all animals were found positive for pcmv, however under specified pathogen free (spf) or designated pathogen-free (dpf) conditions pcmv-free animals were obtained [25] , indicating that selection of pcmv-free animals by caesarean delivery and spf breeding is possible. on the other hand, due to reactivation of the baboon cmv after pig cardiac xenotransplantation with pharmaceutical immunosuppression a lethal outcome in some cases was observed despite prophylactic treatment with the antiviral drugs ganciclovir or valganciclovir [26] . the auckland island pigs, already used in clinical islet cell transplantation were shown to be free of pcmv [27] . as mentioned above, pcmv can be eliminated easily by caesarean delivery and dpf or spf breeding of the herd [23, 25] . in addition, early weaning of the piglet from the sow can eradicate cmv [28] . to be on the safer site, a treatment with the antiviral drugs ganciclovir, cidofovir, foscarnet, acyclovir, valaciclovir, a prodrug of acyclovir, or valganciclovir can be included into the elimination protocol ( fig. 1) [29] . concerning vaccination against hcmv, despite the urgent need for allotransplant recipients, no success was reported, although first attempts to use the major envelope glycoprotein gb have demonstrated efficacy against hcmv infection and on hcmv-induced disease [30e32]. immunization studies with the gb protein of pcmv should be performed in pigs. porcine lymphotropic herpesvirus (plhv) 1, 2, and 3 are common porcine viruses, however the prevalence and importance of these viruses for xenotransplantation is not well studied [33] . phylogenetic analyses showed that all three phlv clustered together with ruminant gammaherpesviruses, but the plhv-3 is more distantly related to plhv-1 and plhv-2 [34] . the transmission of phlv in pigs is not well understood. plhv may be fig . 1 . schematic presentation of the proposed virus elimination program. the original herd was screened for the presence of a putative zoonotic virus (grey, positive animals; pink, negative animals). negative animals (that may be actually positive, but below the detection limit) were selected and using caesarean delivery, and treatment with antiviral drugs and/or vaccines, truly negative animals could be obtained and used for further breeding and xenotransplantation. transmitted by pre-partum cross-placental vertical transfer and post-partum horizontal transmission, however, cross-placental transfer is not the common way [35] . between 26% up to 88% of animals in different herds in germany, ireland, france, spain and the united states were infected with one of the plhv [34e38]. in contrast to pcmv, early weaning cannot eradicate phlv [39] . circoviruses belong to the smallest viruses replicating autonomously in mammalian cells [40] . porcine circovirus 1 (pcv1) has not been linked with any disease, whereas pcv2 is the causing agent of post-weaning multisystemic wasting syndrome (pmws), a multifactorial disease in pigs [41] . this means that the presence of the virus is necessary for the disease but requires additional factors. the onset of the disease and the severity of the symptoms are influenced by the status of the immune system and genetic predisposition [41] . characteristic clinical signs of pmws are wasting, respiratory dysfunction, enlargement of inguinal lymph nodes, diarrhoea and a generalised depletion of lymphocytes. although humans ingest pcv-contaminated foods and are exposed to pcv through other sources, serological evidence indicated that no transmission of pcv2 to humans took place [42] . also, a contamination of a human vaccine with pcv was shown not to transmit pcv to humans [43, 44] . retrospective testing confirmed the presence of pcv1 dna in rotarix, an oral live-attenuated human rotavirus vaccine for children, beginning with the initial stages of its development and in vaccine lots used in clinical studies conducted preand post-licensure [44] . when human cell lines have been infected with pcv1 and pcv2, pcv1 persisted in most cell lines without causing any visible changes, while pcv2-transfected cells showed a cytopathogenic effect [45] . most importantly, the infection was non-productive [44, 45] . porcine endogenous retroviruses are the result of a transspecies transmission of a retrovirus and integration into the genome of all pigs. perv-a and perv-b are polytropic viruses, infecting also human cells (for review see [46] ). perv-c infects only pig cells, it is present in most, but not all pigs. whereas transformed human cells lacking some intracellular restrictions factors such as abopec can be infected easily, human primary cells can be infected only by high-titre perv-a and recombinant perv-a/c after adaptation on human cells leading to an increased number of transcription factor binding sites in their long terminal repeats [47e50]. since perv-c are present in many pigs, but not all, the selection of pigs not carrying perv-c proviruses automatically prevents generation of high-titre recombinant perv-a/ c. until now, no transmission of pervs was observed, neither in first clinical trials enrolling more than 200 patients, nor in preclinical pig to non-human primate transplantation, nor in infection experiments in small animals or non-human primates with or without pharmaceutical immunosuppression (for review see [46] ). however, most of the patients in the clinical trials were not exposed for a long time to the xenotransplant and with some exceptions associated with parallel kidney allotransplantations, no pharmaceutical immunosuppression was applied. in addition, no transmission of perv was observed in numerous pig to nonhuman primate transplantations [51] . however, it is meanwhile clear that non-human primates are not a suitable model to study this question since non-human primates carry e in contrast to humans e a mutated receptor for perv allowing only infection at a low efficiency [52] . therefore, the question, whether pervs may be transmitted during xenotransplantation is still open and an elimination of infectious proviruses is advised, since retroviruses may induce tumours and immunodeficiencies. pervs are closely related to other gammaretroviruses such as the feline leukaemia virus (felv), the murine leukaemia virus (mulv) and the koala retrovirus (korv) [53] . all these related viruses induce severe immunodeficiencies and tumours in the infected host. gammaretroviruses used as vectors in gene therapy for the treatment of a severe combined immunodeficiency in children were shown to induce leukaemia in the patients [54] , indicating that transspecies transmission of gammaretroviruses into human may induce tumour development. highly sensitive and specific methods have been developed to screen for the prevalence and expression of perv in donor pigs. whereas expression of perv in german landrace pigs is relatively low, expression in yucatan minipigs is high [55] . in these animals viral protein may be detected in different tissues of the animals [56] and infectious virus was found released from stimulated peripheral blood mononuclear cells (pbmcs) [55] . crossing of landrace pigs with minipigs increased the expression rate of perv [55] . when g€ ottingen minipigs were screened, a relatively low expression and no release of virus particles were observed [4] . the elimination programs for hev and pcmv were described in detail above [5, 29] . the elimination programs for other microorganisms (with exception of perv, see below) will be similarly, including screening of the donor pigs using specific and sensitive methods, treatment with antiviral drugs and immunisation (fig. 1) . for some porcine microorganisms effective vaccines are available, for example for circoviruses (ingelvac circoflex ® ) and mycoplasma (ingelvac circoflex ® ). several other vaccines are available to treat pigs and prevent transmission of the infectious agents (table 2) [57]. however, vaccination is only necessary, when the infectious agent is present in the pig herd and pose a risk for the transplant recipient. in the case of bacteria it has to be considered whether the donor pig (and in some cases also the recipient) should be treated with antibiotics. since pervs are present in the genome of all pigs and cannot be eliminated by the way other microorganisms can be eliminated easily, other strategies were developed to prevent transmission of these infectious agents. first, animals with a low copy number of perv and a low expression rate should be selected. second, animals not carrying perv-c should be selected in order to prevent perv-a/ c recombination. third, transgenic pigs have been generated expressing small interfering rna, specifically inhibiting the expression of perv and therefore lowering the probability of perv transmission [58e61].since in the genome of pigs multiple perv proviruses were found, sometimes more than 150, an elimination of all perv copies by genome editing using zinc finger nucleases (zfns) may be difficult. we selected zfn specific for a sequence of the highly conserved pol region of the virus. however first attempts to eliminate pervs in the pig genome failed, obviously due to the strong toxic effect when zfns were cutting the genome at multiple sites and therefore destabilising the genome [62] . a lower concentration of the zfn and a gradual elimination may be alternative strategies. in addition, new attempts using crispr/cas9 (clustered regularly interspaced short palindromic repeats/crisprassociated) for gene editing should be undertaken with the goal to eliminate at least the replication competent proviruses. fourth, and last but not least, transmission of pervs may be prevented by a vaccine. when immunising different animal species (goats, rats, mice, hamster, guinea pigs) with the recombinant transmembrane envelope protein p15e and the surface envelope protein gp70 of perv, always effective neutralising antibodies were induced [63e66]. the results of the immunisation with the transmembrane envelope protein are of great interest. in all cases neutralising antibodies were obtained and one epitope recognised was localised in the membrane proximal external region (mper), the other in the fusion peptide proximal region [63, 64] . the epitope in the mper was not only localised similarly as a corresponding epitope recognised by antibodies broadly neutralising hiv-1 isolated from hiv-1 infected individuals in the transmembrane envelope protein gp41 of hiv-1, but despite the evolutionary difference between perv and hiv-1 three identical amino acids were detected in the epitopes [63, 64] . immunisation with the surface envelope protein gp70 induced higher titres of neutralising antibodies compared with immunisation with the transmembrane envelope protein p15e [65] . since there is no animal model which allows analysing the efficiency of the perv vaccine in vivo, the related felv was studied. cats were immunised with recombinant p15e and gp70 of felv. neutralising antibodies were detected and the epitopes recognised were very similarly located compared with the epitopes recognised by the antibodies neutralising perv [67, 68] . when the immunised cats were challenged with infectious felv, the animals were protected, suggesting that in the case of perv the vaccine may also work [69] . these data indicate that immunisation of the transplant recipient may be successful in the case other prevention strategies do not work. elimination programs: screening, selection, treatment and isolation of donor pigs transmission of infection with human allografts: essential considerations in donor screening the international xenotransplantation association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetesechapter 2: source pigs microbiological safety of the first clinical pig islet xenotransplantation trial in new zealand screening pigs for xenotransplantation: prevalence and expression of porcine endogenous retroviruses in g€ ottingen minipigs xenotransplantation and hepatitis e virus extended microbiological characterization of g€ ottingen minipigs in the context of xenotransplantation: vertical transmission of hepatitis e virus hepatitis e virus: animal reservoirs and zoonotic risk monitoring for potentially xenozoonotic viruses in new zealand pigs suggestions for the diagnosis and elimination of hepatitis e virus in pigs used for xenotransplantation ribavirin efficiently suppresses porcine nidovirus replication hepatitis e vaccine development: a 14 year odyssey assessment of the crossprotective capability of recombinant capsid proteins derived from pig, rat, and avian hepatitis e viruses (hev) against challenge with a genotype 3 hev in pigs overview: cytomegalovirus and the herpesviruses in transplantation cytomegalovirus infections in solid organ transplantation: a review diseases of swine experimental transplacental transmission of porcine cytomegalovirus human cytomegalovirus productively infects porcine endothelial cells in vitro human fibroblasts are permissive for porcine cytomegalovirus in vitro entry of human cytomegalovirus into porcine endothelial cells depends on both the cellular vascular origin and the viral strain detection of infectious baboon cytomegalovirus after baboon-to-human liver xenotransplantation porcine cytomegalovirus and coagulopathy in pig-to-primate xenotransplantation activation of cytomegalovirus in pig-to-primate organ xenotransplantation porcine cytomegalovirus infection is associated with early rejection of kidney grafts in a pig to baboon xenotransplantation model results of life-supporting galactosyltransferase knockout kidneys in cynomolgus monkeys using two different sources of galactosyltransferase knockout swine herpesvirus infections in xenotransplantation: pathogenesis and approaches prevention, detection, and management of early bacterial and fungal infections in a preclinical cardiac xenotransplantation model that achieves prolonged survival microbiological safety of the first clinical pig islet xenotransplantation trial in new zealand reduction of consumptive coagulopathy using porcine cytomegalovirus-free cardiac porcine grafts in pig-to-primate xenotransplantation xenotransplantation and porcine cytomegalovirus (pcmv). xenotransplantation reanalysis of transvax immunogenicity developing a vaccine against congenital cytomegalovirus (cmv) infection: what have we learned from animal models? where should we go next? future virol vaccine prevention of maternal cytomegalovirus infection gene expression of porcine lymphotrophic herpesvirus-1 in miniature swine with posttransplant lymphoproliferative disorder a novel porcine gammaherpesvirus methods for the exclusion of circoviruses and gammaherpesviruses from pigs approaching virus safety in xenotransplantation: a search for unrecognized herpesviruses in pigs infection of pigs in ireland with lymphotropic gamma-herpesviruses and relationship to postweaning multisystemic wasting syndrome characterization of the dna polymerase loci of the novel porcine lymphotropic herpesviruses 1 and 2 in domestic and feral pigs early weaning of piglets fails to exclude porcine lymphotropic herpesvirus molecular biology of porcine circovirus: analyses of gene expression and viral replication the serological evidence in humans supports a negligible risk of zoonotic infection from porcine circovirus type 2 porcine circovirus diseases detection of porcine circovirus type 1 in commercial pig vaccines using polymerase chain reaction investigation of a regulatory agency enquiry into potential porcine circovirus type 1 contamination of the human rotavirus vaccine, rotarix: approach and outcome infection studies on human cell lines with porcine circovirus type 1 and porcine circovirus type 2 infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses genetic alterations of the long terminal repeat of an ecotropic porcine endogenous retrovirus during passage in human cells comparison of porcine endogenous retroviruses infectious potential in supernatants of producer cells and in cocultures perv infection of human peripheral blood mononuclear cells (pbmcs) the number of a u3 repeat box acting as an enhancer in long terminal repeats of polytropic replication-competent porcine endogenous retroviruses dynamically fluctuates during serial virus passages in human cells xenotransplantation of islet cells: what can the nonhuman primate model bring for the evaluation of efficacy and safety? suboptimal porcine endogenous retrovirus infection in non-human primate cells: implication for preclinical xenotransplantation koala retroviruses: characterization and impact on the life of koalas insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of scid-x1 distribution and expression of porcine endogenous retroviruses in multi-transgenic pigs generated for xenotransplantation expression of porcine endogenous retroviruses (perv) in different organs of a pig production of transgenic pigs that express porcine endogenous retrovirus small interfering rnas knockdown of porcine endogenous retrovirus (perv) expression by perv-specific shrna in transgenic pigs long-term effects of perv-specific rna interference in transgenic pigs inhibition of porcine endogenous retroviruses by rna interference: increasing the safety of xenotransplantation attempts to knock out multiple porcine endogenous retrovirus (perv) sequences in the pig genome by zinc finger nucleases (zfn) neutralizing antibodies against conserved domains of p15e of porcine endogenous retroviruses (pervs): basis for a vaccine for xenotransplantation? development of sensitive methods for detection of porcine endogenous retrovirus-c (perv-c) in the genome of pigs increased titers of neutralizing antibodies after immunization with both envelope proteins of the porcine endogenous retroviruses (pervs) novel neutralising antibodies targeting the n-terminal helical region of the transmembrane envelope protein p15e of the porcine endogenous retrovirus (perv) neutralising antibodies against the transmembrane protein of feline leukaemia virus (felv) increased neutralizing antibody response after simultaneous immunization with leucogen and the feline leukemia virus transmembrane protein immunization with the transmembrane protein of a retrovirus, feline leukemia virus: absence of antigenemia following challenge supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ijsu. 2015 .08.032. key: cord-273705-0oyzg5tq authors: duffy, mark a; chen, qi; zhang, jianqiang; halbur, patrick g; opriessnig, tanja title: impact of dietary spray-dried bovine plasma addition on pigs infected with porcine epidemic diarrhea virus date: 2018-08-29 journal: transl anim sci doi: 10.1093/tas/txy088 sha: doc_id: 273705 cord_uid: 0oyzg5tq experimental data suggest that the addition of spray-dried plasma (sdp) to pig feed may enhance antibody responses against certain pathogens and negatively impact virus survival. the benefit of sdp on escherichia coli infection is well documented. the aim of this study was to determine the effect of bovine sdp (bovsdp) in the pig diet on acute porcine epidemic diarrhea virus (pedv) infection. a total of 16 3-wk-old conventional crossbred pigs were used and divided into three groups. treatments included 1) a negative control group fed a commercial diet and sham inoculated with commercial liquid porcine plasma (n = 3), 2) a positive control group fed a commercial diet and inoculated with pedv-spiked porcine plasma (pedv; n = 8), and 3) a third group of pigs fed the commercial diet with inclusion of 5% spray-dried bovine plasma and inoculated with pedv-spiked porcine plasma (bovsdp; n = 5). although clinical signs associated with pedv infection were mild in the bovsdp group, two of eight pigs in the pedv group developed moderate clinical disease and had to be euthanized. the pedv igg and iga antibody levels and prevalence rates were significantly (p < 0.05) higher in the pedv–bovsdp group compared with the pedv group at 7 d postinoculation. the average fecal pedv rna shedding time was 7.2 ± 1.0 d for the pedv–bovsdp group and 9.3 ± 1.1 d for the pedv group with an overall time to clearance of pedv shedding of 11 d for pedv–bovsdp pigs and at least 14 d for pedv pigs, which was not different (p = 0.215). the results indicate that addition of bovsdp induced an earlier anti-pedv antibody response in pigs experimentally infected with pedv thereby reducing clinical disease and the amount and duration of viral shedding during acute pedv infection. coronaviridae. pedv isolates can be divided into two genogroups: g1, which encompasses a majority of strains isolated before 2010, and g2, which contains more recently identified strains (huang et al., 2013) . pedv was first discovered in north america in april 2013 and since its emergence has caused substantial losses to the u.s. pork industry. the clinical disease associated with pedv is characterized by watery diarrhea and vomiting in all ages of pigs and high mortality rates in young pigs (pensaert and debouck, 1978) . an intact intestinal mucosa, which prevents the entrance of agents across the epithelium, is a first line of protection with great importance in weaned pigs that, due to separation from their dam and comingling with other pigs, are under high stress and exposed to numerous pathogens they have not encountered before (pitman and blumberg, 2000; boudry et al., 2004; pie et al., 2004; bailey et al., 2005; moretó and pérez-bosque, 2009 ). producers commonly add spraydried plasma (sdp) of porcine or bovine origin to weaned pig diets as it has been shown to promote growth (grinstead et al., 2000) and aids in combating common postweaning pathogens such as escherichia coli (torrallardona et al., 2003; bosi et al., 2004) . it is well recognized that the addition of sdp to pig feed enhances the immune response and decreases pathogen loads compared with pigs without plasma access through feed. for instance, plasma protein supplements modulated the mucosal immune response in organized and diffuse gut-associated lymphoid tissue, which is accompanied by a reduction of proinflammatory cytokine production (bosi et al., 2004; nofrarias et al., 2006; pérez-bosque et al., 2008) . a canadian case-control study investigating factors associated with mortality due to porcine circovirus type 2 (pcv2) found that nursery rations were more likely to contain sdp in control herds compared with clinically affected case herds (dewey et al., 2006) ; however, alternatively the observed differences may be completely unrelated to the sdp inclusion in the diets. in addition, the biological neutralization activity of antibodies against common pig pathogens present in sdp was conserved and may contribute to the biosafety of commercially available sdp (polo et al., 2013) . previously, it has also been shown that sdp has an intrinsic effect reducing survival of pedv under in vitro conditions (quist-rybachuk et al., 2015) . after the introduction of pedv into the united states, concerns on possible contribution of porcine-based sdp in rapid farm-to-farm transmission were raised (pasick et al., 2014) despite studies clearly demonstrating that pedv is inactivated during the spray-drying process (opriessnig et al., 2014; pujols and segales, 2014; gerber et al., 2014b) . to eliminate any possible risk of pig pathogen spread via sdp while retaining its benefits as part of a pig diet, pork producers may switch from porcine-origin sdp toward bovine-origin sdp (bovsdp). the purpose of this study was to determine whether there is any benefit of adding bovsdp to a diet of pigs during acute pedv infection. the experiment was approved by the iowa state university institutional animal care and use committee (iacuc approval number 2-14-7742-s). sixteen, 2-wk-old, crossbred, colostrum-fed pigs were selected from a commercial pedv-free herd. the farm of origin was tested on a monthly basis for pedv rna on representative fecal samples and for pedv antibodies on selected serum samples and had no history of clinical diarrhea. the farm of origin was also free of porcine reproductive and respiratory syndrome virus (prrsv), influenza a virus (iav), and mycoplasma hyopneumoniae. before shipment to the research facility, the pigs received a one dose commercial pcv2 vaccine (merck animal health, inc.). the pigs were transported to the research facility at iowa state university, randomly assigned to one of three groups of three to eight pigs, and housed in separate rooms on solid concrete floors (table 1) . upon arrival of the pigs in the research facility, they were tested negative for pedv antibodies in serum and pedv rna in fecal samples. the experimental design is summarized in figure 1 . briefly, the pigs were inoculated with pedv at 3 wk of age (day postinoculation or dpi 0), and all pigs were necropsied at dpi 14. starting with arrival in the research facility and for the duration of the study, all pigs were fed the same standard commercial corn-soybean meal-dried whey-based diet ( table 2 ) except for the diet of the pedv-bovsdp group, which was supplemented with 5% spray-dried commercial bovine plasma replacing soy protein concentrate on an equal total lysine basis ( figure 1 ). the commercial spray-dried bovine plasma (lot #s510011001) was produced in a manufacturing plant located in kansas, united states, and submitted to commercial spray-drying conditions including a minimum outlet temperature of 80 °c throughout substance (throughout the entire particle's mass). the control diet did not contain any bovsdp. for the pedv inoculation, the passage 7 pedv g2b isolate 13-19338e (chen et al., 2014) at viral concentration of 10 3.0 50% tissue culture infectious dose per milliliter was used. in brief, pedv was propagated on vero cells (atcc ccl-81) with minimal essential medium supplemented with tryptose phosphate broth (0.3%), yeast extract (0.02%), trypsin 250 (5 µg/ml), gentamicin (0.05 mg/ml), penicillin (100 unit/ml), streptomycin (100 µg/ ml), and amphotericin (0.25 µg/ml) as previously described (chen et al., 2014) . commercial liquid porcine plasma negative for pedv rna was used as diluent to adjust the pedv inoculum (pedv and pedv-bovsdp groups) or saline (negative control [neg] group) to a total volume of 10 ml for each pig. inoculation was performed orally by the same team for all pigs. the pigs were held in vertical recumbence between the legs of the holder while the second person administered the material by slowly dripping the inoculum into the mouth of the pig using a syringe on dpi 0 when the pigs were 3 wk old. after pedv inoculation, all pigs were monitored daily for signs of illness such as lethargy, vomiting, or diarrhea. rectal temperatures were taken every other day on each pig, and the fecal consistency score ranging from 0 = normal, 1 = semisolid, 2 = pasty, and 3 = liquid was determined (gerber et al., 2016) . all pigs were weighed at dpi −6 and again at dpi 14, and the adg was calculated. to verify the presence of pedv iga and igg antibodies, blood samples were collected in serum separator tubes on dpi 0, 7, and 14 and centrifuged at 4,000 rpm for 10 min at 4 °c. all serum samples were tested for the presence of anti-pedv igg or iga by an "in-house" indirect elisa based on the spike 1 gene of a prototype u.s. pedv strain similar to the one used as challenge virus in this study (gerber et al., 2014a; gerber and opriessnig, 2015) . a sample with a sample-to-positive (s/p) ratio equal to or greater than 0.2 was considered positive for pedv igg, and a sample with an s/p ratio equal to or greater than 0.13 was considered positive for pedv iga. to determine fecal pedv shedding, rectal swabs were collected from all pigs on dpi 6 and from dpi 1 to 14 using polyester swabs and stored in 5-ml plastic tubes containing 1-ml sterile saline. total nucleic acids were extracted from fecal swab suspensions using the magmax pathogen rna/dna kit and an automated nucleic acid extraction system (thermo scientific kingfisher flex, thermo fisher scientific, pittsburgh, pa) according to the instructions of the manufacturer figure 1 . experimental design. blood was collected at arrival and dpi 0, 7, and 14, and rectal swabs were collected 6 d before challenge, on dpi 1 and daily thereafter. rectal temperatures were taken every other day starting with 1 d before pedv inoculation. translate basic science to industry innovation (opriessnig et al., 2014) . presence of pedv rna was determined by using a quantitative real time rt pcr that was set up using path-id multiplex one-step rt-pcr kit (thermo fisher scientific), and amplifications were performed on the applied biosystems 7500 fast real-time pcr system thermocycler and the accompanying software (opriessnig et al., 2014) . on dpi 14, all pigs were killed by intravenous pentobarbital overdose (fatal-plus, vortech pharmaceuticals ltd, dearborn, mi) and necropsied. tissues collected from pigs at necropsy included eight sections of small intestines, three sections of large intestine, and one section of mesenteric lymph node. tissues were immediately put in 10% buffered formalin and routinely processed for histopathology and assessed for lesions by a veterinary pathologist blinded to the treatment status. presence and degree of atrophic enteritis were scored ranging from 0 = normal to 3 = severe. to determine presence and amount of pedv antigen in tissue sections, a pedv immunohistochemical stain was performed on intestines from all pigs as described (stevenson et al., 2013) . the amount of pedv antigen was scored by a veterinary pathologist blinded to the treatment status with 0 = no signal, 1 = 1% to 10% staining, 2 = 11% to 50% staining, and 3 = greater than 50% staining. the statistical software used was jmp pro 11. analysis of variance (anova) was used for cross-sectional assessment of the average daily weight gain and continuous measures including viral shedding, iga, and igg antibody levels. the pedv genomic copy numbers per milliliter of fecal suspension were log transformed before statistical analysis. if a significant (p < 0.05) difference was detected, pairwise testing using the tukey-kramer adjustment was performed to determine which groups were different. daily rectal temperature data were analyzed with multivariate anova. non-repeated measures of necropsy and histopathology data were assessed using nonparametric kruskal-wallis anova. if a nonparametric anova test was significant (p < 0.05), then wilcoxon tests were used to assess the differences between pairs of groups. differences in incidence of clinical scores were evaluated by using the fisher's exact test. for fecal shedding of pedv rna, an area under the curve (auc) was calculated for each animal individually and differences between groups were assessed by unpaired t-test (data passed shapiro-wilks normality test). there was no difference in rectal temperatures among the groups over time, and none of the pigs developed elevated rectal temperatures during the experiment. diarrhea was not observed in the neg group while individual pigs in the pedv group and pedv-bovsdp group had fluid-to-pasty-to-semisolid feces between dpi 2 and 6 with no significant differences among groups. in addition, vomiting was occasionally observed in single pigs during this time. by dpi 9, two pedv pigs became lethargic and had reduced appetite. one of these pigs developed mild pasty diarrhea. due to welfare concerns, these pigs were euthanized between dpi 11 and 13. the adg is summarized in table 1 . overall, there were numerical differences among groups for the adg: neg pigs had the highest adg, and pedv pigs had the lowest adg; however, the differences were not significant. all pigs were negative for pedv iga and igg antibodies at arrival, and the neg pigs remained seronegative for the duration of the study (figures 2 and 3) . three of five pedv-bovsdp pigs had detectable anti-pedv iga antibodies in serum by dpi 7, and all pigs in this group and five per eight pedv pigs were positive by dpi 14. group mean iga levels were higher (p < 0.011) for pedv-bovsdp pigs compared with pedv pigs at dpi 7 ( figure 2 ). group mean igg levels were also higher (p < 0.021) in pedv-bovsdp pigs at dpi 7 compared with the pedv pigs ( figure 3 ). all pedv-infected pigs in both groups had anti-igg antibodies by dpi 14 (figure 3 ). all pigs were pedv rna negative at dpi −6, and pedv rna was not detected in any of the neg pigs throughout the study. fecal shedding of pedv rna was first detected in a pedv pig at dpi 1 and shedding in this group lasted until termination of the study at dpi 14 (figures 4 and 5) . pigs in the pedv-bovsdp group shed pedv from dpi 2 to 11 (figures 4 and 5) . the prevalence of pcr positive pedv-bovsdp pigs compared with the pedv group was higher (p = 0.021) at dpi 2, while it was lower (p = 0.028) at dpi 12. the average shedding period was 9.3 ± 1.1 for pedv pigs and 7.2 ± 1.0 for pedv-bovsdp pigs, which was not different (p = 0.215). similarly, the cumulative fecal viral rna shedding was not different (p = 0.384) between pedv pigs and pedv-bovsdp pigs. in the two pigs that were euthanized due to welfare reasons, in addition to the standard set of enteric tissues, sections of liver, lung, spleen, tonsil, heart, and kidney were also collected and assessed to rule out any concurrent systemic infection. one of these two pigs had moderate diffuse atrophic enteritis associated with pedv antigen as determined by immunohistochemical stains (score 2). this pig also had the highest amount of pedv rna measured (log 10 9.5 genomic copies per fecal swab at dpi 6 and figure 2 . mean group anti-pedv iga elisa s/p ratios ± sem at days 0, 7, and 14 after pedv inoculation (dpi) in the different treatment groups and number of positive pigs/total number of pigs in the group for each day. the results of the two pigs that were killed on dpi 11 and 13 were included at dpi 14. an s/p ratio greater than 0.13 was considered positive. different superscripts ( a,b ) indicate significantly different means at a given day. statistical analysis was performed by one-way anova followed by pairwise testing using the tukey-kramer adjustment if p < 0.05. the statistical software used was jmp pro 11. the number of elisa positive pigs per total number of pigs per group is listed next to each group mean. log 10 8.9 at dpi 11) when it was killed. the other pig had its peak pedv shedding at dpi 6 (log 10 6.3 pedv genomic copies), but its shedding decreased to log 10 5.6 genomic copies by dpi 13. no other lesions were seen in these two pigs. the remaining pigs were necropsied at the scheduled time at dpi 14, and lesions or pedv antigen were not seen in any of these pigs suggesting that the pedv infection and associated lesions had resolved. pedv has become a major economic concern for north american pig producers since it was first . mean group log 10 amount of pedv rna fecal samples in pedv and pedv-bovsdp pigs at different days after pedv infection and number of positive pigs per total number of pigs in the group for each day. an "a" on a given day indicates significant (p < 0.05) different group means. statistical analysis was performed by one-way anova followed by pairwise testing using the tukey-kramer adjustment if p < 0.05. the statistical software used was jmp pro 11. 443. mean group anti-pedv igg elisa s/p ratios ± sem at days 0, 7, and 14 after pedv inoculation in the different treatment groups and number of positive pigs per total number of pigs in the group for each day. the results of the two pigs that were killed on dpi 11 and 13 were included at dpi 14. an s/p ratio greater than 0.2 was considered positive. different superscripts ( a,b ) indicate significantly different means at a given day. the number of elisa positive pigs per total number of pigs per group is listed next to each group mean. identified in the united states in 2013 (stevenson et al., 2013; chen et al., 2014) . the lack of highly effective vaccines and the relative ineffectiveness of common treatment methods have led to a search for alternative methods of treating and preventing outbreaks. the inclusion of sdp into the diets of young pigs has been shown to have health benefits for other diseases. this study was designed to determine whether there is any benefit to adding bovsdp to the diet during pedv infection. supplementing feed rations of pigs, fish, poultry, cats, and dogs with sdp as protein source is performed on a regular basis. sdp is a protein-rich product obtained from blood from healthy animals (cattle or pigs) at slaughter (torrallardona, 2010; pérez-bosque et al., 2016) . in pet food, sdp is a preferred binder in canned food products due to its high-protein content and its physicochemical properties (polo et al., 2005; rodriguez et al., 2016) . porcine sdp was first introduced as protein source for pigs during the early 1990s (cole and sprent, 2001 ) and since has been used widely in the diet of weaned pigs (torrallardona, 2010) . benefits of adding porcine sdp include improvement of weight gain mainly due to increased feed intake and reduction of incidence and severity of diarrhea after weaning (adewole et al., 2016) . comprehensive information on the effect of sdp obtained in 75 trials involving over 12,000 pigs has been summarized (torrallardona, 2010) . in this study, the pedv challenge was performed after an acclimation period of 1 wk to minimize stress from weaning and transport to the new facility. successful pedv challenge was confirmed by detecting pedv rna in fecal swabs. all pigs (100%) from the bovsdp-pedv group shed pedv in fecal samples from dpi 2 to 6, whereas between 25% and approximately 87% of the animals in pedv group shed the virus during that time. these results highlight that a higher number of animals excreted pedv during the early stages of infection in bovsdp-pedv group. differences in the challenge dose or challenge process can be ruled out. the pedv inoculum was prepared in a similar manner and at the same time for both groups by a single person and stored on ice until challenge of each group. the challenge was conducted by the same personnel for both groups with approximately 20 min between the two groups. the obtained results could have been by chance due to the group sizes. alternatively, the bovsdp-pedv animals could be more prone to excrete pedv in the early stages in the infection. unlike in suckling pigs that are very susceptible to pedv infection, only 2 per 13 pedv-infected pigs (both from the pedv challenged groups) had to be killed due to the severity of clinical signs. this is expected and comparable with other trials infecting 3-wk-old pigs (crawford et al., 2015) . in the current study, the pedv igg and iga antibody responses were more rapid in the bovsdp group compared with the pedv group. specifically, by dpi 7, 60% of the bovsdp pigs were anti-igg and figure 5 . mean group log 10 amount of pedv rna in pcr positive pigs in fecal samples at different days after pedv infection. an "a" at a given day indicates significant (p < 0.05) different group means. statistical analysis was performed by one-way anova followed by pairwise testing using the tukey-kramer adjustment if p < 0.05. the statistical software used was jmp pro 11. iga positive compared with 12.5% of the pedv pigs. of note, while systemic iga antibodies were measured, mucosal iga levels were not determined, and it is therefore unknown how the addition of the bovsdp affected the gut immunity. in a previous study, a good correlation of iga levels in serum and feces was found , and as fecal samples and gut mucosa are more difficult to process during routine lab work, serum iga levels are commonly tested. explanations for the earlier humoral immune response in the bovsdp group may include acceleration of the clinical course by the dietary supplement; however, a more rapid immune response due to earlier replication of the virus in more pigs unrelated to the diet modification is also possible. serum anti-pedv ig in pig serum has been demonstrated to neutralize infectivity of pedv (hofmann and wyler, 1989) , and bovine plasma could have a similar neutralizing activity, which was not further assessed in this study. besides the presence of possible neutralizing antibodies in sdp, other plasma compounds such as peptides (anderson and anderson, 2002) could contribute to the benefits seen with sdp addition to a diet. virus shedding in the bovsdp group was 2.1 d shorter than in the pedv group. the fecal pedv rna shedding in this group was terminated by dpi 11. a previous study has shown that pedvinfected pigs shed infectious pedv capable of horizontal transmission for 14 to 16 d after infection (crawford et al., 2015) . this is similar to what was observed in the pedv-infected pigs without bovsdp in the diet in this study. the data from this trial indicate a beneficial effect of bovsdp on acute pedv infection, which is similar to previous reports using the prrsv infection model (pujols et al., 2011) . specifically, pigs fed bovsdp were able to clear virus shedding 2.1 d sooner than non-bovsdp pigs. the addition of bovsdp to the diet resulted in faster and stronger pedv antibody responses and reduced pedv shedding time compared with pigs with no bovsdp in the diet. limitations of this study include the usage of one single virus titer and the low numbers of pigs tested. in addition, two pedv pigs had to be removed early from the study, which could have impacted the outcomes. a larger study with higher numbers of pigs per group comparing porcine and bovine-derived sdp with multiple necropsy days should be conducted to further confirm the possible benefits of sdp in pedv-infected pigs. gut health of pigs: challenge models and response criteria with a critical analysis of the effectiveness of selected feed additives-a review the human plasma proteome: history, character, and diagnostic prospects the development of the mucosal immune system pre-and post-weaning: balancing regulatory and effector function spray-dried plasma improves growth performance and reduces inflammatory status of weaned pigs challenged with enterotoxigenic escherichia coli k88 weaning induces both transient and long-lasting modifications of absorptive, secretory, and barrier properties of piglet intestine isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states protein and amino acid requirements of weaner pigs evaluation of porcine epidemic diarrhea virus transmission and the immune response in growing pigs postweaning mortality in manitoba swine detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa detection of immunoglobulin (ig) a antibodies against porcine epidemic diarrhea virus (pedv) in fecal and serum samples the spray-drying process is sufficient to inactivate infectious porcine epidemic diarrhea virus in plasma increased frequency of porcine epidemic diarrhea virus shedding and lesions in suckling pigs compared to nursery pigs and protective immunity in nursery pigs after homologous re-challenge effects of a whey protein product and spray-dried animal plasma on growth performance of weanling pigs quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (pedv) origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states dietary plasma proteins, the intestinal immune system, and the barrier functions of the intestinal mucosa effects of spray-dried porcine plasma and plant extracts on intestinal morphology and on leukocyte cell subsets of weaned pigs porcine epidemic diarrhea virus rna present in commercial spray-dried porcine plasma is not infectious to naive pigs investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in canada a new coronavirus-like particle associated with diarrhea in swine dietary plasma proteins modulate the immune response of diffuse gut-associated lymphoid tissue in rats challenged with staphylococcus aureus enterotoxin spray dried plasma as an alternative to antibiotics in piglet feeds, mode of action and biosafety weaning is associated with an upregulation of expression of inflammatory cytokines in the intestine of piglets first line of defense: the role of the intestinal epithelium as an active component of the mucosal immune system neutralizing antibodies against porcine circovirus type 2 in liquid pooled plasma contribute to the biosafety of commercially manufactured spray-dried porcine plasma functional properties of spray-dried animal plasma in canned petfood commercial spray-dried porcine plasma does not transmit porcine circovirus type 2 in weaned pigs challenged with porcine reproductive and respiratory syndrome virus survivability of porcine epidemic diarrhea virus (pedv) in bovine plasma submitted to spray drying processing and held at different time by temperature storage conditions sensitivity of porcine epidemic diarrhea virus (pedv) to ph and heat treatment in the presence or absence of porcine plasma effect of spray-dried plasma on food intake and apparent nutrient digestibility by cats when added to a wet pet food recipe emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences spray dried animal plasma as an alternative to antibiotics in weanling pigs-a review. asian-aust effect of fishmeal replacement with spray-dried animal plasma and colistin on intestinal structure, intestinal microbiology, and performance of weanling pigs challenged with escherichia coli k99 this study was funded by the apc, inc. additional support was provided by the biotechnology and biological sciences research council (bbsrc) institute strategic programme grant awarded to the roslin institute (bb/j004324/1; bbs/e/d/20241864). we thank dr. huigang shen for assistance with sample testing and dr. priscilla gerber for assistance with the statistical analysis. conflict of interest statement. none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. key: cord-297669-22fctxk4 authors: proudfoot, chris; lillico, simon; tait-burkard, christine title: genome editing for disease resistance in pigs and chickens date: 2019-06-25 journal: anim front doi: 10.1093/af/vfz013 sha: doc_id: 297669 cord_uid: 22fctxk4 nan for thousands of years, humans have used selective breeding to improve desirable traits in both livestock and companion animals. in livestock, targeted breeding has been common practice since the british agricultural revolution of the 18th century, with measurable production traits such as feed conversion in cattle or wool production in sheep actively selected for. in the late 20th century, genomic selection was added to the livestock breeding tool box; by reading specific locations in the genome and assigning them to measurable production traits, faster improvement in livestock production efficiency has been achieved. one of the inherently difficult production traits to measure is resistance to a specific disease, as animals with less severe symptoms or pathology may simply have been exposed to less pathogen. experimental infections guaranteeing equal pathogen exposures are expensive and require large numbers of animals for genetic association studies, making them ethically questionable. genome editing offers new opportunities to livestock breeding for disease resistance, allowing the direct translation of laboratory research into disease-resistant or resilient animals. made? genome editors are custom enzymes that allow scientists to cut the dna strands in the nucleus of a cell at a specific position. the researcher can then influence how the dna is repaired, introducing very precise genetic changes at a target locus in their species of interest. this technology has been revolutionary and provides exciting possibilities for the production of livestock resistant to viral diseases. such opportunities are particularly pertinent given state efforts to improve global food security and reduce food waste throughout the production chain. the most prominent editor technology today, crispr/cas, uses a 20 nucleotide rna guide to target its enzyme component to a designated locus in the genome. the probability of off-target cutting with a high fidelity cas enzyme is very low, because with four potential base combinations at each of the 20 nucleotides there are over one trillion unique guide combinations. once the enzyme has cut the dna strands, the predominant repair pathway in most cells is nonhomologous end joining, an error-prone process which often introduces small insertions or deletions into the genetic code at the break site. if the target is within a gene, such perturbations can result in a disruption to the function of that gene, potentially leading to a loss of protein function. this can be very useful to basic science as it allows researchers to discover functions associated with novel genes. for many applications, a more precise change to the genome is required. to that end, scientists regularly make an alternative dna repair process called homology-directed repair. to do this, researchers provide a novel dna sequence alongside the crispr/cas reagents, whereby the cellular repair machinery uses the new dna as a template when fixing the break. this approach facilitates the introduction of defined implications • genome editing technology enlarges the tool box of trait-selective breeding. • methods for genome editing have developed over the past decades, making the technology more efficient and specific. • technology to generate edited pigs and chickens is developing alongside genome editors to generate animals faster and more affordable. • for two major pig diseases, it has been shown that resistant animals can be generated that are refractory to infection. in chickens there are promising laboratory results but no genome-edited, resistant chickens yet. • genome editing allows us to overcome bottlenecks in trait-selective breeding and allows the incorporation of genetic traits from other breeds, related species, or laboratory results. • two major hurdles still to be faced prior to the implementation of this promising technology are consumer acceptance and the regulatory framework. changes at the genomic target locus and has sufficient refinement to alter a single nucleotide, allowing precise modification of gene function. finally, by introducing a pair of editors, it is possible to generate two concurrent dna breaks on the same chromosome. the cellular repair machinery then joins the ends of the cut sites, promoting the deletion of the intervening sequence. all the editor reagents introduced to the cell are rapidly degraded, with only the alteration to the genomic sequence remaining to be propagated following cell division. genome editing has been applied to a wide variety of agricultural species including salmonids, poultry, and ruminants. however, due to its global economic value, relatively short generation time, and multiparous nature, the most edited livestock species to date is the pig. there are two main methods widely used for the generation of edited pigs: cloning of edited fibroblasts or direct injection of the zygotes with editor reagents. both work well, and each has specific advantages. in cloning, fibroblast cells can be maintained in the lab for prolonged periods. this allows researchers to introduce editor reagents into the cultured cells typically by lipofection, electroporation, or microinjection. editing events in each cell of a population can be characterized and individual cells with the desired alteration to their genome selected for the cloning process, whereby the fibroblast cell is fused with an enucleated oocyte shell in a process called somatic cell nuclear transfer ( figure 1a ). the reconstituted "zygote" is then transferred to a recipient gilt or sow (carlson et al., 2012) . despite the benefit of being able to prescreen the donor cells, cloning is generally inefficient with hundreds of reconstituted zygotes being transferred to a single recipient. cloning also yields reduced litter sizes when compared with standard breeding and offspring often demonstrate reduced viability. as an alternative to cloning, newly fertilized zygotes can be directly microinjected with genome-editing reagents and transferred immediately back to the oviduct of a recipient animal ( figure 1b ). in contrast to cloning, this approach (lillico et al., 2013) results in the efficient establishment of pregnancies and robust litters. however, without the prescreening of cells that is routine in cloning, offspring from direct zygote manipulation inevitably encompasses a range of editing outcomes, since selection of a specific edit is not possible. porcine zygotes can also be generated by maturation of oocytes extracted from slaughterhouse-derived ovaries and in vitro fertilization. unfortunately, in vitro fertilization in pigs often results in polyspermy, rendering the resulting embryo inviable. however, in this controlled environment, editing rates can be increased and costs and animal use reduced. an emerging alternative to these proven methods could be the use of surrogate sires. as a first step towards this goal, pigs have been edited to remove a gene required for male fertility, generating an empty spermatagonial stem cell niche in the testis . spermatogonial stem cells can be isolated and cultured in vitro, opening the possibility to edit and characterize these cells before transfer to a recipient (park et al., 2019 ) ( figure 1c ). genetic modification of poultry poses unique challenges due to the very different physiology of the avian egg compared with a mammalian oocyte. as a result, isolation and transfer of a chicken yolk is not practical. one approach that has been taken is in ovo electroporation of editing reagents, which allowed the analysis of gene function in the neural crest (gandhi et al., 2017) . however, others reported that electroporation resulted in mosaicism with editing limited to a subset of cells as the chicken embryo is already much further developed when an egg is laid compared with a zygote (veron et al., 2015) ( figure 1d ). as a result, it is unlikely that this approach could be efficiently utilized to generate edited birds. an alternative approach involves sperm transfection-assisted gene editing, whereby sperm are lipofected with editing reagents before use in artificial insemination (cooper et al., 2017 ) ( figure 1e ). however, advances in chicken stem cell technology show the greatest promise for genome editing in chicken. primordial germ cells (stem cells that eventually develop into germ cells) can be isolated from the blood of developing chicks in ovo and cultured in vitro. as with mammalian fibroblasts, these cells can be edited and selected in vitro before transfer into the bloodstream of a stage-matched recipient where they migrate to and populate the developing gonad. the chicken embryo is accessed through an opening in the egg shell, which is sealed again until the chicken hatches. genome editing in primordial germ cells has been successfully demonstrated by a number of groups (park et al., 2014; taylor et al., 2017; idoko-akoh et al., 2018) and one group has generated modified birds (park et al., 2014) . the founder birds generated from this editing method are chimeric due to the presence of preexisting germ cells. the resulting offspring generated from breeding with the founders will be a mixture of edited or nonedited. recipient chicken embryos devoid of germ cells are currently being developed that will significantly increase the efficiency of this process (m. mcgrew, unpublished results) ( figure 1f ). genome editors will undoubtedly have a significant role on the generation of disease-resistant animals as exemplified below. it is important to note that currently the technology is limited to modifying a single gene or a snp with large effects; however, disease resistance in many cases is likely to be a polygenic trait. multiplexing technology is under development such that in the future polygenic traits could be altered in a single step. progress so far? porcine reproductive and respiratory syndrome virus. porcine reproductive and respiratory syndrome (prrs) is arguably the most economically important pig disease worldwide. the causative agent of prrs is an arterivirus, named prrs virus (prrsv), that affects pigs of all ages but most importantly causes late-term abortions and stillbirth in sows and severe respiratory disease in piglets with severe morbidity and high mortality. prrsv also incapacitates the pig's immune response, providing an ideal breeding ground for severe secondary infections, mostly by bacteria, which leads to increased use of antibiotics. prrsv exclusively infects cells of the monocyte/macrophage lineage and two macrophagespecific proteins, cd163 and cd169, were identified as receptors for the virus: cd169 acting on the surface of the cells and cd163 inside the internalizing transport vesicles (calvert et al., 2007; van gorp et al., 2008) . the virus was thought to attach to cd169 to be taken up into the cells; however, genome-edited pigs lacking cd169 were not resistant to prrsv infection (prather et al., 2013) . cd163 on the other hand is thought to act through a key-lock interaction with the virus to allow it to escape from the internalizing transport vehicles into the cytosol where it replicates. cd163 consists of nine globular domains, organized like beads on a string, with domain 5 determined to mediate the key-lock interaction allowing viral entry into pig cells (van gorp et al., 2010) . using genome editing to generate pigs lacking cd163 whitworth et al. showed for the first time that this approach could be used to produce livestock resistant to important viral diseases, in this case prrs (whitworth et al., 2016) . cd163 is known to have a range of important biological functions in homeostasis, inflammation, and immune responses. as a refinement on functional knock out of the entire cd163 protein, editing reagents were designed to remove only domain 5 leaving the remainder of the protein intact. the resulting animals were completely resistant to prrsv infection and maintained the biological functions associated with the remaining domains of cd163 (burkard et al., 2017; burkard et al., 2018) . porcine epidemic diarrhea virus/transmissible gastroenteritis virus. the two coronaviruses porcine epidemic diarrhea virus (pedv) and transmissible gastroenteritis virus (tgev) both cause severe diarrhea in preweaned piglets and are associated with high morbidity and mortality. in vitro host-pathogen studies identified aminopeptidase n as the receptor for tgev and a potential receptor for pedv (delmas et al., 1992; li et al., 2007) . the use of genome editing to generate pigs lacking aminopeptidase n successfully showed that pigs resistant to tgev infection could be generated. however, the edited animals remained susceptible to pedv infection (whitworth et al., 2019) . aminopeptidase n is important for peptide digestion in the small intestine and knockout mice were shown to have delayed mammary gland development. in humans, aminopeptidase n defects are associated with different types of leukemia and lymphoma. therefore, further investigation into the potential consequences of the absence aminopeptidase n in pigs is warranted as it may affect the overall health and/or productivity of the animals. african swine fever virus. african swine fever virus (asfv) causes a severe hemorrhagic disease in domestic pigs (sus scrofa domesticus) and wild boars (sus scrofa ferus) with high mortality in pigs of all ages. asfv is highly contagious and can be transmitted by soft ticks of the ornithodoros genus. it was identified in and contained to africa with occasional transmission around the strait of gibraltar into portugal and spain. in 2007 an introduction of the virus into the caucasus region showed that the virus does not solely rely on ticks for transmission in the wild, as transport of contaminated material and direct contact between animals have been shown to be major routes of disease dissemination. since then, the virus has spread across eastern europe and russia and was recently found in western europe and china. asfv poses a huge risk to the pig industry worldwide and is a limiting factor to a sustainable pig industry in many parts of africa. interestingly, asfv also infects wild suids, such as warthogs (phacocherus africanus) and bushpigs (potamocherus porcus), without causing overt disease. such infected wild suids are thought to act as a reservoir of the virus in africa. in the late stages of asfv infection, a cytokine storm, i.e., an overreaction of the immune system, is observed, which is thought to strongly contribute to the lethal outcome of disease. a comparison of the warthog and domestic pig genomes identified differences in the rel-like domain-containing protein a (rela, also known as p65) protein, which is involved in nf-κb cytokine signaling, was thought to underlie the different responses of the related species to asfv infection (palgrave et al., 2011) . researchers used genome editing to convert a key region of the encoded domestic pig protein sequence to the warthog equivalent (lillico et al., 2016) . data on susceptibility of the edited animals to asfv infection have yet to be reported. in this instance, it is important to differentiate between disease resistance, the ability of an animal to suppress the establishment and/or development of an infection, and disease resilience, where an infected host manages to maintain an acceptable level of productivity despite challenge pressure. should these pigs prove to be resilient to asfv infection it is likely that their use may not be permitted in many jurisdictions, since they could act as reservoirs of infection. however, in environments where the disease is endemic use of such animals could be beneficial. avian leucosis virus. avian leukosis virus infection results in inappetence, diarrhea, weight loss, a reduction in eggs laid, and often causes tumor formation in the chicken. the virus is divided into six subgroups, with the avian leucosis virus subgroup j (alv-j) shown to be responsible for major disease outbreaks in china. the cellular receptor of alv-j was identified to be the chicken sodium/hydrogen exchanger 1 protein on the cell surface. chicken somatic cell lines have been edited to introduce changes to this gene-conferring resistance to avian leucosis virus in vitro (lee et al., 2017) . despite cells showing resistance to alv-j infection, no edited chickens have been produced to date. in both mice and humans, a lack of the sodium/hydrogen exchanger 1 protein is associated with severe neurological disease; however, targeted changes to single amino acids may retain biological functions of the protein in chicken while resulting in disease resistance. avian influenza virus. in chickens, disease resistance to avian influenza is at the top of the wish list due to the serious impact on chicken health but also the risk of transmission to humans. similarly, influenza a is also one of the diseases on the resistance wish list for pigs, as they can act as an intermediate host-aiding virus adaptation to humans. the acidic leucine-rich nuclear phosphoprotein-32a (anp32a) was found to play a key role in avian influenza virus replication in both chicken and water fowl. although the virus polymerase protein readily interacts with the avian anp32a, the human version of the same protein supports only limited replication of the viral genome. it has been demonstrated in vitro that deletion of a small region of chicken anp32a can prevent replication of avian influenza virus (long et al., 2016; long et al., 2019) . although the functional consequence of edited anp32a has yet to be demonstrated in vivo, such approaches offer exciting opportunities that have the potential to benefit both industry and animal welfare. as exemplified above, currently many gene editing approaches focus on targeting host genes involved in mediating entry of the virus, with a special focus on receptors. however, as the example for avian influenza shows, host genes play an important role in other steps of the pathogen replication cycle and also provide editing targets for disease resilience or resistance. more in-depth host-pathogen interaction studies, including genome-wide editing studies in vitro, will no doubt produce a variety of further candidate genes for genetic disease resistance. an alternative antipathogen approach pursued for decades is the generation of transgenic livestock, expressing antiviral or antibacterial agents, such as enzymes or small interfering rnas. genome editing can be used to improve the integration efficiency of these transgenes at specific locations in the genome; however, the discussion of transgenic disease-resistant animals is beyond the scope of this review. how does genome editing fit within existing selective breeding structures and how will it be regulated? selective breeding has generated highly productive, robust animals that are adapted to a modern production environment. livestock production is dynamic, with evolving challenges such as climate change and disease outbreaks coupled with societal pressure to reduce antimicrobial use. selective breeding for disease resistance has proven difficult, as outbreaks are often sporadic and resistant/resilient animals often difficult to identify. in circumstances where a genetic trait for disease resistance can be identified in the breeding population, then selection through the selective breeding can be achieved. a good example of this is pigs with resistance to f18 type enterotoxigenic e. coli. association studies revealed that a polymorphism in the fucosyl transferase 1 gene conferred resistance to these bacteria. there was initial concern that selection for the locus figure 2 . genetic resistance to disease and how genome editing can help integrate traits into highly productive lines. (a) genetic resistance to disease may be present in a small percentage of production animals and genetic selection for these animals may be associated with the risk of inbreeding, productivity loss, or the risk of losing other desirable traits. genome editing allows integration of the disease-resistance trait into a wider selection of pigs, ensuring genetic variability and maintenance of desirable traits. (b) genetic resistance to disease may be present in an indigenous or less productive breed. crossbreeding would result in productivity loss and the risk of losing other desirable traits, such as fur color. genome editing allows for incorporation of genetic disease resistance into highly bred lines without losing productivity. (c) genetic resistance may be observed in a closely related species, e.g., wild boar or wild suids in the case of the domestic pig. integration into highly bred domestic pig lines would only be possible by genome editing. (d) resistance genes may be identified in laboratory research but not in highly bred lines, making integration into those productive animals only possible using genome editing. harboring this gene may counterselect for another gene associated with stress resistance. however, this proved not to be the case and genetic selection for the favorable fucosyl transferase 1 allele has been integrated into many pig-breeding programs (coddens et al., 2008) . this was possible, in part, because the favorable allele was present at sufficient prevalence (in most studies between 5% and 10%) in the breeding population to allow for selection while avoiding inbreeding. in circumstances where an allelic variant associated with a resistant phenotype is present at a much lower frequency, it may prove difficult to incorporate effective selection into a standard breeding regime without the risk of inbreeding and related longer-term productivity loss (figure 2a) . genome editing has the potential to contribute in such circumstances, allowing the direct introgression of a beneficial allele into the offspring of diverse, highly productive animals. similarly, disease-resistance traits associated with less productive indigenous breeds are unlikely to be introduced to highly productive populations by standard crossbreeding as this would result in a significant set-back in productivity, abrogating decades or even centuries of advances made through genetic selection ( figure 2b ). in circumstances where resistance or resilience is observed in a related species, crossbreeding is simply not possible. genome editing could bridge these gaps. one example of this is resilience of wild suids to african swine fever virus while domestic pigs can suffer from severe disease. it is not possible to crossbreed these species, so introduction of the genetics underlying resilience is not possible by this route. genetic comparison can be used to identify the functional differences underlying such traits, and genome editing employed to introduce appropriate variants into domestic pigs ( figure 2c ). finally, with a good understanding of host-pathogen interactions, novel genetics that has not been observed in live animals can be created and tested for efficacy in a laboratory environment. this was the case for both the cd163/prrsv and apn/tgev examples in pigs and would be the case for the anp32a/ influenza and the alv-j resistance in chicken, described above. in such circumstances, integration through genome editing presents a practical route to benefit from the findings ( figure 2d ). it is imperative that in such circumstances thorough phenotypic characterization of the edited animals be carried out as deletion of all or part of a functional protein could result in a loss of (systemic) biological function. a second measure worthy of consideration before embarking on an editing project is whether the gene is located within a locus that has been actively selected in breeding programs. this could indicate whether a potential target is associated with known production traits. this approach has been taken for prrsv-resistant pigs, with evaluation as to whether the cd163 gene locus has been selected for in pig breeding programs (johnsson et al., 2018) . overall, genome editing holds vast promise for the future production of animals resistant or resilient to disease, improving productivity and animal welfare while reducing food waste throughout the production chain. through reduction of primary and secondary infections, it should also be possible to reduce antimicrobial use in livestock production. technical improvements in the generation of genome editing animals will undoubtedly reduce the cost implications of this technology. the two major hurdles still to be faced prior to implementation of this promising technology are consumer acceptance and the regulatory framework. approval of edited animals for human consumption relies on national and multinational legislation, which is currently at early stages. encouragingly, some international jurisdictions such as argentina and brazil have about the authors dr. chris proudfoot is research fellow at the roslin institute/university of edinburgh since 2011. his work centers on generation of genome-modified livestock, with particular emphasis on genome editors, to improve disease resistance or to accurately model human disease. dr. proudfoot has worked extensively with zfns, talens, and crispr/cas9 to produce a variety of edited animals. he was a member of the team that produced the first edited livestock using this method. dr. simon lillico is a research associate at the roslin institute/university of edinburgh. he joined the institute on an industrial collaboration to produce highvalue therapeutic proteins in hens eggs and then applied his expertise in lentiviral transgenesis to generate livestock models of human diseases. the rapid expansion of the field of genome editors over the last 7 yr has made practicable genome modifications which had previously been unattainable. dr. lillico has been at the forefront of application of these editors to livestock, creating either disease-resistant/resilient strains, or accurate models of human disease. dr. christine tait-burkard is an assistant professor at the roslin institute/university of edinburgh since 2017 in the departments of genetics and genomics and infection and immunity. her research focuses on understanding host-pathogen interactions on a cellular and genetic level, developing new in vitro tools for virus research, improving and developing easy-to-use diagnostics, and devising strategies to combat viral disease in livestock in general and pigs in particular. she employs genome editing and genetic selection to generate animals genetically resistant to viral disease. corresponding author: christine.burkard@roslin.ed.ac.uk already ruled that modifications, such as the prrsv-resistant pig, that do not have any new genetic information integrated into the animal, will be exempt from regulation. precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd163 srcr5 domain are fully resistant to both prrsv genotypes while maintaining biological function pigs lacking the scavenger receptor cysteine-rich domain 5 of cd163 are resistant to porcine reproductive and respiratory syndrome virus 1 infection cd163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses efficient talen-mediated gene knockout in livestock the possibility of positive selection for both f18(+)escherichia coli and stress resistant pigs opens new perspectives for pig breeding generation of gene edited birds in one generation using sperm transfection assisted gene editing (stage) aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev optimization of crispr/cas9 genome editing for loss-of-function in the early chick embryo high fidelity crispr/cas9 increases precise monoallelic and biallelic editing events in primordial germ cells precise gene editing of chicken na+/h+ exchange type 1 (chnhe1) confers resistance to avian leukosis virus subgroup j (alv-j) porcine aminopeptidase n is a functional receptor for the pedv coronavirus live pigs produced from genome edited zygotes mammalian interspecies substitution of immune modulatory alleles by genome editing species difference in anp32a underlies influenza a virus polymerase host restriction avian anp32b does not support influenza a virus polymerase and influenza a virus relies exclusively on anp32a in chicken cells species-specific variation in rela underlies differences in nf-κb activity: a potential role in african swine fever pathogenesis successful genetic modification of porcine spermatogonial stem cells via an electrically responsive au nanowire injector generation of germline ablated male pigs by crispr/cas9 editing of the nanos2 gene targeted gene knockout in chickens mediated by talens an intact sialoadhesin (sn/siglec1/cd169) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus efficient talen-mediated gene targeting of chicken primordial germ cells sialoadhesin and cd163 join forces during entry of the porcine reproductive and respiratory syndrome virus identification of the cd163 protein domains involved in infection of the porcine reproductive and respiratory syndrome virus crispr mediated somatic cell genome engineering in the chicken gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus resistance to coronavirus infection in amino peptidase n-deficient pigs we acknowledge financial support from the biotechnology and biological science research council (bbsrc) (bb/ r004463/1, bb/n015339/1) and the bbsrc institute strategic programme grant funding to the roslin institute (bbs/ e/d/20002172 and bbs/e/d/20002174). key: cord-277487-jgbjxgh1 authors: graham, simon p.; mclean, rebecca k.; spencer, alexandra j.; belij-rammerstorfer, sandra; wright, daniel; ulaszewska, marta; edwards, jane c.; hayes, jack w. p.; martini, veronica; thakur, nazia; conceicao, carina; dietrich, isabelle; shelton, holly; waters, ryan; ludi, anna; wilsden, ginette; browning, clare; bialy, dagmara; bhat, sushant; stevenson-leggett, phoebe; hollinghurst, philippa; gilbride, ciaran; pulido, david; moffat, katy; sharpe, hannah; allen, elizabeth; mioulet, valerie; chiu, chris; newman, joseph; asfor, amin s.; burman, alison; crossley, sylvia; huo, jiandong; owens, raymond j.; carroll, miles; hammond, john a.; tchilian, elma; bailey, dalan; charleston, bryan; gilbert, sarah c.; tuthill, tobias j.; lambe, teresa title: evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored covid-19 vaccine candidate chadox1 ncov-19 date: 2020-06-20 journal: biorxiv doi: 10.1101/2020.06.20.159715 sha: doc_id: 277487 cord_uid: jgbjxgh1 clinical development of the covid-19 vaccine candidate chadox1 ncov-19, a replication-deficient simian adenoviral vector expressing the full-length sars-cov-2 spike (s) protein was initiated in april 2020 following non-human primate studies using a single immunisation. here, we compared the immunogenicity of one or two doses of chadox1 ncov-19 in both mice and pigs. whilst a single dose induced antigen-specific antibody and t cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in sars-cov-2 neutralising titres. as sars-cov-2 began to spread around the world at the beginning of 2020 several vaccine platform technologies were employed to generate candidate vaccines. several use replicationdeficient adenoviral (ad) vector technology and express the sars-cov-2 spike (s) protein. the first phase i clinical study of an ad5-vectored vaccine has been reported 1 , chadox1 ncov-19 (azd1222) phase i trials (nct04324606) began in april 2020 with phase ii and iii trials (nct04400838) started soon thereafter, and an ad26-vectored vaccine is expected to enter phase i shortly. typically, only one dose of ad-vectored vaccines has been administered in early preclinical challenge studies or clinical studies against emerging or outbreak pathogens 2-5 . rhesus macaques immunised with a single dose of chadox1 ncov-19 were protected against pneumonia but there was no impact on nasal virus titers after high dose challenge to both the upper and lower respiratory tract 6 . to increase antibody titres and longevity of immune responses, a booster vaccination may be administered. homologous prime-boost immunisation resulted in higher antibody titres including neutralising antibodies and a trend towards a lower clinical score in a mers-cov challenge study 7 . here, we set out to test the immunogenicity of either one or two doses of chadox1 ncov-19 in mice and pigs, to further inform clinical development. 'prime-boost' vaccinated inbred (balb/c) and outbred (cd1) mice were immunised on 0 and 28 days post-vaccination (dpv), whereas, 'prime-only' mice received a single dose of chadox1 ncov-19 on day 28. spleens and serum were harvested from all mice on day 49 (3 weeks after boost or prime vaccination). analysis of sars-cov-2 s protein-specific murine splenocyte responses by ifnγ elispot assay showed no statistically significant difference between the prime-only and primeboost vaccination regimens, in either strain of mouse ( figure 1a ). intracellular cytokine staining (ics) of splenocytes ( figure 1b) showed, in both mouse strains, that the response was principally driven by cd8 + t cells. the predominant cytokine response of both cd8 + and cd4 + t cells was expression of ifn-γ and tnf-α, with negligible frequencies of il-4 + and il-10 + cells, consistent with previous data suggesting adenoviral vaccination does not induce a dominant th2 response 8, 9 . there were no signficant differences in cd4 + and cd8 + t cell cytokine responses between prime-only and primeboost mice. prime-only and prime-boost pigs were immunised on 0 dpv and prime-boost pigs received a second immunisation on 28 dpv. blood samples were collected weekly until 42 dpv to analyse immune responses. ifn-γ elispot analysis of porcine peripheral blood mononuclear cells (pbmc) showed responses on 42 dpv (2 weeks after boost) that were significantly greater in the prime-boost pigs compared to prime-only animals (p < 0.05; figure 1c ). the prime-boost 42 dpv responses were greater than responses observed in either group on 14 dpv, but inter-animal variation meant this did not achieve statistical significance. ics analysis of porcine t cell reponses showed a dominance of th1-type cytokines (similar to the murine response) but with a higher frequency of s-specific cd4 + t cells compared to cd8 + t cells ( figure 1d ). however, cd4 + and cd8 + t cell cytokine responses did not differ significantly between vaccine groups or timepoints (14 vs. 42 dpv). sars-cov-2 s protein-specific antibody titres in serum were determined by elisa using recombinant soluble trimeric s (fl-s) and receptor binding domain (rbd) proteins. a significant increase in fl-s binding antibody titres was observed in prime-boost balb/c mice compared to their prime-only counterparts (p < 0.01), however, the difference between vaccine groups for cd1 mice was not significant (figure 2a ). antibody responses were evaluated longitudinally in pig sera by fl-s and rbd elisa. compared to pre-vaccination sera, significant fl-s specific antibody titres were detected in both prime-only and prime-boost groups from 21 and 14 dpv, respectively (p < 0.01; figure 2b ). fl-s antibody titres did not differ signifcantly between groups until after the boost, when titres in the prime-boost pigs became significantly greater with an average increase in titres of > 1 log10 (p < 0.0001). rbd-specific antibody titres showed a similar profile with significant titres in both groups from 14 dpv (p < 0.05) and a further significant increase in the prime-boost pigs from 35 dpv onwards which was greater than the prime-only pigs (p < 0.0001; figure 2c ). sars-cov-2 neutralising antibody responses were assessed using a virus neutralisation test (vnt; figure 2d ) and pseudovirus-based neutralisation test (pvnt; figure 2e ). after the prime immunisation, sars-cov-2 neutralising antibody titres were detected by vnt in 14 and 28 dpv sera from 2/3 prime-boost and 2/3 prime-only pigs. two weeks after the boost (42 dpv), neutralising antibody titres were detected and had increased in all prime-boost pigs, which were significantly greater than the earlier timepoints and the titres measured in the prime-only group (p < 0.01). in agreement with this analysis, serum assayed for neutralising antibodies using the pvnt revealed that antibody titres in 42 dpv prime-boost pig sera were significantly greater than earlier timepoints and the prime-only group (p < 0.001). statistical analysis showed a highly significant correlation between pvnt and vnt titres (spearman's rank correlation r = 0.86; p < 0.0001). in this study, we utilised both a small and a large animal model to evaluate the immunogenicity of either one or two doses of a covid-19 vaccine candidate, chadox1 ncov-19 (now known as azd1222). small animal models have variable success in predicting vaccine efficacy in larger animals but are an important stepping stone to facilitate prioritisation of vaccine targets. in contrast, larger animal models, such as the pig and non-human primates, have been shown to more accurately predict vaccine outcome in humans [10] [11] [12] . the mouse data generated in this study suggested that the immunogenicity profile was at the upper end of a dose response curve, which may have saturated the immune response and largely obscured our ability to determine differences between prime-only or prime-boost regimens. we have developed the pig as a model for generating and understanding immune responses to vaccination against human influenza [13] [14] [15] and nipah virus 16, 17 .the inherent heterogeneity of an outbred large animal model is more representative of immune responses in humans. extensive development of reagents to study immune responses in pigs in recent years has extended the usefulness and applicability of the pig as a model to study infectious disease. these data demonstrate the utility of the pig as a model for further evaluation of the immunogenicity of chadox1 ncov-19 and other covid-19 vaccines. we show here that t cell responses are higher in pigs that received a prime-boost vaccination when compared to prime only at day 42, whilst comparing responses 14 days after last immunisation demonstrates the prime-boost regimen trended toward a higher response. in addition, chadox1 ncov-19 immunisation induced robust th1-like cd4 + and cd8 + t cell responses in both pigs and mice. this has important implications for covid-19 vaccine development as virus-specific t cells are thought to play an important role in sars-cov-2 infection [18] [19] [20] [21] [22] . while no correlate of protection has been defined for covid-19, recent publications suggest that neutralising antibody titres may be correlated with protection in animal challenge models 23, 24 . a single dose of chadox1 ncov-19 induces antibody responses, but we demonstrate here that antibody responses are significantly enhanced after homologous boost in one mouse strain and to a greater extent in pigs. however, it is likely that a combination of neutralising antibodies and antigen-specific t cells would act in synergy to prevent and control infection, as we have recently shown in the context of influenza vaccination 13, 25 . whilst human immunogenicity and clinical read-outs are a critically meaningful endpoint, studies in small animals and pigs will help prioritise candidates to be tested in humans. further clinical studies are needed to assess immunogenicity after prime-boost vaccination and the impact on clinical efficacy and durability of the immune response. mouse and pig studies were performed in accordance with the uk animals (scientific procedures) act 1986 and with approval from the relevant local animal welfare and ethical review body (mice -project license p9808b4f1, and pigs -project license pp1804248). the principles of the 3r's were applied for the duration of the study to ensure animal welfare was not unnecessarily compromised. vero e6 cells were grown in dmem containing sodium pyruvate and l-glutamine (sigma-aldrich, poole, uk), 10% fbs (gibco, thermo fisher, loughborough, uk), 0.2% penicillin/streptomycin (10,000 u/ml; gibco) (maintenance media) at 37 °c and 5% co2. sars-cov-2 isolate england-2 stocks were grown in vero e6 cells using a multiplicity of infection (moi) of 0.0001 for 3 days at 37 °c in propagation media (maintenance media containing 2% fbs). sars-cov-2 stocks were titrated on vero e6 cells using mem (gibco), 2% fcs (labtech, heathfield, uk), 0.8% avicel (fmc biopolymer, girvan, uk) as overlay. plaque assays were fixed using formaldehyde (vwr, leighton buzzard, uk) and stained using 0.1% toluidine blue (sigma-aldrich). all work with live sars-cov-2 virus was performed in acdp hg3 laboratories by trained personnel. the propagation, purification and assessment of chadox1 ncov-19 titres were as described previously 7 . a synthetic dna, encoding the spike (s) protein receptor binding domain (rbd; amino acids 330-532) of sars-cov-2 (genbank mn908947), codon optimised for expression in mammalian cells (idt technology) was inserted into the vector popinttgneo incorporating a c-terminal his6 tag. recombinant rbd was transiently expressed in expi293™ (thermo fisher scientific, uk) and protein purified from culture supernatants by immobilised metal affinity followed by a gel filtration in phosphate-buffered saline (pbs) ph 7.4 buffer. a soluble trimeric s (fl-s) protein construct encoding residues 1-1213 with two sets of mutations that stabilise the protein in a pre-fusion conformation (removal of a furin cleavage site and the introduction of two proline residues; k983p, v984p) was expressed as described 26 . the endogenous viral signal peptide was retained at the nterminus (residues 1-14), a c-terminal t4-foldon domain incorporated to promote association of monomers into trimers to reflect the native transmembrane viral protein, and a c-terminal his6 tag included for nickel-based affinity purification. similar to recombinant rbd, fl-s was transiently expressed in expi293™ (thermo fisher scientific) and protein purified from culture supernatants by immobilised metal affinity followed by gel filtration in tris-buffered saline (tbs) ph 7.4 buffer. for analysis of t cell responses in pigs, overlapping 16mer peptides offset by 4 residues based on the predicted amino acid sequence of the entire s protein from sars-cov-2 wuhan-hu-1 isolate (ncbi reference sequence: nc_045512.2) were designed and synthesised (mimotopes, melbourne, australia) and reconstituted in sterile 40% acetonitrile (sigma-aldrich) at a concentration of 3 mg/ml. three pools of synthetic peptides representing residues 1-331 (pool 1), 332-748 (pool 2) and 749-1273 (pool 3) were prepared for use to stimulate t cells in ifn-γ elispot and intracellular cytokine staining (ics) assays. for analysis of t cell responses in mice, overlapping 15mer peptides offset by 11 residues were designed and synthesised (mimotopes) and reconstituted in sterile 100% dmso (sigma-aldrich) at a concentration of 100 mg/ml. two peptide pools spanning s1 region (pool 1: 1 to 77 and 317-321, pool 2:78-167) and 2 peptide pools spanning s2 region (pool 3:166 to 241, pool 4:242 to 316) were used for stimulating splenocytes for ifn-γ elispot analysis, and single pools of s1 (pool 1 and pool 2) and s2 (pool 3 and pool 4) were used to stimulate splenocytes for ics. mice: inbred female balb/colahsd (balb/c) (envigo) and outbred crl:cd1 (cd1) (charles river) of at least 6 weeks of age were randomly allocated into 'prime-only' or 'prime-boost' vaccination groups (balb/c n=5 and cd1 n=8). prime-boost mice were immunised intramuscularly with 10 8 infectious units (iu) (6.02x10 9 virus particles; vp) chadox1 ncov-19 and boosted intramuscularly four weeks later with 1 × 10 8 iu chadox1 ncov-19. prime-only mice received a single dose of 10 8 iu chadox1 ncov-19 at the same time prime-boost mice were boosted. spleens and serum were harvested from all animals a further 3 weeks later. pigs: six 8-10-week-old, weaned, female, large white-landrace-hampshire cross-bred pigs from a commercial rearing unit were randomly allocated to two treatment groups (n = 3): 'prime-only' and 'prime-boost'. both groups were immunised on day 0 with 1 × 10 9 iu (5.12 × 10 10 vp) chadox1 ncov-19 in 1 ml pbs by intramuscular injection (brachiocephalic muscle). 'prime-boost' pigs received an identical booster immunisation on day 28. blood samples were taken from all pigs on a weekly basis at 0, 7, 14, 21, 28, 35 and 42 dpv by venepuncture of the external jugular vein: 8 ml/pig in bd sst vacutainer tubes (fisher scientific) for serum collection and 40 ml/pig in bd heparin vacutainer tubes (fisher scientific) for peripheral blood mononuclear cell (pbmc) isolation. mice: antibodies to sars-cov-2 fl-s protein were determined by performing a standardised elisa on serum collected 3-weeks after prime or prime-boost vaccination. maxisorp plates (nunc) were coated with 100 ng/well fl-s protein overnight at 4°c, prior to washing in pbs/tween (0.05% v/v) and blocking with blocker casein in pbs (thermo fisher scientific) for 1 hour at room temperature (rt). standard positive serum (pool of mouse serum with high endpoint titre against fl-s protein), individual mouse serum samples, negative and an internal control (diluted in casein) were incubated for 2 hours at rt. following washing, bound antibodies were detected by addition of alkaline phosphatase-conjugated goat anti-mouse igg (sigma-aldrich), diluted 1/5000 in casein, for 1 hour at rt and detection of anti-mouse igg by the addition of pnpp substrate (sigma-aldrich). an arbitrary number of elisa units were assigned to the reference pool and od values of each dilution were fitted to a 4-parameter logistic curve using softmax pro software. elisa units were calculated for each sample using the od values of the sample and the parameters of the standard curve. pigs: serum was isolated by centrifugation of sst tubes at 1300 × g for 10 minutes at rt and stored at -80°c. sars-cov-2 rbd and fl-s specific antibodies in serum were assessed as detailed previously 26 with the exception of the following two steps. the conjugated secondary antibody was replaced with goat anti-porcine igg hrp (abcam, cambridge, uk) at 1/10,000 dilution in pbs with 0.1% tween20 and 1% non-fat milk. in addition, after the last wash, a 100 µl of tmb (one component horse radish peroxidase microwell substrate, biofx, cambridge bioscience, cambridge, uk) was added to each well and the plates were incubated for 7 minutes at rt. a 100 µl of biofx 450nmstop reagent (cambridge bioscience) was then added to stop the reaction and microplates were read at 450nm. end-point antibody titres (mean of duplicates) were calculated as follows: the log10 od was plotted against the log10 sample dilution and a regression analysis of the linear part of this curve allowed calculation of the endpoint titre with an od of twice the average od values of 0 dpv sera. the ability of pig sera to neutralise sars-cov-2 was evaluated using virus and pseudovirus neutralisation assays. for both assays, sera were first heat-inactivated (hi) by incubation at 56 °c for 2 hours. virus neutralization test (vnt): starting at a 1 in 5 dilution, two-fold serial dilutions of sera were prepared in 96 well round-bottom plates using dmem containing 1% fbs and 1% antibiotic-antimycotic (gibco) (dilution media). 75 μl of diluted pig serum was mixed with 75 μl dilution media containing approximately 64 plaque-forming units (pfu) sars-cov-2 for 1 hour at 37 °c. vero e6 cells were seeded in 96-well flat-bottom plates at a density of 1 × 10 5 cells/ml in maintenance media one day prior to experimentation. culture supernatants were replaced by 100 µl of dmem containing 10% fcs and 1% antibiotic-antimycotic, before 100 µl of the virus-sera mixture was added to the vero e6 cells and incubated for six days at 37 °c. cytopathic effect (cpe) was investigated by brightfield microscopy. cells were further fixed and stained as described above, and cpe scored. each individual pig serum dilution was tested in quadruplet on the same plate and no sera/sars-cov-2 virus and no sera/no virus controls were run concurrently on each plate in quadruplet. wells were scored for cytopathic effect and neutralisation titres expressed as the reciprocal of the serum dilution that completely blocked cpe in 50% of the wells (nd50). researchers performing the vnts were blinded to the identity of the samples. pseudovirus neutralisation test (pvnt): lentiviral-based sars-cov-2 pseudoviruses were generated in hek293t cells incubated at 37 °c, 5% co2. cells were seeded at a density of 7.5 x 10 5 in 6 well dishes, before being transfected with plasmids as follows: 500 ng of sars-cov-2 spike, 600 ng p8.91 (encoding for hiv-1 gag-pol), 600 ng csflw (lentivirus backbone expressing a firefly luciferase reporter gene), in opti-mem (gibco) along with 10 µl pei (1 µg/ml) transfection reagent. a 'no glycoprotein' control was also set up using carrier dna (pcdna3.1) instead of the sars-cov-2 s expression plasmid. the following day, the transfection mix was replaced with 3 ml dmem with 10% fbs (dmem-10%) and incubated at 37 °c. at both 48 and 72 hours post transfection, supernatants containing pseudotyped sars-cov-2 (sars-cov-2 pps) were harvested, pooled and centrifuged at 1,300 x g for 10 minutes at 4 °c to remove cellular debris. target hek293t cells, previously transfected with 500 ng of a human ace2 expression plasmid (addgene, cambridge, ma, usa) were seeded at a density of 2 × 10 4 in 100 µl dmem-10% in a white flat-bottomed 96-well plate one day prior to harvesting of sars-cov-2 pps. the following day, sars-cov-2 pps were titrated 10-fold on target cells, with the remainder stored at -80 °c. for pvnts, pig sera were diluted 1:20 in serum-free media and 50 µl was added to a 96-well plate in quadruplicate and titrated 4fold. a fixed titred volume of sars-cov-2 pps was added at a dilution equivalent to 10 6 signal luciferase units in 50 µl dmem-10% and incubated with sera for 1 hour at 37 °c, 5% co2. target cells expressing human ace2 were then added at a density of 2 x 10 4 in 100 µl and incubated at 37 °c, 5% co2 for 72 hours. firefly luciferase activity was then measured with brightglo luciferase reagent and a glomax-multi + detection system (promega, southampton, uk). pseudovirus neutralization titres were expressed as the reciprocal of the serum dilution that inhibited luciferase expression by 50% (ic50). mice: single cell suspension of mouse spleens were prepared by passing cells through 70 μm cell strainers and ack lysis (thermo fisher) prior to resuspension in complete media (mem supplemented with 10% fcs, pen-step, l-glut and 2-mercaptoethanol). for analysis of ifn-γ production by elispot assay, splenocytes were stimulated with s peptide pools at a final concentration of 2g/ml on ipvh-membrane plates (millipore) coated with 5g/ml anti-mouse ifn-γ (clone an18; mabtech). after 18-20 hours of stimulation, ifn-γ spot forming cells (sfc) were detected by staining membranes with anti-mouse ifn-γ biotin mab (1 µg/ml; clone r46a2, mabtech) followed by streptavidin-alkaline phosphatase (1 µg/ml) and development with ap conjugate substrate kit (bio-rad). for analysis of intracellular cytokine production, cells were stimulated with 2 μg/ml s peptide pools, media or cell stimulation cocktail (containing pma-ionomycin, biolegend), together with 1 μg/ml golgiplug (bd biosciences) and 2 μl/ml cd107a-alexa647 for 6 hours in a 96-well u-bottom plate, prior to placing at 4 o c overnight. following surface staining with cd4-buv496, cd8-percp-cy5.5, cd62l-bv711, cd127-bv650, cd44-apc-cy7 and live/dead aqua (thermo fisher), cells were fixed with 10% neutral buffered formalin (containing 4% paraformaldehyde) and stained intracellularly with tnf--af488, il-2-pe-cy7, il-4-bv605, il-10-pe and ifn-γ-e450 diluted in perm-wash buffer (bd biosciences). sample acquisition was performed on a fortessa (bd) and data analysed in flowjo v9 (treestar). an acquisition threshold was set at a minimum of 5000 events in the live cd3 + gate. antigen-specific t cells were identified by gating on live/dead negative, doublet negative (fsc-h vs fsc-a), size (fsc-h vs ssc), cd3 + , cd4 + or cd8 + cells and cytokine positive. total sars-cov-2 s specific cytokine responses are presented after subtraction of the background response detected in the media stimulated control spleen sample of each mouse, prior to summing together the frequency of s1 and s2 specific cells. pigs: pbmcs were isolated from heparinised blood by density gradient centrifugation and cryopreserved in cold 10% dmso (sigma-aldrich) in hi fbs 17 . resuscitated pbmc were suspended in rpmi 1640 medium, glutamax supplement, hepes (gibco) supplemented with 10 % hi fbs (new zealand origin, life science production, bedford, uk), 1% penicillin-streptomycin and 0.1% 2-mercaptoethanol (50 mm; gibco) (crpmi). to determine the frequency of sars-cov-2 s specific ifn-γ producing cells, an elispot assay was performed on pbmc from 0, 14, 28 and 42 dpv. multiscreen 96-well plates (mahas4510; millipore, fisher scientific) were pre-coated with 1 µg/ml anti-porcine ifn-γ mab (clone p2g10, bd biosciences) and incubated overnight at 4 °c. after washing and blocking with crpmi, pbmcs were plated at 5 × 10 5 cells/well in crpmi in a volume of 50 µl/well. pbmcs were stimulated in triplicate wells with the sars-cov-2 s peptide pools at a final concentration of 1 µg/ml/peptide. crpmi alone was used in triplicate wells as a negative control. after 18 hours incubation at 37 °c with 5% co2, plates were developed as described previously 17 . the numbers of specific ifn-γ secreting cells were determined using an immunospot ® s6 analyzer (cellular technology, cleveland, usa). for each animal, the mean 'crpmi only' data was subtracted from the s peptide pool 1, 2 and 3 data which were then summed and expressed as the mediumcorrected number of antigen-specific ifn-γ secreting cells per 1 x 10 6 pbmc. to assess intracellular cytokine expression pbmc from 14 and 42 dpv were suspended in crpmi at a density of 2 × 10 7 cells/ml and added to 50 µl/well to 96-well round bottom plates. pbmcs were stimulated in triplicate wells with the sars-cov-2 s peptide pools (1 µg/ml/peptide). unstimulated cells in triplicate wells were used as a negative control. after 14 hours incubation at 37 °c, 5% co2, cytokine secretion was blocked by addition 1:1,000 bd golgiplug (bd biosciences) and cells were further incubated for 6 hours. pbmc were washed in pbs and surface labelled with zombie nir fixable viability stain (biolegend), cd4-percp-cy5.5 mab (clone 74-12-4, bd bioscience) and cd8β-fitc mab (clone ppt23, bio-rad antibodies). following fixation (fixation buffer, biolegend) and permeabilization (permeabilization wash buffer, biolegend), cells were stained with: ifn-γ-af647 mab (clone cc302, bio-rad antibodies, kidlington, uk), tnf-α-bv421 mab (clone mab11, biolegend), il-2 mab (clone a150d 3f1 2h2, invitrogen, thermo fisher scientific) and il-4 bv605 mab (clone mp4-25d2, biolegend) followed by staining with anti-mouse igg2a-pe-cy7 (clone rmg2a-62, biolegend). cells were analysed using a bd lsrfortessa flow cytometer and flowjo x software. total sars-cov-2 s specific cytokine positive responses are presented after subtraction of the background response detected in the media stimulated control pbmc sample of each pig, prior to summing together the frequency of s-peptide pools 1-3 specific cells. graphpad prism 8.1.2 (graphpad software, san diego, usa) was used for graphical and statistical analysis of data sets. anova or a mixed-effects model were conducted to compare responses over time and between vaccine groups at different time points post-vaccination as detailed in the results. neutralising antibody titre data were log transformed before analysis. neutralising antibody titre data generated by the vnt and pvnt assays were compared using spearman nonparametric correlation. p-values < 0.05 were considered statistically significant. were immunised on day 0 and 28 with chadox1 ncov19 (prime-boost) or chadox1 ncov19 on day 28 (prime-only); pigs (n=3) were immunised with chadox1 ncov-19 on days 0 and 28 (primeboost), or only on day 0 (prime-only). to analyse sars-cov-2 s-specific t cell responses, all mice were sacrificed on day 49 for isolation of splenocytes and pigs were blood sampled longitudinally to isolate pbmc. following stimulation with sars-cov-2 s-peptides, responses of murine splenocytes (a) and porcine pbmc (c) were assessed by ifn-γ elispot assays. using flow cytometry, cd4 + and cd8 + t cell responses were characterised by assessing expression of ifn-γ, tnf-α, il-2, il-4 and il-10 (mice; b) and ifn-γ, tnf-α, il-2 and il-4 (pigs; d). each data point represents an individual mouse/pig with bars denoting the median response per group/timepoint. : sars-cov-2 s protein-specific antibody responses following chadox1 ncov-19 primeonly and prime-boost vaccination regimens in mice and pigs. inbred balb/c (n=5) and outbred cd1 (n=8) were immunised on day 0 and 28 with chadox1 ncov19 (prime-boost) or chadox1 ncov19 on day 28 (prime-only), whereas, pigs were immunised with chadox1 ncov-19 on days 0 and 28 (prime-boost), or only on day 0 (prime-only). to analyse sars-cov-2 s protein-specific antibodies in serum, all mice were sacrificed on day 49 and pigs were blood sampled weekly until day 42. antibody units or end-point titres (ept) were assessed by elisa using recombinant sars-cov-2 fl-s for both mice (a) and pigs (b), and recombinant s protein rbd for pigs (c). sars-cov-2 neutralising antibody titres in pig sera were determined by vnt, expressed as the reciprocal of the serum dilution that neutralised virus infectivity in 50% of the wells (nd50; d) , and pvnt, expressed as reciprocal serum dilution to inhibit pseudovirus entry by 50% (ic50; e). each data point represents an individual mouse/pig sera with bars denoting the median titre per group. a single dose of chadox1 mers provides broad protective immunity against a variety of mers-cov strains. biorxiv chadox1 ncov-19 vaccination prevents sars-cov-2 pneumonia in rhesus macaques. biorxiv a single dose of chadox1 mers provides protective immunity in rhesus macaques antigen encoded by vaccine vectors derived from human adenovirus serotype 5 is preferentially presented to cd8+ t lymphocytes by the cd8α+ dendritic cell subset immunization with an adenovirus-vectored tb vaccine containing ag85a-mtb32 effectively alleviates allergic asthma the pig: a model for human infectious diseases large animal models for vaccine development and testing the contribution of non-human primate models to the development of human vaccines comparison of heterosubtypic protection in ferrets and pigs induced by a single-cycle influenza vaccine immunogenicity and protective efficacy of seasonal human live attenuated cold-adapted influenza virus vaccine in pigs aerosol delivery of a candidate universal influenza vaccine reduces viral load in pigs challenged with pandemic h1n1 virus vaccine development for nipah virus infection in pigs bovine herpesvirus-4-vectored delivery of nipah virus glycoproteins enhances t cell immunogenicity in pigs. vaccines (basel) targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid-19 patients clinical and immunological features of severe and moderate coronavirus disease 2019 transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients presence of sars-cov-2 reactive t cells in covid-19 patients and healthy donors. medrxiv sars-cov-2 infection protects against rechallenge in rhesus macaques dna vaccine protection against sars-cov-2 in rhesus macaques vaccination with viral vectors expressing np, m1 and chimeric hemagglutinin induces broad protection against influenza virus challenge in mice a serological assay to detect sars-cov-2 seroconversion in humans this study was supported by engineering sarah gilbert and teresa lambe are named on a patent application covering chadox1 ncov-19. the remaining authors declare no competing interests. the funders played no role in the conceptualisation, design, data collection, analysis, decision to publish, or preparation of the manuscript. correspondence and material requests to professor simon p. graham (simon.graham@pirbright.ac.uk) and professor teresa lambe (teresa.lambe@ndm.ox.ac.uk). key: cord-288101-pij16jaa authors: li, jun-yu; yong, yan-hong; gong, dong-liang; shi, lin; wang, xiao-min; gooneratne, ravi; yadnyavalkya, patil; ju, xiang-hong title: proteomic analysis of the response of porcine adrenal gland to heat stress date: 2019-02-28 journal: research in veterinary science doi: 10.1016/j.rvsc.2018.11.004 sha: doc_id: 288101 cord_uid: pij16jaa abstract heat stress (hs) and its associated pathologies are major challenges facing the pig industry in southern china, and are responsible for large economic losses. however, the molecular mechanisms governing the abnormal secretion of hs-responsive hormones, such as glucocorticoids, are not fully understood. the goal of this study was to investigate differentially expressed proteins (deps) in the adrenal glands of pigs, and to elucidate changes in the immune neuroendocrine system in pigs following hs. through a functional proteomics approach, we identified 1202 peptides, corresponding to 415 proteins. of these, we found 226 deps between heat-stressed and control porcine adrenal gland tissue; 99 of these were up-regulated and 127 were down-regulated in response to hs. these deps included proteins involved in substrate transport, cytoskeletal changes, and stress responses. ingenuity pathway analysis was used to identify the subcellular characterization, functional pathway involvement, regulatory networks, and upstream regulators of the identified proteins. functional network and pathway analyses may provide insights into the complexity and dynamics of hs-host interactions, and may accelerate our understanding of the mechanisms of hs. rising global temperatures have been accompanied by increased interest in researching the detrimental effects of heat stress (hs) on the swine industry. pigs enter a state of hs when the ambient temperature exceeds their thermal neutral zone (16-22°c for adult pigs) (coffey et al., 1995) . due to their high production of metabolic heat, accelerated fat deposition, and lack of sweat glands, pigs there are more sensitive to hs than many other mammals (d'allaire et al., 1996) . heat stress in pigs not only decreases food intake and body weight gain, but also has immunosuppressive effects, all of which may result in large economic losses for the swine industry (cruzen et al., 2015; pearce et al., 2013) . for instance, hs is estimated to cost the us swine industry losses of over $300 million each year (st-pierre et al., 2003) . understanding the stress-associated mechanisms involved in immune system function and the increased susceptibility of livestock to heat-related illness is more important now than ever, as the majority of emerging animal diseases are zoonotic and can potentially threaten public health. when exposed to a high-temperature environment, the central nervous system of mammals, including livestock, engages in physiological responses that result in the activation of the hypothalamic-pituitary-adrenal (hpa) axis and the sympatho-adrenal axis. the predominant hormone regulating the synthesis and secretion of adrenal glucocorticoids is adrencocorticotropic hormone (acth) (minton, 1994) . in pigs, cattle, and sheep, both corticotropin-releasing hormone and vasopressin regulate the secretion of acth, suggesting that these two proteins interact to enhance acth secretion. acth acts on the adrenal gland with the circulation, it to induces the expression and secretion of glucocorticoids, which suppress the production of cytokines and other pro-inflammatory mediators, including tnf-α, interferon-γ, il-1β, il-11, il-12, il-8, and prostaglandins (wilckens and rijk, 1997) . glucocorticoids also facilitate the release of anti-inflammatory mediators, such as transforming growth factor-α, il-10, and il-4, and have apoptotic effects and strong anti-proliferative properties in immune cells (visser and nagelkerken, 2002) . ultimately, cytokines activate the release of glucocorticoids, which in turn suppress cytokine synthesis fin a negative feedback loop (barrat et al., 2002; haddad et al., 2002) . thus, it is of interest to understand how higher core temperatures alter adrenal function. isobaric tag for relative and absolute quantification (itraq) is a powerful quantitative proteomics technique. in recent years, several proteomic studies have explored protein expression in a number of porcine cells and tissues, including pulmonary alveolar macrophages (lu et al., 2013) , mesenchymal stem cells (huang et al., 2015) , liver (liu et al., 2016) , heart (cabrera et al., 2012) , and intestine (colladoromero et al., 2015) . nevertheless, no large-scale proteomic analysis to date has examined the molecular complexes or pathways involved in the pathogenesis of the hpa axis. the purpose of the present study was to investigate protein expression in the adrenal gland of pigs in response to hs, and to elucidate potential changes in that occur in the endocrine system under hs. pigs were maintained and studied in accordance with the national institutes of health (nih) guidelines for the care and use of laboratory animals, and all protocols were approved by the guangdong ocean university animal care and use committee, china. six castrated bama miniature pigs (sus scrofadomestica) 3 months, weighing 30-40 kg were obtained from the bama miniature pigs breeding farm in the guangxi zhuang autonomous region of china. the six pigs were randomly divided into a heat stress group (3 barrows, ha) and a control group (3 barrows, ca). control pigs were housed with an ambient temperature of 28 ± 3°c, and the relative humidity was kept at approximately 90%. pigs assigned to the heat stress treatment were kept at 35 ± 1°c(maintained using an artificial climate chamber) in a manmade climate room, with a relative humidity of approximately 90%. all pigs were given access to water ad libitum. diet (see diet composition table) was formulated according to the recommended nutrient allowances for this breed of pig and the feeding was done twice a day, in the morning as well as in the evening. pigs were euthanized by a head-only electric stun tong apparatus on the 7th day under heat stress, followed by manual exsanguination. immediately after slaughter, adrenal tissue was removed and weighed. subsequently, tissues were washed with pbs to remove any blood and contaminants on the tissue surface. adrenal tissue was placed into sterile tubes and snap frozen in liquid nitrogen three pigs were used in the control group and three pigs were used in the heat-stressed group. the adrenal tissues of the 3 pigs in each of the groups were pooled together to form one pooled sample and utilized as one sample for further analyses. once in the laboratory, frozen specimens were stored at −80°c until biochemical and molecular analyses were performed. frozen samples of adrenal tissue from all pigs in the two groups were crushed in a mortar containing liquid nitrogen. the powder (approximately 100 mg per sample) was transferred to a sterile tube containing 1 ml lysis buffer (lb; containing 7 m urea, 2 m thiourea, 4% chaps, 40 mm tris-hcl, ph 8.5, 10 mm dithiothreitol, dtt). tissue homogenate was further disrupted using an ultrasonic cell disruptor (vcx130, usa) at 20% power output for 10 min, cycling between 2 s on and 4 s off. afterwards, the lysate was centrifuged at 25,000 ×g for 30 min at 4°c, and the supernatant was collected for protein quantification. protein concentration was measured using the pierce bca protein assay kit (thermo scientific, usa). protein digestion was performed as per the fasp procedure described by wisniewski, zougman et al. (wiśniewski et al., 2009) ; and the resulting peptide mixture was labeled using the itraq reagent-4plex multiplex kit (ab sciex, framingham, usa), according to the manufacturer's instructions. after 2 h of incubation at room temperature, labeled samples were mixed at equal ratios. subsequently, labeled peptides were combined and fractionated by strong cation exchange (scx) chromatography (han et al., 2015) and desalted on c18 cartridges (66872-u; sigma, st. louis, mo, usa). the dried peptide mixture was reconstituted and acidified with 2 ml buffer a (10 mm kh 2 po 4 in 25% of acn, ph 3.0) and loaded onto a column (4.6 × 250 mm). peptides were eluted at a flow rate of 1 ml/ min with a gradient of 0%-5% buffer b (2 m kcl, 10 mm kh 2 po 4 in 25% of acn, ph 2.7) for 5 min, 5-10% buffer b for 10-15 min, 10%-30% buffer b for 25-35 min, and 30%-50% buffer b for 35-50 min. the elution was monitored by absorbance at 214 nm, and fractions were collected every 1 min. the tryptic peptides were extracted, and the peptide mixtures were concentrated by speed vac centrifuge to dryness, and were again dissolved with 2% acetonitrile (acn) in 0.1% formic acid before lc-ms/ms analysis. the fractions from 2.3 were subjected to liquid chromatography-tandem mass spectrometry (lc-ms/ms) analysis. initially, samples were loaded onto pre-columns (180 μm × 20 mm; 5 μm-c18; waters, usa). peptide mixtures were separated on analytical columns (100 μm × 100 mm; 1.7 μm-c18; waters, usa) at a flow rate of 300 nl/ min over 60 min. thermo easy-nlc is a binary buffer system used for high performance liquid chromatography (hplc), consisting of 0.1% formic acid (buffer a) and 80% acetonitrile (acn) in 0.1% formic acid (buffer b). the related liquid phase gradient was as follows: 0-40 min with 5% to 35% buffer b; 40-45 min with 35%-80% buffer b; and 45-50 min with 80% buffer b. peptides eluted by hplc were directly injected into a q-exactive mass spectrometer (thermo fisher scientific). data were acquired in the positive ion mode with a selected mass range of 300-1800 mass/charge (m/z). q-exactive survey scans were obtained at 70,000 (m/z 200) and 17,500 (m/z 200), with the resolution for higher-energy collisional dissociation (hcd) spectra and maximum ion injection times fixed at 20 ms and 60 ms, respectively. dynamic exclusion (40.0 s duration) was used. ms/ms data were collected using the top 10 most abundant precursor ions. the normalized collision energy was 30 ev, and the underfill ratio defined as 0.1%. the instrument was operated with peptide recognition mode enabled. protein identification was performed using the mascot 2.3.02 search engine (matrix science, london, uk). according to the relative abundance of different itraq tags, the peptides derived from different groups were quantified by the scaffold software, and represent the ratio of one group to another. the relative quantification of the protein is calculated by using the relative quantification of the peptide, which is expressed as the average ratio. to determine the deps in the adrenal gland between the hs and the control groups, the average ratio of identified proteins was calculated by proteinpilot based on the weighted average log ratios of the peptides. the deps were further analyzed for significant down-or upregulation, which was not determined by the size of the ratio but was calculated by software perseus. a cutoff level of significance of 5% (or p < 0.05) was chosen as a criterion. gi numbers of all significantly regulated proteins and some unaltered proteins were imported into the ingenuity pathway analysis software (ipa, www.ingenuity.com) for bioinformatics analysis based on published reports and databases such as gene ontology, uniport and trembl. the canonical pathways and proteins interaction network of the deps were analyzed using the ipa. statistical analysis was performed using spss statistics 22.0. differences analysis in physiology indices between the ha group and the ca group were performed using a t-test, and p < 0.05 was taken to indicate statistical signifcance. we measured the body temperature, heart rate and respiratory rate of pigs at days 1, 3 and 6. it was found that body temperature and heart rate were increased significantly in heat-stressed pigs at days 1 and 6. (p < 0.05), however, the difference between body temperature and heart rate was not significant at 3 days. compared to controls, the respiratory rate, too increased substantially in pigs under heat stress at days 1, 3 and 6 (p < 0.01) (see table 1 ). the sample size used for this was 3 pigs each in control and hs groups respectively. we used the itraq method to identify proteins differentially expressed in the adrenal gland between the heat stress and control groups. we identified 226 differentially expressed proteins (deps), of which 99 were up-regulated and 127 were down-regulated in the ha group vs. the ca group. table 2 provides information about 18 key deps, 9 upregulated and 9 downregulated (see table 2 ). all the deps have been provided in the supplementary table 1. all protein and peptide identifications were obtained by database searching and stringent data filtering. the lc-ms/ms analysis produced 21,879 spectra, corresponding to 1202 unique peptides; 415 the effect of heat stress on the body temperature, heart rate and respiratory rate at days 1, 3, and 6. we found that heat stress increased the body temperature (bt), heart rate (hr) and respiratory rate (rr) of pigs significantly. the sample size used for this was 3 pigs each in control and hs groups respectively. the asterisk '*' and double-asterisk '**' denote a significant difference between stressed pigs and control pigs on the same day (p < 0.05 and 0.01, respectively). the units of the above indices are centigrade (°c), beats per minute and breaths per minute, respectively. proteins were identified at a false discovery rate (fdr) of ≤0.01 (fig. 1a) . the molecular weights of the identified deps ranged from 0 to 20 kd (n = 39), 20-40kd (n = 57), 40-60kd (n = 48), 60-80kd (n = 38), 80-100kd (n = 14), or > 100 kd (n = 30) (fig. 1b) . in addition, the identified deps had high peptide coverage; 85% of deps had > 10% sequence coverage, and 52% of deps had > 20% sequence coverage (fig. 1c) . 79.65% of the identified deps were represented by three or more peptides (fig. 1d) . to gain functional insights into the cellular proteome, gene ontology annotation was used to determine the subcellular localization of the 226 identified deps. the 226 deps identified from adrenal gland tissue altered by heat stress localized to various subcellular regions: high density lipoprotein particles (1.9%), extracellular area (15.0%), endoplasmic reticulum (7.9%), protein-lipid complexes (2.3%), cytoplasmic lipoprotein particles (2.3%), triglyceride-rich lipoprotein particles (1.9%), serosa (2.8%), platelet alpha particles (2.8%), endoplasmic reticulum inherent (1.9%), extracellular matrix (4.2%), chromatin (2.3%), pigment granules (5.6%), extracellular space (2.3%), serosa integrity (2.3%), neuronal processes (10.7%), secretory granules (5.1%), cytoplasmic vesicles (15.4%), and cytoskeleton (13.3%). (fig. 2) . gene identifications of the identified deps (supplementary table 1 ) were converted to human geninfo identifier (gi) numbers, since the pig genome database has poor annotations compared to the human genome, and because many proteins were unassigned or uncharacterized. to better understand these 226 deps, we used ingenuity pathways analysis (ipa) tool to examine canonical pathways; the top 20 enriched pathways are shown (fig. 3) , including pathways related to inflammation and immunity, such as 'acute phase response signaling' and 'il-12 signaling and production in macrophages'. the deps identified in adrenal gland tissue during heat stress by itraq were clustered according to different functions. four functional groups were found: diseases and disorders, molecular and cellular functions, physiological system development, and toxicity functions were significantly enriched (p ≤ 0.05; fig. 4 ). the 226 deps from adrenal gland under heat stress, which correspond to 24 diseases and disorders (fig. 4a) , included proteins that are related to neurological disease, psychological disease, metabolic disease, skeletal and muscular disorders, hereditary disorders, hematological disease, immunological disease, inflammatory disease, inflammatory response, respiratory disease, dermatological disease and conditions, connective tissue disorders, infectious disease, cardiovascular disease, cancer, and endocrine system disorders. these 226 deps were assigned to 25 molecular and cellular function groups (fig. 4b) , including cell death and survival, molecular transport, cellular growth and proliferation, cellular assembly and organization, cellular function and maintenance, cellular movement, lipid metabolism, small molecule biochemistry, free radical scavenging, protein degradation, protein synthesis, and cell morphology. these deps were also enriched in 18 physiological system developmental functions groups (fig. 4c) , including tissue development, nervous system development and function, organ morphology, organismal development, embryonic development, hematological system development and function, immune cell trafficking, and tissue morphology. finally, these deps were enriched in 9 toxicity function groups (fig. 4d) , including renal necrosis/cell death, liver hyperplasia/ hyperproliferation, kidney failure, cardiac inflammation, and heart failure. proteins that changed significantly in the adrenal gland under heat stress were mapped to 13 functional networks (fig. 5) . the main networks of interest correspond to (1) cell assembly and tissue, cellular function and maintenance (fig. 5a); (2) cell migration, intercellular signal interactions (fig. 5b) small molecule biochemistry (fig. 5d ). proteins that are present in these pathways and identified as up-regulated deps in our analysis are depicted in shades of red; those that were identified as down-regulated deps are shown in green. proteins in the network but not identified as deps in our study are depicted in white. we also predicted the upstream regulators of adrenal deps by ipa analysis and found that cytokines, kinases, chemical agents, chemical kinase activators, mature micrornas, and growth factor were activators of these deps, as an example, chemokine(c-c motif)ligand 5, curcumin and brain derived neurotrophic factor while cytokines, mature microrna, auxins, transcription regulators, and chemicals were inhibitors of these deps, such as, il-6, ccaat enhancer-binding protein β and dexamethasone etc. these predicted upstream regulators of deps responsive to heat stress may have an important role in regulating hormone secretion and signal transduction in pigs. . the previous research of our laboratory found that: the analysis of plasma cortisol levels in bama miniature pigs revealed that the levels increased with the duration of heat stress. although there was no significant difference in the cortisol levels of control and heat-stressed pigs on the first day. however, at subsequent time points, cortisol levels were significantly higher in heat-stressed pigs compared with those in the control animals on the 7th day (ju et al., 2014) . in the present study, we used a method combining proteomics and bioinformatics to identify proteins that are differentially expressed in the adrenal gland of pigs under heat stress, and to elucidate molecular pathways and cellular functions that might mediate the heat stress response through these deps. we identified a total of 226 deps in the pig adrenal gland under heat stress conditions. ipa analysis software was used to analyze the cell localization, molecular function, signal pathway, regulatory network, and upstream regulators of these deps, which laid the foundation to elucidate mechanism of heat stress and stress-induced immunosuppression. the function of tubulin is mainly to interact with microtubule-associated proteins and related proteins that activate microtubule structures and maintain microtubule polymerization and depolymerization, modulate cell morphology, and are involved in cell division, cell movement, and transport of intracellular substances. cytoskeletal changes are associated with trans-cellular membrane trafficking. together, β-tubulin and α-tubulin (table 2 ) participate in the formation of microtubules, the integrity of which is essential for the segregation of chromosomes during cell division, the maintenance of cell shape, and the intracellular trafficking of macromolecules and organelles. changes in β-tubulin and vimentin levels have been detected in sars-cov (jiang et al., 2005) and infectious bursa disease virus (ibdv) (zheng et al., 2008a) . β-tubulin was one of the deps identified in this study, and its expression was approximately 2.2-fold up-regulated in adrenal tissue under heat stress, suggesting that differentially expressed cytoskeletal proteins could promote stress response in the adrenal gland. further large-scale studies are required to understand the roles and interrelation of β-tubulin and vimentin in porcine heat stress response. the heat shock protein (hsp) response is a highly conserved cellular response to external stress in all species. hsps take part in antigen presentation, intracellular trafficking, and apoptosis, and acting as molecular chaperones by assisting nascent polypeptides in assuming their proper conformations (khar et al., 2001) . several hsps were identified as down-regulated deps in this study, including hsp27 and hsp60. several members of the hsp family are expressed on the surfaces of cells; these can stimulate immune effector cells directly or can play crucial roles in antigen cross-priming. in this situation, hsps act as shuttle molecules for exogenous antigens and can directly stimulate t cells by prompting apc cytokine secretion (kotsiopriftis et al., 2005) . hsps appear to play a part of the innate immune response since the emergence of phagocytes in early multicellular organisms, and were commandeered for adaptive immune responses with the appearance of immune specificity (srivastava, 2002) . hsp70 is an endogenous ligand for the toll-like receptors (tlrs) that bind to microorganism-and tumor-specific antigens, then combine with major histocompatibility complexes (mhc) i and ii, which activate tumor-specific pathogens and t cells (han et al., 2009 ). mortaz found that high temperature induced mouse bone marrow mast cells to release hsp70, and the secretion of hsp70 in turn activated the toll-like receptor 4(tlr4) pathway (mortaz et al., 2006) . the expression of hsp70 is enhanced under stress conditions, and is generally considered to act through its role as a molecular chaperone, but recent reports indicate that hsp70 also modulates inflammatory responses by inhibiting activation of the inflammatory transcription factor, nuclear factor-kappa b (zheng et al., 2008b) . in addition, hsp70 may directly interfere with cell death pathways, such as those involved in apoptosis and necrosis (yenari et al., 2005) . in this study, hsp70 was up regulated(1.52 fold reduction) in porcine adrenal gland tissue under heat stress, further indicating the role of hsp70 in adrenal gland injury and highlighting its relevance to inflammatory responses. hsp60 has direct modulatory effects on inflammatory cells, and can activate monocytes and granulocytes to produce inflammatory cytokines, tumor necrosis factor-α (tnf-α), il-12 and il-6 (wells and malkovsky, 2000) . hsp60 can also specifically bind tlrs, especially tlr4, which is involved in human and rat atherosclerotic lesions (grundtman et al., 2011) . hsp27 belongs to a family of small heat shock proteins, can affect protein assembly, and may also participate in protein degradation. this conclusion follows directly from data suggesting that heat stress reduces the expression of heat shock proteins, handicapping their ability to induce a protective immune response when immunocytes are confronted with foreign entities. antigen presentation by hsps activates the innate and adaptive immune systems to initiate an acute response to stress factors, and suppression of hsps obstructs this response. these properties allow hsps to be used in immunotherapy in novel ways, which could lead to a greater understanding of how heat stress modulate the immune response, and why heat stress induces immunosuppression in pigs afflicted by post weaning multi-system wasting syndrome (pmws). histone h2a was significantly down-regulated in porcine adrenal gland under heat stress. ipa analysis shows that histone h2a plays a fig. 5 . ingenuity pathway analysis of proteins significantly altered in heat stressed pigs. red, up-regulated proteins; green, down-regulated proteins significantly; white, proteins known to be in the network but were not identified in our study. the colour depth shows the magnitude of the change in protein expression level. the shapes are suggestive of the molecular class(i.e., protein family). lines connecting the molecules indicate the relationship between molecules. dashed lines demonstrate indirect interactions, and solid lines demonstrate direct interactions. the arrow styles demonstrate specific molecular relationships and the directionality of the interaction. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) role in cancer, infectious diseases, reproductive system diseases, liver diseases, and immune diseases. however, in a bovine proteome study, histone h2a was considered a new natural immune molecule, and was down-regulated in neutrophils of immunosuppressed dairy cows. the immunological function of neutrophils releases a series of dna, histones, and antimicrobial peptides, forming a microbial kill 'trap' (lippolis and reinhardt, 2008; john and lippolis, 2005; kimura et al., 2006) . histone h2a belongs to a group of conserved eukaryotic cationic proteins that are involved in antibacterial activity, and function in dna folding. further studies are needed to determine if down-regulation of h2a expression in the adrenal gland under heat stress conditions is related to immunosuppression. annexin (anxa) is a calcium-dependent phospholipid-binding protein widely found in eukaryotes. phosphatidylserine (ps) is transferred from the inside of cell membrane to the outside after cell apoptosis. in the presence of calcium ions, anxa5 has a high affinity for ps, and can specifically bind to ps exposed on cell membrane. thus, anxa5 specifically recognizes apoptotic cells, and acts as a novel molecular probe to detect apoptosis. anxa5 is one of the most widely distributed and abundant members of the anxa family, and is involved in the anti-coagulation activity, calcium channel activity, protein kinase inhibitory activity, and many other important physiological processes. anxa5 is also closely related to inflammatory responses and cell stress (gauer et al., 2013; lokman et al., 2011) . anxa1, 2, and 11, were down-regulated deps identified in our study. these proteins participate in cell function and maintenance, intercellular communication and interaction, cell survival and death, cell migration, neurological diseases, and other processes. whether these down-regulated proteins are involved in the regulation of heat stress-induced apoptosis is still unclear (gu et al., 2014) , and must be explored in further studies. cui performed similar studies on heat stressed pig livers and found that chronic heat stress caused a reduction in the weight of the liver. they also found 45 proteins in the liver that were differentially abundant between heatstressed pigs and control pigs. proteins found were responsible for heat shock and oxidative stress response, cellular apoptosis, metabolism, signal transduction, cytoskeleton and immune system responses. their research found that heat caused an innate immune response whereas the voluntary diet restriction resulted in stress and cellular apoptosis (cui et al., 2016) . in summary, in this study we identified deps in the porcine adrenal gland under heat stress were characterized, and we constructed a comprehensive network of protein regulation in the porcine adrenal gland in response to heat stress. although many significantly differentially regulated proteins and pathways are closely related to the symptoms or pathological responses of heat stress, further functional investigations ought to be carried out to facilitate our understanding of the pathogenic mechanisms and molecular responses of pigs to heat stress. further work may identify novel therapeutic targets or lead to the development of new methods of preventing heat stress. in conclusion, we identified 226 deps, of which 99 were up-regulated and 127 were down-regulated, from pig adrenal gland tissue under heat stress. among these 226 deps, tubulin and heat shock protein (hsp) such as hsp70, hsp60, hsp27, and histone h2a might be involved in the regulation of heat stress. ingenuity pathways analysis (ipa) tool examined canonical pathways related to inflammation and immunity, such as 'acute phase response signaling' and 'il-12 signaling and production in macrophages'. our expression profiles provide new insights into the key proteins involved in heat stress in the pig. supplementary data to this article can be found online at https:// doi.org/10.1016/j.rvsc.2018.11.004. in vitro generation of interleukin 10-producing regulatory cd4+ t cells is induced by immunosuppressive drugs and inhibited by t helper type 1 (th1)-and th2-inducing cytokines altered expression of mitochondrial electron transport chain proteins and improved myocardial energetic state during late ischemic preconditioning feeding growing-finishing pigs to maximize lean growth rate quantitative proteomics and bioinformatic analysis provide new insight into the dynamic response of porcine intestine to salmonella typhimurium effects of long term heat stress in utero or during finishing on pork carcass composition chronic heat stress induces immune response, oxidative stress response, and apoptosis of finishing pig liver: a proteomic approach sow mortality associated with high ambient temperatures membrane modulates affinity for calcium ion to create an apparent cooperative binding response by annexin a5 heat shock protein 60 and immune inflammatory responses in atherosclerosis heat stress induces apoptosis through transcription-independent p53-mediated mitochondrial pathways in human umbilical vein endothelial cell cytokines and neuro-immune-endocrine interactions: a role for the hypothalamic-pituitary-adrenal revolving axis soluble b and t lymphocyte attenuator possesses antitumor effects and facilitates heat shock protein 70 vaccine-triggered antitumor immunity against a murine tc-1 cervical cancer model in vivo itraqbased quantitative analysis of hippocampal postsynaptic density-associated proteins in a rat chronic mild stress model of depression proteomic analysis of porcine mesenchymal stem cells derived from bone marrow and umbilical cord: implication of the proteins involved in the higher migration capability of bone marrow mesenchymal stem cells quantitative analysis of severe acute respiratory syndrome (sars)-associated coronavirus-infected cells using proteomic approaches implications for cellular responses to virus infection proteomic survey of bovine neutrophils heat stress upregulation of toll-like receptors 2/4 and acute inflammatory cytokines in peripheral blood mononuclear cell (pbmc) of bama miniature pigs: an in vivo and in vitro study induction of stress response renders human tumor cell lines resistant to curcumin-mediated apoptosis: role of reactive oxygen intermediates effect of recombinant bovine granulocyte colony-stimulating factor covalently bound to polyethylene glycol injection on neutrophil number and function in periparturient dairy cows heat shock protein 90 expression in epstein-barr virus-infected b cells promotes gammadelta t-cell proliferation in vitro centennial paper: proteomics in animal science itraq-based proteomic analysis reveals alterations in the liver induced by restricted meal frequency in a pig model the role of annexin a2 in tumorigenesis and cancer progression twodimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (itraq) labeling approac function of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system in models of acute stress in domestic farm animals acetylsalicylic acid-induced release of hsp70 from mast cells results in cell activation through tlr pathway the effects of heat stress and plane of nutrition on metabolism in growing pigs interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses economic losses from heat stress by us livestock industries 1 expression of the il-10 receptor on human monocytes is moderately suppressed by glucocorticoids heat shock proteins, tumor immunogenicity and antigen presentation: an integrated view glucocorticoids and immune function: unknown dimensions and new frontiers universal sample preparation method for proteome analysis antiapoptotic and anti-inflammatory mechanisms of heat-shock protein protection proteomics analysis of host cells infected with infectious bursal disease virus anti-inflammatory effects of the 70 kda heat shock protein in experimental stroke not applicable. the authors of this study have no conflicts of interest. jl conceived the study designed the experiments, prepared the samples for mass spectrometry, analyzed the mass spectrometry data, conducted the experimental work created the figures and wrote the manuscript. yy participated in the enrichment analysis, created the pathway figure, aided in revising the manuscript. dg, ls and pt analyzed the mass spectrometry data, participated in the enrichment analysis, participated in writing and revision of the manuscript. rg obtained and analyzed flow cytometry data and aided revising the manuscript. xwang was responsible for animal care xj provided the financial support, and designed the experiments and revised the manuscript. all authors have confirmed the final version of the manuscript. key: cord-003447-kbpvt5on authors: atherstone, c.; galiwango, r. g.; grace, d.; alonso, s.; dhand, n. k.; ward, m. p.; mor, s. m. title: analysis of pig trading networks and practices in uganda date: 2018-08-02 journal: trop anim health prod doi: 10.1007/s11250-018-1668-6 sha: doc_id: 3447 cord_uid: kbpvt5on east africa is undergoing rapid expansion of pig rearing, driven by increasing pork consumption. introduction and expansion of pig production systems in this biodiverse landscape may create new risks, including zoonotic pathogen transmission. historically, biosecurity measures have primarily been focused at farm level, ignoring the important function pig traders fulfill between farmers and consumers. this study interviewed pig traders operating at uganda’s only registered pork abattoir to describe their characteristics, business practices, biosecurity practices, and pig health management and reporting practices. all the traders were male, and nearly all (90.5%) relied on pig trading as their primary source of income. most of the pigs brought for processing at the slaughterhouse were purchased from smallholder farms (87.3%). in addition, there was a significant difference in the high price paid per kilogram at farm gate by region (p = 0.005). high prices paid at farm gate were associated with holiday periods (p < 0.001), harvest season (p < 0.001), and drought (p < 0.001). traders preferred buying live pigs from male farmers (88.9%) because they were considered the final decision makers and owned the pigs being sold. all pig traders were aware of clinical signs indicating a pig was sick. this study has provided baseline information on pig trader practices in uganda. improvements in local pork slaughterhouses and markets will benefit not only pig traders in accessing consistent customers but also individual pig farmers by increasing their market access. finally, given their role as a link between farmers and consumers, traders would benefit from targeted inclusion in disease control and prevention strategies. electronic supplementary material: the online version of this article (10.1007/s11250-018-1668-6) contains supplementary material, which is available to authorized users. the domestic pig population in uganda, currently estimated at 3.2 million, plays an essential economic and social role in a country, in which 70% of households derive some or all of their livelihoods directly from livestock (uganda bureau of statistics 2008). pig keeping has grown in popularity as a livelihood activity due to their high reproduction rates, rapid weight gain, potential to provide quick financial returns, and rising demand for pork. in the past 50 years, pork consumption has increased more than 20-fold, from an estimated annual per capita consumption of 0.14 kg in 1962 to 3.37 kg in 2013. pork currently accounts for more than a third of the annual per capita meat consumption (food and agriculture organization (fao) 2011) . further, total pork consumption is projected to increase by 184% between 2000 and 2030 in uganda due to human population growth (food and agriculture organization 2011) . the increase in pork consumption, while exceptionally rapid in uganda, is not unique in the region. the democratic republic of congo's total consumption of pork is expected to increase by 100%, tanzania by 32%, and kenya by 25% between 2000 and 2030 (food and agriculture organization 2011) . introduction and expansion of pig production systems in these biodiverse landscapes may create new risks, including pathogen transfer from pigs to humans (ocaido et al. 2013; wilson 2017; food and agriculture organization (fao) 2012; atherstone et al. 2015; hamill et al. 2013; food and electronic supplementary material the online version of this article (https://doi.org/10.1007/s11250-018-1668-6) contains supplementary material, which is available to authorized users. agriculture organization 2017). of particular public health interest is the role of pigs in the zoonotic transmission of emerging pathogens to people (atherstone et al. 2015; vergara-alert et al. 2017; middleton and westbury 2002; ma et al. 2008; kobinger et al. 2011; conlan et al. 2012; mccormack and allworth 2002; abubakar et al. 2004; marsh et al. 2011) . as pig traders form an important link between pig farms and pork customers, research informing their knowledge, attitudes, and practices is essential. african swine fever (asf) is considered the major infectious disease constraint to pig production in africa (penrith et al. 2013) , and as such, research to date has focused on this infection. penrith and vosloo reported that outbreaks of asf in new areas of africa have almost all been associated with movement of domestic pigs and pig products (penrith and vosloo 2009 ). in uganda, several studies to characterize practices associated with the occurrence and spread of asf identified the collection of pigs and pig products from farms (kabuuka et al. 2014) , distribution of infected pork by traders , pig movements due to restocking and trade (kalenzi atuhaire et al. 2013; nantima et al. 2015) , free range movement of pigs on farms (nantima et al. 2015) and trade of live pigs and pig products (tejler and teijler 2012) as risk factors. these studies focused on pig farmers and their perception of trading and pig movements. a limited number of studies have targeted pig traders, but again, these were restricted to knowledge and practices related to asf transmission and control (dione et al. 2016; chenais et al. 2015; muhangi et al. 2014) . despite the link between disease spread and pig movement, little is known about broader trading practices, motivations for buying, and patterns of purchases and sales of pigs in uganda. given pig traders' important role in supplying pork for a rapidly expanding consumer base and linking farmers with consistent markets, a better understanding of their practices and motivations around purchasing, transportation, and pig health management is needed. this would assist in developing policies that specifically support traders and the important functions they serve while identifying suitable interventions to ensure a safe, reliable pork supply. therefore, the objectives of this study were to (1) describe pig trader characteristics, trading practices, biosecurity practices, pig health management, and reporting practices and (2) map source locations of pigs purchased to supply pork through the major abattoir in uganda. wambizzi cooperative society limited is located in nalukolongo, southwestern kampala, uganda's capital city. wambizzi was selected as it is the only registered pig abattoir in uganda and has many pig traders supplying live pigs to meet the urban demand for pork. as a registered slaughterhouse, carcasses processed at wambizzi are visually inspected by kampala city council authority (kcaa) meat inspectors and stamped bfit for human consumption.^pork processed at the abattoir is sold in the greater kampala area to pubs, pork joints, hotels, butchers, supermarkets, and private organizations (non-governmental organizations, missions, and private individuals). the slaughterhouse has a capacity of 200 pigs/ day, but the supply of pigs fluctuates substantially throughout the year (roesel et al. 2016) . according to slaughterhouse records, an average of 60,078 kg of pork was processed monthly from april 2014 to may 2015. using the estimated annual per capita consumption of 3.37 kg of pork (food and agriculture organization (fao) 2011) and the 2014 kampala population of 1.5 million (uganda bureau of statistics 2016), wambizzi produced roughly 14.3% of the pork consumed in kampala during this time frame. pig slaughtering also occurs in backyards and slaughter slabs to supply informal, roadside pork joints and butcheries. pig traders privately operating from wambizzi use the abattoir facility to slaughter their pigs and have their pork inspected and stamped to meet requirements in the formal marketplace. traders buy live pigs from farms/markets and aggregate them into groups for transport to the abattoir. during this interim period (farm gate to abattoir), pigs are under the ownership and care of traders. once processed, traders sell the pork to their own customers in the sales building adjacent to the evisceration and inspection building. while membership with the abattoir is not required, traders pay a fee per pig (6000 ugx, i.e., 1.67 usd in 2017) to use the abattoir facilities (roesel et al. 2016 ). pig traders were interviewed between october 2015 and october 2016, during periods corresponding with national holidays when the sale and consumption of pork increases (roesel et al. 2016; ouma et al. 2015) . there is no formal register of pig traders operating at wambizzi. in preparation for this research, a member of the research team with prior experience working with pig traders informally questioned traders on site over several days to develop a more recent estimate of the number of traders operating from wambizzi. based on this, the total number of traders operating at wambizzi was estimated at 60. thus, we aimed to interview 60 traders over the course of the study. a variety of methods were used to recruit participants, including direct approach and active-snowballing. traders arriving at the abattoir were approached by a local member of the research team and initially asked about their interest in learning about the study. if the trader expressed interest, information on the scope and purpose of the research was provided orally. traders who indicated that they were willing to participate in the research study were asked to give written consent to be interviewed. if the trader was unable to give written consent due to physical impairment or illiteracy, their thumbprint was provided in place of signature. additional traders were identified by asking participants who had completed the interview for the name and contact information of other pig traders operating from wambizzi. furthermore, we observed trader brands on pigs at slaughter (e.g., number or letter carved on the animal at the time of purchase) and asked participants who had completed the interview if they could identify the trader who supplied the pig. the enumerator then contacted these newly identified pig traders to invite their participation in the research study. a local enumerator with previous experience working with pig traders in uganda was recruited and trained for data collection. a structured questionnaire was adapted from previous research conducted with pig traders by the international livestock research institute (ilri) in uganda under the smallholder pig value chains development project (cgiar livestock and fish research program 2015) . the questionnaire captured information on pig trader characteristics, live pig buying practices, transportation practices, and pig health management. traders also reported the sub-counties where they had purchased live pigs over the 12 months prior to the interview date. the questionnaire was developed in english and translated into the local language (luganda). the questionnaire comprised primarily closed-ended questions to keep the interview to a maximum of 45 min. openended questions regarding buying practices and clinical signs observed in pigs were included, with answers recorded exactly as the interviewee stated. the full questionnaire is included in the supplementary materials. the slaughter process started at 4 am each morning and preceded the selling of pork from 8 am to 10 am. the enumerator was on site by 6 am each morning to identify pig traders previously not interviewed. however, to ensure that the interview did not conflict with pig trader's business, most interviews took place between 10 am and noon each day. data from the questionnaires was entered into epi info 7.1 (centers for disease control, atlanta, ga, usa). following data cleaning, data was exported to spss 24.0 (ibm corp., armonk, ny, usa) for analysis. standard descriptive analysis was performed for categorical and quantitative variables describing pig trader characteristics, live pig purchasing practices, transportation practices, and pig health management. population pyramid style graphs were prepared to compare pork demand and pork farm gate prices by months. source locations (reported to the sub-county level) were entered into microsoft excel and checked for spelling accuracy. the subcounties were then joined to the centroid of each sub-county polygon in the 2014 global administrative unit layers for uganda (food and agriculture organization, rome, italy) using arcgis 10.2 (environmental systems research institute, redlands, ca, usa). the number of pig traders operating in each sub-county was mapped using graduated symbols. two binary outcome variables of interest were explored further, namely high price paid at farm gate over the last year (1/0) and low price paid at farm gate over the last year (1/0). reasons given for prices paid were recoded into binary variables (yes/no) and used as explanatory variables. univariable binomial logistic regression analyses were conducted to evaluate the associations of the binary explanatory variables with both the outcome variables. explanatory variables with a p value < 0.15 were included in two multivariate regression models for high or low price paid to evaluate associations after adjusting for other variables in the model. in addition, non-parametric analyses were conducted for four quantitative variables (high price paid per kilogram, low price paid per kilogram, number of live pigs bought during high demand weeks, and number of live pigs bought during low demand weeks) to identify significant differences by operating region (kruskal-wallis test), number of districts (mann-whitney u test), and number of regions (mann-whitney u test) traders purchased live pigs from. operating region was identified based on the location pig traders reported purchasing live pigs in. because price and number of pigs purchased were not identified by individual districts and many pig traders operated in multiple regions, operating region was binned into three categories: central only, eastern only, and all other regions (including western, northern, and responses that covered multiple regions). number of regions and number of districts a pig trader operated in were recoded into two responses: above median and below median. independent variables with significant differences (p < 0.05) were subject to post-hoc pairwise comparisons to identify which specific responses(s) were significantly different from each other. a total of 63 interviews were conducted with pig traders operating from wambizzi between october 2015 and october 2016. no traders declined participation in the study. pig trader characteristics are shown in table 1 . all pig traders interviewed were male and ranged in age from 28 to 60 years (median 38 years; first quartile (q1) = 34, third quartile (q3) = 47). the median number of years working as a pig trader was 12 years (range 3 months-36 years; q1 = 8, q3 = 18.75). a large proportion (41.3%; 26/63) of participants had not completed primary school. most pig traders were engaged in trading as their primary source of income (90.5%; 57/63) and described their business operation as fixed (96.8%; 61/ 63), meaning that they had established locations for buying live pigs and selling pork. proximity to pork customers was the primary reason for having a fixed business operation (79.4%; 50/63) with most traders supplying pigs solely to wambizzi (93.7%; 59/63). when asked about other pig traders operating in their areas, almost all the traders had competition for live pigs in their buying areas (98.4%; 62/63). almost two thirds of the pig traders were not members of a trading group or cooperative (63.5%; 40/63). buying and transportation practices are shown in table 2 . most traders sourced their pigs directly from smallholder farms (87.3%; 55/63). only one trader reported purchasing pigs at a livestock market. when pig traders were asked about whom they prefer to purchase pigs from at the farm, 88.9% (56/63) preferred buying from men rather than from women. reasons offered by traders who preferred to purchase from men included that men were the following: the decision makers on the farm, faster decision makers, and owned the pigs being sold. further analysis to understand this gender preference was not possible because the number of responses for women were all less than 5. lorries (trucks) were the most common type of vehicle used to transport pigs to the abattoir (60.3%; 38/63). the majority of traders rented the vehicles they used to transport pigs (90.5%; 57/63). vehicles were cleaned after each use (95.2%; 60/63) using both water and laundry washing powder (98.4%; 62/63). none of the pig traders reported using bleach or any other type of disinfectant to clean their vehicles. the pig waste (feces, urine, bedding) left in the vehicle after transporting the pigs was most commonly heaped at wambizzi for crop farmers to collect and use for compost in their gardens (54%; 34/63). june and december were frequently identified as months with high customer demand for pork, whereas february and september were associated with low customer demand (fig. 1) . during months when demand for pork was low and high, respectively, traders bought a median of 28.5 pigs (range 3-140 pigs/week; q1 = 20; q3 = 45) and 77.5 pigs (range 5-260 pigs/week; q1 = 50; q3 = 120) per week. figure 2 shows the months traders associated with paying high or low farm gate prices to purchase pigs. the median reported high prices at farm gate was 6000 ugx/kg (range 6000−8000 ugx/kg; 3590 ugx = 1 usd as of january 2017). when low prices were paid at farm gate, the median was 5000 ugx/kg (range 5000-7500 ugx/kg). reasons for paying high and low prices at farm gate are shown in table 3 . in multivariate logistic regression, holiday period, crop/coffee harvesting season, and drought were significantly associated with high price paid, whereas drought, school fees due time, and sick pigs were significantly associated with low price paid. figure 3 shows the source locations of pigs purchased on the day of interview and preceding 12 months. pig traders reported buying live pigs in one to eight districts (median 3) across one to three regions (median 1) in uganda. thirty-six percent of traders purchased live pigs only in the central region (n = 23) and 27% of traders purchased live pigs only in the eastern region (n = 17). farm gate prices by operating region as well as the number of districts/regions that a pig trader operates in are outlined in table 4 . pig traders operating in one region paid significantly higher prices per kilogram at farm gate than traders operating in two to three regions (p = 0.001). the number of live pigs purchased per week by operating region and number of districts/regions that a trader operates in is shown in table 5 . during months when demand for pork was low, the region(s) a pig trader operated in to purchase live pigs was significantly associated with the number of pigs purchased (p = 0.014). pig traders operating in only central region purchased a significantly higher number of pigs during low demand months than traders operating in only eastern region (p < 0.001). traders operating in only the eastern region purchased a significantly lower number of pigs during low demand months than traders operating in all other regions (p = 0.002). all the pig traders reported recognizing clinical signs indicating a pig was sick. when asked to list these signs, the most commonly stated signs were dropping of ears (46%; 29/63), reddening of ears (44.4%; 28/63), straightening of the tail (31.7%; 20/63), and weakness or difficulty standing (31.7%; 20/63). traders typically did not report pigs considered to be sick to anyone (92.1%; 58/63). if there was reporting, the trader informed a meat inspector on site at wambizzi (80%; 4/5) or a veterinary officer (20%; 1/5). if sick pigs were observed while under the traders' care, 77.8% of the traders did nothing to care for the sick pigs (49/63). if action was taken, the sick pig was slaughtered at wambizzi and the meat sold (14.3%; 9/63). this is the first study to describe ugandan pig trader characteristics and business practices around live pig buying, transportation, and health management. the prices paid to farmers for their pigs were associated with the number of regions and districts a pig trader operates in. in addition, pig traders reported paying higher prices during holiday periods and harvest season (crops/coffee). traders preferred buying live pigs from male farmers because they considered them the final decision makers and owned the pigs being sold. finally, we found that all pig traders checked for clinical signs in pigs that indicated the animal was sick. pig traders who operate in only one region paid, on average, higher prices per kilogram to farmers for their pigs. considering that such traders are likely to travel shorter distances to source their pigs, compared to those traders that work in more regions, this may suggest that the distance travel outcome variables with less than five responses were not including in the univariable analysis b p value = 0.089. explanatory variable removed from multivariable logistic regression model fig. 3 source locations of live pigs on the day of interview and preceding 12 months, as reported by 63 pig traders interviewed at wambizzi cooperative society slaughterhouse, kampala, uganda, 2015 to source pigs has an impact on the price paid for the pigs, with traders traveling less offering higher prices to farmers. however, another possibility may be that traders who operate in multiple regions are large-scale traders, buying pigs in bulk, and therefore paying lower prices. nevertheless, given the importance of pigs as an asset within smallholder farming households, it is advantageous when pig farmers can secure more income from the sale of their pigs. given that almost half the traders in this study operated in four or more districts in uganda, pigs are traveling large distances from farm to slaughterhouse. reducing the distance pigs' travel for processing is both an animal welfare and a disease mitigating practice, especially for limiting the dissemination of asf (tejler 2012) . thus, there is a need for locally regulated slaughter facilities and/or improved transport infrastructure throughout the country to reduce the distance traveled from farm to slaughterhouse. centralized slaughter facilities would also help address the lack of consistent market access, a commonly cited constraint among pig farmers (ouma et al. n.d.; wabacha et al. 2004; kagira et al. 2010; muhanguzi et al. 2012a ). moreover, traders in this study reported that the primary reason for maintaining a fixed business operation was proximity to pork customers. local slaughter facilities would provide a centralized location for traders to access customers. this study also found that holiday periods, harvest season, and drought were the most commonly cited reasons trader paid high prices at farm gate. other studies have noted that smallholder farmers keep pigs as a source of cash in times of need (dione et al. 2014; deka et al. 2007; gichohi et al. 1988) . when pig traders need to source live pigs around harvest season, they pay more for these pigs because farmers have recently sold their crops and, therefore, do not need the additional cash generated from the sale of a pig. the situation is a little different around the holidays. an increase in pig sales and pork consumption during festive seasons is well documented (roesel et al. 2016; dione et al. 2014; adams et al. 2012; kambashi et al. 2014) . it is possible that traders have a harder time sourcing the number of pigs they need at the holiday period or that farmers know of the increased holiday demand and raise their prices. the higher prices paid for pigs around holidays are inducements for farmers to time their pig rearing activities to take advantage of the economic benefit of having pigs ready for sale according to holiday periods. free ranging, tethering, and feeding of crop residues and grasses to pigs are common practices among smallholder pig farmers in east africa chenais et al. 2015; tejler 2012; kagira et al. 2010; dione et al. 2014; muhanguzi et al. 2012b; nantima et al. 2016) . in all these production systems, drought would reduce the amount and quality of feed available for pigs and, therefore, would reduce the number of pigs suitable for sale. it is also possible that farmers intentionally chose not to rear grower and fattener pigs during known seasons of drought. it will be important to address live pig supply issues, whether at holiday periods, drought, or from other causes, to ensure a consistent pork supply so that consumers are able to access the quality and quantity of pork they demand. at the farm level, this research found that pig traders have a strong preference to buy live pigs from male pig farmers. a study in kenya found that the decision to keep pigs was made by men (simiyu and foeken 2013) . because of the large financial investment, technical knowledge required to raise pigs and role as sole decision makers in their home, men maintained control of pigs and leveraged this control for any financial decisions made over the animals (simiyu and foeken 2013) . these subtle cultural values around livestock ownership and household financial decision making come into play in accessing markets for livestock and livestock products. women tend to face more challenges than men in accessing and benefiting from markets, especially more formal markets (kristjanson et al. 2010) . furthermore, given women's traditional responsibility for household food security, their level of control over decisions about whether to sell or consume the family's animal products, as well as over how to use any income obtained from the sale of animal foods, could greatly determine the nutritional well-being of household members (kristjanson et al. 2010) . while there is clearly a need for support of women at the household level in accessing markets for their livestock, this research shows that there is also a need to work with pig traders to enable female pig farmers to access consistent markets for their pigs. in this study, all the traders interviewed observed clinical signs they described as indicating a pig was sick. the frequently observed clinical signs such as reddening of the ears, dropping of ears, and weakness or affected movements are consistent with clinical signs of asf (chenais et al. 2015; tejler 2012; dione et al. 2014) . despite recognition of these signs as indicators of sickness, traders failed to report these suspected cases to the proper authorities. similar findings have been reported in previous research in uganda chenais et al. 2015; muhangi et al. 2014; nantima et al. 2016) and are consistent with studies conducted in indonesia (leslie et al. 2016) . given that traders play an essential role in transporting pigs, there is a need to develop policies and strategies to integrate pig traders into disease reporting and disease mitigating strategies without fear of recrimination or detriment to their business. in addition, given the pressures pig traders are under to meet quality standards of pork customers, pig traders would benefit from training on disease mitigating strategies including safe and hygienic slaughter practices, perhaps through an industry association or group. this would also address the gap between traders admitting that they are responsible for conducting their business in support of disease prevention but do not perceive themselves as key actors in the control of disease (dione et al. 2016 ). there are several limitations to this study. first, responses to the questionnaire are subject to recall bias. this is especially true of answers around the number and location of pigs purchased over the 12 months prior to the interview. however, the number of pigs for which traders provided a source location (n = 7185) was considerably less than the number of pigs processed at the slaughterhouse (n = 19,021 for july 2011-june 2012) (roesel et al. 2016 ). it appears that when the pig traders were unsure of actual numbers, they underreported, or only reported the location they sourced the pigs without any accompanying number of pigs purchased. self-reported locations are likely to be reliable as the traders used communitybased scouts to identify pigs for sale. we purposely interviewed pig traders during periods when demand for pork was historically high and theorized that this would mean that more pig traders would be bringing pigs in for processing. however, it is possible that there are pig traders that only operate sporadically and would have been missed in this study. we worked with the pig traders at wambizzi to identify other pig traders to interview. we also cataloged the brands on each pig being processed, as each trader has a unique symbol to identify their pigs once they have been processed. every effort was made to identify all potential research participants. previous research, undertaken with the management at wambizzi, stated that there were 20 pig traders regularly operating from the premises (roesel et al. 2016) . given this, our study team managed to identify three times the number of pig traders. further analysis beyond descriptive analyses was hindered by the low number of responses for certain variables. for example, we were unable to analyze why pig traders prefer buying live pigs from men rather than women. given the priority of gender empowerment in uganda and the significance of livestock in alleviating poverty for women and children, it is important to identify ways to support women in accessing markets for their pigs. more detailed interviews and focus groups may shed additional light on trader buying decisions. this study has provided baseline information on pig trader practices in uganda. the prices paid at farm gate for pigs are affected by the number of regions and districts a trader operates in to procure pigs. given the animal welfare and disease transmission implications of pigs traveling over multiple districts and regions from farm to slaughterhouse, consideration should be given to establishment of local pork slaughterhouses and markets and improvements to transport infrastructure. furthermore, pig traders prefer buying live pigs from male farmers. for women to overcome the challenges of accessing formal livestock markets, there is a need for additional research to identify how women can access pork markets in uganda, particularly if pig traders are involved. finally, this research shows that pig traders are observing sick pigs but fail to report these sick pigs. historically, disease control interventions have been focused on farm level biosecurity. given their role as a link between farmers and consumers, traders would benefit from targeted inclusion in disease control and prevention strategies. isolation and molecular identification of nipah virus from pigs animal biosecurity in the mekong: future directions for research and development proceedings of an international workshop assessing the potential role of pigs in the epidemiology of ebola virus in uganda uganda pig value chain assessment tool_kap traders attitudes and practices related to african swine fever within smallholder pig production in northern uganda serologic study of pig-associated viral zoonoses in laos assam's pig sub-sector: current status, constraints and opportunities participatory assessment of animal health and husbandry practices in smallholder pig production systems in three high poverty districts in uganda risk factors for african swine fever in smallholder pig production systems in uganda qualitative analysis of the risks and practices associated with the spread of african swine fever within the smallholder pig value chains in uganda food and agriculture organization. mapping supply and demand for animal-source foods to 2030. animal production and health working paper pigs and public health risks. agriculture and consumer protection department food and agriculture organization (fao). faostat. 2011. food and agriculture organization (fao). pig sector kenya pig production and consumption in relation to oil-seed cake production and utilisation in kenya domestic pigs as potential reservoirs of human and animal trypanosomiasis in northern tanzania drivers and risk factors for circulating african swine fever virus in uganda characteristics of the smallholder free-range pig production system in western kenya epidemiological overview of african swine fever in uganda smallholder pig production systems along a periurban-rural gradient in the western provinces of the democratic republic of the congo replication, pathogenicity, shedding, and transmission of zaire ebolavirus in pigs livestock and women ' s livelihoods: a review of the recent evidence. livest. women ' s livelihoods a rev. recent evid live pig markets in eastern indonesia: trader characteristics, biosecurity and implications for disease spread the pig as a mixing vessel for influenza viruses: human and veterinary implications ebola reston virus infection of pigs: clinical significance and transmission potential emerging viral infections in australia experimental nipah virus infection in pigs and cats practices in the pig value chain in uganda; implications to african swine fever transmission factors that influence pig production in central uganda-case study of nangabo sub-county, wakiso district factors that influence pig production in central uganda-case study of nangabo sub-county, wakiso district risk factors associated with occurrence of african swine fever outbreaks in smallholder pig farms in four districts along the uganda-kenya border enhancing knowledge and awareness of biosecurity practices for control of african swine fever among smallholder pig farmers in four districts along the kenya-uganda border food safety and zoonotic hazards in pig value chains in east africa smallholder pig value chain assessment in uganda: results from producer focus group discussions and key informant interviews characterization of smallholder pig production systems in uganda: constraints and opportunities for engaging with market systems 196-characterization of smallholder pig production systems in uganda: constraints and opportunities for engaging with market systems review of african swine fever: transmission, spread and control: review article african swine fever virus eradication in africa fit for human consumption? a descriptive study of wambizzi pig abattoir i'm only allowed to sell milk and eggs': gender aspects of urban livestock keeping in eldoret, kenya outbreaks of african swine fever in domestic pigs in gulu district outbreaks of african swine fever in domestic pigs in gulu district the national livestock census report livestock susceptibility to infection with middle east respiratory syndrome coronavirus characterisation of smallholder pig production in kikuyu division, central kenya zoonoses and antimicrobial resistance in kenya and malawi acknowledgements the authors would like to thank the management of wambizzi cooperative society who facilitated this research and the pig traders operating at wambizzi for their involvement in this study. this research benefited from the technical input and tools developed by ilri uganda colleagues ben lukuyu, emily ouma, michel dione, and kristina roesel.funding funding for this study was provided by the cgiar research program on agriculture for nutrition and health, led by the international food policy research institute. statement of animal rights this human study has been approved by the appropriate ethics committees and has therefore been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments. the authors declare that there is no conflict of interest. key: cord-282849-ve8krq78 authors: stebler, rosa; carmo, luís p.; heim, dagmar; naegeli, hanspeter; eichler, klaus; muentener, cedric r. title: extrapolating antibiotic sales to number of treated animals: treatments in pigs and calves in switzerland, 2011–2015 date: 2019-09-20 journal: front vet sci doi: 10.3389/fvets.2019.00318 sha: doc_id: 282849 cord_uid: ve8krq78 to evaluate the contribution of antimicrobial use in human and veterinary medicine to the emergence and spread of resistant bacteria, the use of these substances has to be accurately monitored in each setting. currently, various initiatives collect sales data of veterinary antimicrobials, thereby providing an overview of quantities on the market. however, sales data collected at the level of wholesalers or marketing authorization holders are of limited use to associate with the prevalence of bacterial resistances at species level. we converted sales data to the number of potential treatments of calves and pigs in switzerland for the years 2011 to 2015 using animal course doses (acd). for each authorized product, the number of potential therapies was derived from the sales at wholesaler's level and the acd in mg per kg. for products registered for use in multiple species, a percentage of the sales was attributed to each authorized species according to their biomass distribution. we estimated a total of 5,914,349 therapies for pigs and 1,407,450 for calves in 2015. using the number of slaughtered animals for that year as denominator, we calculated a treatment intensity of 2.15 therapies per pig and 5.96 per calf. between 2011 and 2015, sales of veterinary antimicrobials decreased by 30%. the calculated number of potential therapies decreased by 30% for pigs and 15% for calves. an analysis of treatment intensity at antimicrobial class level showed a decrease of 64% for colistin used in pigs, and of 7% for macrolides used in both pigs and calves. whereas the use of 3rd and 4th generation cephalosporins in calves decreased by 15.8%, usage of fluoroquinolones increased by 10.8% in the same period. corresponding values for pigs were −16.4 and +0.7%. this is the first extrapolation of antimicrobial usage at product level for pigs and calves in switzerland. it shows that calves were more frequently treated than pigs with a decreasing trend for both number of therapies and use of colistin, macrolides and cephalosporins 3rd and 4th generations. nonetheless, we calculated an increase in the usage of fluoroquinolones. altogether, this study's outcomes allow for trend analysis and can be used to assess the relationship between antimicrobial use and resistance at the national level. to evaluate the contribution of antimicrobial use in human and veterinary medicine to the emergence and spread of resistant bacteria, the use of these substances has to be accurately monitored in each setting. currently, various initiatives collect sales data of veterinary antimicrobials, thereby providing an overview of quantities on the market. however, sales data collected at the level of wholesalers or marketing authorization holders are of limited use to associate with the prevalence of bacterial resistances at species level. we converted sales data to the number of potential treatments of calves and pigs in switzerland for the years 2011 to 2015 using animal course doses (acd). for each authorized product, the number of potential therapies was derived from the sales at wholesaler's level and the acd in mg per kg. for products registered for use in multiple species, a percentage of the sales was attributed to each authorized species according to their biomass distribution. we estimated a total of 5,914,349 therapies for pigs and 1,407,450 for calves in 2015. using the number of slaughtered animals for that year as denominator, we calculated a treatment intensity of 2.15 therapies per pig and 5.96 per calf. between 2011 and 2015, sales of veterinary antimicrobials decreased by 30%. the calculated number of potential therapies decreased by 30% for pigs and 15% for calves. an analysis of treatment intensity at antimicrobial class level showed a decrease of 64% for colistin used in pigs, and of 7% for macrolides used in both pigs and calves. whereas the use of 3rd and 4th generation cephalosporins in calves decreased by 15.8%, usage of fluoroquinolones increased by 10.8% in the same period. corresponding values for pigs were −16.4 and +0.7%. this is the first extrapolation of antimicrobial usage at product level for pigs and calves in switzerland. it shows that calves were more frequently treated than pigs with a decreasing trend for both number of therapies and use of colistin, macrolides and cephalosporins 3rd and 4th generations. nonetheless, we calculated an increase in the usage of fluoroquinolones. altogether, this study's outcomes allow for trend analysis and can be used to assess the relationship between antimicrobial use and resistance at the national level. keywords: antibiotics, antimicrobial consumption, course dose, pigs, calves introduction use of antimicrobials contributes to the emergence and spread of resistant bacteria in both humans and animals. as early as in the 1960's concerns arose in relation to therapeutic, preventive and growth-promoting treatments in food-producing animals. the fact that most antibiotic classes are administered to treat infections in both humans and animals was one of the major concerns (1, 2) . monitoring antimicrobial usage is therefore a prerequisite to assess the impact of antibiotic treatments on the selection and spread of bacterial resistances. in order to achieve that goal, a number of programs monitoring sales and/or usage of antimicrobials have been established both at national level, for example in switzerland [arch-vet; (3)], denmark (4), and international level [esvac project of the european medicines agency; (5) ]. these programs do not only aim at the identification of trends in sales and usage of antimicrobial classes but should also allow establishing a link with changes observed in resistance monitoring programs, thereby providing a basis for risk assessment and evaluation of regulatory interventions (6) . in order to assess the association between antimicrobial use and resistance, it is of crucial relevance to obtain consumption data at species or, when possible, production type level; there are several species and production type-specific factors that can impact on the relationship between use and resistance. those factors include age at treatment, age and weight at slaughter, products available per species or production type, and especially production structures (7) (8) (9) . antimicrobial sales data are defined as the minimal standard for monitoring programs by the world organization for animal health [office international des epizooties, oie; (10) ]. they can be collected at either the manufacturer, wholesaler or pharmacy level depending on the national distribution routes of the products. sales data are useful to evaluate long-term trends but do not include information about dose, route of administration, indication or duration of therapy. however, in the context of resistance epidemiology, only data about actual use of antimicrobials collected either at prescription or patient level might deliver the information necessary to establish and evaluate implemented measures. such data can only be currently collected in few countries with advanced collection systems, such as denmark (4) and the netherlands (11) among other european countries. the aacting network is maintaining a list of the various collection systems already in place (www.aacting.org). the collection of data at animal level is the ultimate goal of antimicrobial monitoring systems and, until this is available in all participating countries, alternatives using normalization of sales data by the total weight of the food producing animal population as a denominator have been developed. one such denominator is the population correction unit of the esvac project (12) . other institutions (13) and countries, including canada (14) and switzerland [arch-vet; (3)], have implemented similar methods in their surveillance systems. as usage of antimicrobials is strongly dependent on population structure and repartition between high and low-using species, the normalization by weight may provide information on long-term trends but at the same time, a higher usage in one species will be "diluted" by lower usage species/production types (like dairy cows) with a large contribution to the overall livestock biomass (15) . it is therefore important to measure antibiotic consumption as near as possible to the end users, i.e., to obtain information on species, dosage, duration and whenever possible, indication. the extrapolation of sales data using course doses is an interim measure to data collection at animal level. course dose indicators have been proposed, such as the animal course dose (acd) by the french agency for food, environmental, and occupational health & safety (16) or defined course dose (dcdvet) from the ema (17) . an advantage of acd is its product-specific calculation, therefore better representing national specificities than dcdvet units. acds are established for each product using data from the summary of product characteristics (spc) and contain the necessary detail on both dose and therefore potency, and duration of use. the main aim of this study was to provide for the first time an extrapolation of the available national sales data to the number of treated animals in switzerland. we chose to specifically investigate the treatment of pigs and calves because these are mainly reared and treated in groups via oral application. due to the lack of detailed data about repartition of sales, we made assumptions regarding weight at treatment and repartition of sales data between species using a previously published repartition method. we then defined acd for each product containing antimicrobials authorized in switzerland for use in either pigs or calves and combined this information with national antibiotic sales data to extrapolate the number of potentially treated animals during the years 2011 to 2015. veterinary antibiotic sales data for the years 2011 to 2015 were obtained from the federal office of food safety under a confidentiality agreement. since 2004, sales data are collected in switzerland from marketing authorization holders based on article 35 of the ordinance of veterinary medicines (18) . marketing authorization holders are required to deliver data on every product containing antimicrobials that was sold during a calendar year. products subject to data collection are defined by their atcvet codes (19) as listed in the esvac project (12) . additionally, data on antibiotic products not considered by the esvac project, like sprays or products to treat sensory organs, are also collected. data obtained from the federal office of food safety for this study contained the quantity of active antimicrobial ingredient sold in kilogram for each product and year under investigation. the amount of antimicrobials sold of products authorized for a single species was directly assigned to their target species. for each product authorized for more than one species, a repartition had to be determined. we used two distinct methods: the first one was used for premixes, the latter being legally defined in switzerland as being "veterinary medicinal products used to treat groups of animals and incorporated into either water or feed" [ordinance on authorizations for medicinal products, art. 2; (20) ]. for all of these products, periodic safety update reports (psurs) containing data on species repartition submitted to swissmedic, the swiss agency for therapeutic products, during the years 2007 to 2012, were used. as premixes represented only 28 products from a total of 112 under investigation but between 57.6% (2015) and 67.8% (2011) of the total sales, another repartition method had to be used for oral solutions, oral powders and injectables. this repartition was done according to biomass repartition as described by carmo et al. (21) . briefly, for each product authorized for one or more target species, each target species was assigned a percentage of kg of the total sales representing the proportion of its biomass in the total biomass of the list of authorized species for the product. for the present study, food producing animal population numbers were obtained from the federal office of statistics (www.bfs.admin.ch), number of dogs from the anis database (identitas ag, bern, www.anis. ch) and the number of cats from the swiss association of pet food producers (verband für heimtiernahrung, bern, www.vhn. ch). in analogy with calculations of the population correction unit (pcu) of esvac (12) , the number of slaughtered animals were used for fattening pigs and calves, whereas data for dairy cows, sows, sheep, goats, horses, dogs, and cats represent live animals. throughout the text and in the tables, "pigs" refer to fattening pigs. supplementary table 1 lists the number of animals and the weights used for the biomass repartition. the most likely weight at treatment was sourced from the esvac report (12, 22) . as heavy animals with a rather low treatment intensity, like dairy cows, skew the biomass repartition, we chose to only include them in the calculation when they were either explicitly listed as authorized species ("dairy cows") or, when a withdrawal time for milk was given in the spc of the product. for pigs, we did not include the production stage of piglets or weaners. using the number of animals in different production stages presents some challenges, the most prominent one for pigs being the lack number of acds = total quantity of active ingredient sold in one year (mg) daily dose mg kg × duration of tratment days × weight at treatment (kg) therapeutic intensity in species x = number of acds in species x total number of animals for species x of available data for the repartition of use between piglets and e.g., fattening pigs. only few antimicrobials are primarily used in piglets or weaners, colistin being such an example. for almost all other products authorized for pigs, no data are available to stratify antimicrobial consumption per different age classes using sales data. repartition data will only be available once reporting of all treatments with antimicrobials in switzerland is made mandatory at the end of the year 2019. for this reason and because sales data include the use of antimicrobials by all the age categories of the species for the years under investigation, we used slaughtered numbers of pigs as denominator for the therapeutic incidence in this species. finally, for injectable products authorized without indication of the production stage ("bovines" containing dairy cows and "pigs" representing slaughtered pigs and sows) we used raw data provided by experts for the study by carmo et al. (23) to determine if a use would take place in the particular production stage under consideration. the animal course dose (acd) was calculated for each active pharmaceutical ingredient contained in each product authorized during the years under investigation. data were collected from the authorized summary of product characteristics (24) and entered into an ms excel sheet containing: name of the product, authorization number, list of authorized species, active ingredient(s), dose and duration. doses given in international units were converted to mg using conversion factors listed in the esvac report (12) . whenever the recommended dose was a range, the highest recommended dose and longest duration were chosen to reflect the minimal number of animals potentially treated. moreover, when different doses were authorized for different indications, the most likely indication was chosen. this was the case for products presenting both a prophylactic or metaphylactic indication with different doses and duration. acds were defined per kg and the acd per animal obtained by multiplication with the likely weight at treatment. to take swiss specificities into account, the weight at treatment for pigs was taken from a previous study by schnetzer et al. (25) and the weight for calves based on expert opinion (prof. m. kaske, zurich, personal communication). therapeutic intensity reflects the number of acds per slaughtered animal (pig or calf) in 1 year. for combination products, the number of acds was calculated separately for each active pharmaceutical ingredient. therefore, a single treatment with a combination containing 3 antimicrobials results in 3 acds. acd and intensity were calculated using the following equations: from the year 2011 to 2015, sales of antibiotics for use in food producing animals decreased by 29.8% ( table 1 ). in the same time, the percentage represented by premixes decreased from 67.8 to 57.7%. therefore, measured in kg, antimicrobials sold in premixes made the largest part of yearly sales of antimicrobials for the veterinary medicine. as a consequence, pigs and calves are the most pertinent species among food producing animals to be investigated for use and trend detection. in tonnage sold for use in these species, the decrease in the 5 years under investigation is comparable: 38.4% in pigs and 30.1% in calves. however, normalizing these numbers to the respective biomass of the produced (slaughtered) population reveals a much higher use per kg of biomass for calves (449.9 mg/kg biomass in 2015) than for pigs (77.4 mg/kg biomass). the difference between both species even increased from 4.8-fold higher for calves in 2011 to 5.8 in 2015. normalizing sales data to either the overall biomass of food producing animals or to the biomass of a particular species is a crude estimate of antimicrobials use, not taking dose or duration into account. we therefore calculated the number of course doses (acds) per product and species. a summary of the results is presented in table 2 . the total number of acds was approximately 4.5 times higher in pigs and decreased by 31.7% over the years under investigation, whereas the decrease for calves was 23.0%. normalization to the number of slaughtered animals showed a much slower decrease of 14.9% for calves between 2011 and 2015 compared to 29.6% in pigs. as a result, the difference between both species grew from 2.3-fold in the year 2011 to 2.8-fold in the final year under investigation. not all antibiotics have the same potential impact on resistance selection and consequences for the treatment of both humans and animals. moreover, different products are authorized for distinct conditions in pigs or calves. the repartition of the number of acds per class of antimicrobials was therefore calculated separately for each species for the year 2015. table 3 presents the repartition by antimicrobial class for ingredients sold in premixes and as parenteral injections. in this year, polymyxins (in form of colistin) were the class with the highest potential numbers of acds per pigs, followed by sulfonamides. in calves, the highest number was represented by penicillins (mainly sold as aminopenicillins) followed by tetracyclines. the total number of acds per animal was 4.43 times higher in calves than in pigs. the same calculation was done for injectable products as these may contain antimicrobials of the highest priority [hpcia; (26) ] not available for oral application. for pigs, the highest number of acds per animal in the year 2015 was represented by macrolides, followed by aminoglycosides and fluoroquinolones. for calves (amino)penicillins were the class with the highest number of course doses per animal, followed by macrolides and aminoglycosides. the total number of potential acds per animal for injectable products in the year 2015 was 1.485 for calves and 1.088 for pigs. finally, the evolution of the number of potential acds per animal for hpcias is presented in table 4 . for macrolides used in pigs, a decrease of 22.0% for products sold as premixes was attenuated by a corresponding increase of 11.8% for injectables. this pattern was even more evident in calves where a reduction of 27.1% for premixes was almost completely compensated by an increase of 25.7% in injectables. with respect to the other two classes of hpcias, sales of products containing fluoroquinolones remained stable for pigs (−1.5%) and an increase of 6.4% was observed for the number of potential acds per animal in calves. courses with cephalosporins of the third and fourth generations showed a comparable decrease in pigs (−16.4%) and calves (−15.7%). this is the first study at national level using the acd concept applied to sales of antimicrobials with the objective of extrapolating the number of potentially treated pigs and calves in switzerland. sales of antimicrobials for the veterinary medicine are published at national level since 2005. so far, these data represent the only available source of exhaustive antimicrobial consumption data at national level. sales figures may allow for the recognition of trends, but the lack of information on potency, dose, duration of treatment and repartition per species strongly limits their usefulness. the indicator acd may therefore help to bridge that gap. calculation of acd and repartition of quantities for products authorized for more than one species would not be possible without making assumptions, which might influence the results. the first assumption relates to the weight of the animals. the standard weight has an impact on both the calculation of the species repartition and the acd indicator itself. the impact of using different weights is a topic beyond the scope of this study and the impact on calculations has been studied elsewhere (27) (28) (29) . in this study we used weights at treatment as close as possible to the swiss reality. this should provide the best fitting results, and also guarantees future reproducibility of the method and comparison of results, as these weights are likely to be used when quantifying swiss antimicrobial consumption both at national and international level. this approach is comparable to the one chosen by the esvac project. the method used to stratify antimicrobial consumption by the production types included in the study has some potential bias. as it is based on the total biomass of each animal category, the resulting estimates are highly dependent on the animal demographics and the animal average weight used. this might not always be a representative surrogate of the product repartition by each category. as a reliable repartition is generated by data collected on actual usage, and such data are currently not available in switzerland, we chose an alternative that was applicable at product level that would deliver reproducible results over the years and be as accurate as possible. carmo et al. (21) have compared three different methods to determine species repartition of antimicrobials. the longitudinal study extrapolation method (based on field data) was not applicable at single product level due to the requirement for minimum, mode, and maximum starting values. the biomass distribution was shown to be the method providing the closest results to the extrapolation based on field data, thereby increasing our confidence on the pertinence of the approach we applied. the two main drawbacks of this method are the dependence on defined average weights and country specific animal demographics. however, the method, limited by the data available in the current swiss context, provides a first insight into antimicrobial consumption patterns in different species/production types. in the future, the data collection system is-abv (description available under http://www.aacting.org/matrix/is-abv/?lid=1447) shall provide further insights into these patterns, as well as a basis for comparison with the results from the method and its potential biases. to make our extrapolations as comparable as possible with other projects, we used the same standard weights as in the esvac project (12) . it must also be noticed that the denominators of the indicators presented were based on the number of slaughtered animals only. the weights used for the calculation of the biomass were likely weights at treatment as defined in the esvac project (12) . the use of such a calculation might hinder direct comparisons with other studies and should be taken into consideration when benchmarking these results. when using the biomass as a denominator, the result should be interpreted as an indicator for the amount of active ingredient used per kg of animal produced. likewise, the therapeutic intensity indicates the average number of acds per animal produced/slaughtered. both a high proportion of heavier animals like cows or, alternatively, a high treatment intensity in a species of lower biomass are examples of how animal demographics can bias the results of the stratification approach based on the biomass. the repartition across species is mainly influenced by national production structures. in switzerland, dairy production is an important agricultural sector and therefore dairy cows make a high proportion of the food producing animals' sector (15) . cows represented 49% of the total biomass in the year 2015 and this high proportion leads to an underestimation of the repartition of sales for pigs or calves. this primarily affects the repartition of aminoglycosides and cephalosporins of the third generation, which are antimicrobials frequently used in the treatment of dairy cows. the calculated numbers of acds per animal for these classes presented in table 4 are, therefore, an underestimation. within the same species, biomass repartition could have been used to estimate the use of antimicrobials in different production stages of pigs. however, using piglets, weaners and fattening pigs produced during 1 year introduces the bias of counting a significant but undefined proportion of the animals two or three times. as sales data were only available for one full year, we therefore chose to base our repartition, as well as the denominator for the treatment intensity, on the number of pigs slaughtered during the same year. this indicator is used in this study as a surrogate for all pig production stages. as the numbers of acds represent an extrapolation of usage data based on sales figures, they follow the latter closely. the downward trends in sales is mirrored by the treatment numbers of both calves and pigs. however, differences become evident as soon as additional factors like application route are taken into account. the repartition for pigs in the year 2015 shows that 18% of the active ingredients were used parenterally when based on quantity, whereas they represented 51% of the treatments when using acds. the main reason for this difference lies in the potency of the active ingredients: antimicrobials are used parenterally with a lower dose as there is no loss of active ingredient compared to the lower bioavailability following oral application. another possible reason is the use of more than one acd for parenterally applied combination products as 12 of 71 injectable products investigated were combinations of two active ingredients. whereas, this approach can be disputed as it shows a higher number of "treatments, " we think that the use of acds is better suited to test for associations between antimicrobial use and resistance. converting sales of antimicrobials to number of treatments per animal allows detection of trends that would not be obvious when only assessing the quantity of active ingredients sold. macrolides used to treat calves provide a good example: our results show a clear shift from the oral application in form of premixes toward an increase in the use of injectables. one possible explanation is the increasing availability of macrolide antibiotics with a long duration of action, e.g., tulathromycin, tildipirosin, and gamithromycin. such active compounds combine the easy use of a single application with a long action. moreover, for parenteral applications, both time to maximal concentration and maintenance of active levels are not influenced by the appetite of the animals, therefore guaranteeing the adequate treatment of sick animals with reduced appetite. on the negative side, studies about macrolides used in human medicine convincingly showed a higher level of resistance selection for longer acting molecules (30) . our results show a strong difference in the extrapolated usage of antimicrobials in pigs and calves. this cannot be explained by a single factor as the administration of antimicrobials is driven by medical, economic and also psychosocial factors. crowding effect, stress during transport of very young, not yet immunocompetent animals, partially inadequate colostrum feeding and less than ideal stable climate are among the factors favoring respiratory problems in calves (31, 32) . in the swine industry, some of the abovementioned factors also exist, but the structure and management of pig production limits the risks. management practices like all-in-all-out including disinfection between the batches or integrated production from piglet to finisher can strongly help to reduce antimicrobial usage. in pigs, there are two main periods at risk for treatment with antimicrobials: the first at weaning with around 12 kg body weight and the second at around 25 to 29 kg body weight (25, 33) . in pigs, diarrhea is one of the leading indications for treatment. this is a very unspecific symptom with many different causes, including not only bacterial but also dietary or viral origins. in this context, the availability of vaccines against both circovirus and lawsonia intracellularis infections in the years 2008 to 2010 contributed to the reduction of diarrheal symptoms, and hence, the rather indiscriminate use of antibiotics to treat such symptoms. for calves, respiratory diseases are much more multifactorial and the introduction of various vaccines (against bovine respiratory syncytial, parainfluenza or corona virus) seems not to have had the same positive effect as in the pig industry. several factors hinder a proper comparison of our results with previously published data. to the best of our knowledge, this is the first time that the acd indicator is used at national level in switzerland. as a matter of fact, its use is not currently widespread in other countries, with the exception of france where it was developed. however, the comparison with french data is difficult. no publication presents the french antimicrobial consumption using acds per animal and year as an indicator. the french indicator for exposure to antimicrobials is alea [animal level of exposure to antimicrobials; (16) ]. it is obtained by dividing the effectively treated biomass by the total biomass of the same species. the global alea calculated for the year 2015 in france was 0.488 and represented a decrease of 20.1% compared to 2011. another difficulty is the use of different production categories and standard weights at treatment. for pigs, the french system uses weights up to 350 kg for a specific category of sows and the average for the pig population is set at 105 kg. this is 3.62 times higher than the standard weight at treatment of 29 kg identified in previous swiss studies and used here. the differences in the standard weights at treatment also explain the discrepancies in the antimicrobial consumption for france published, for the same year, in the esvac report (107 mg/pcu) and in the anses report (47 mg/kg). due to the differences in weight and in the categories, and the difficulties in making assumptions and extrapolations, we decided not the compare our figures to the french ones. our data can only be compared with countries where calves are reared for the production of veal meat. besides france and belgium (for which country we could not find adequate data for comparison), this production system also exists in the netherlands. the available report for the year 2015 (34) uses indicators differing from the ones in the present study but still shows a higher treatment intensity in calves compared to pigs. this is in line with the present study, where the antimicrobial use was 2.77-fold higher in calves than in pigs. both examples clearly illustrate the need to harmonize methodologies at international level in order to discuss data collected in different countries. such discussions currently take place within the aactng network (www.aacting.org). this first study of the number of treatments of pigs and calves extrapolated from yearly sales shows both similarities and differences between the two species under consideration. whereas, the sales by species and the number of extrapolated treatments both decreased in a similar way, the difference in the number of treatments per animal between pigs and calves differed over the years under investigation. given that the applied method is based on the extrapolation of sales figures, a similar decrease at species level was to be expected. however, the use of course doses allows to further investigate trends in the patterns of antimicrobial treatments. in our study, this was very clear for the class of macrolides, for which the decreases in oral use were partly (pigs) or completely (calves) compensated by the application of long acting injectables. we, therefore, recommend the use of extrapolated treatment numbers when no exhaustive collection of usage data is in place. the concept of acds can also complement the collection of antimicrobial consumption data at species level allowing for their validation using sales data. all datasets generated for this study are included in the manuscript/supplementary files. rs did all the calculations presented in this work. lc helped with the repartition of sales between species, expert advice and biomass distribution. raw sales data and advice regarding their use was provided by dh. the study was designed by cm and supervised by ke and hn. joint committee on the use of antibiotics in animal husbandry and veterinary medicine world health organization. the medical impact of the use of antimicrobials in food animals arch-vet 2017: bericht über den vertrieb von antibiotika in der veterinärmedizin in der schweiz use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in denmark european surveillance of veterinary antimicrobial consumption. sales of veterinary antimicrobial agents in 30 european joint fao/oie/who expert workshop on non-human antimicrobial usage and antimicrobial resistance: scientific assessment the antimicrobial resistome in relation to antimicrobial use and biosecurity in pig farming, a metagenome-wide association study in nine european countries association between antimicrobial usage, biosecurity measures as well as farm performance in german farrow-to-finish farms factors associated with high antimicrobial use in young calves on dutch dairy farms: a casecontrol study antimicrobial resistance: monitoring the quantities of antimicrobials used in animal husbandry monitoring of antimicrobial resistance and antibiotic usage in animals in the netherlands in 2017 trends in the sales of veterinary antimicrobial agents in nine european countries annual report on antimicrobial agents intended for use in animals canadian antimicrobial resistance surveillance system comparison of the sales of veterinary antibacterial agents between 10 european countries anses. suivi des ventes de médicaments vétérinaires contenant des antibiotiques en france en 2015. anses -agence nationale du médicament vétérinaire european surveillance of veterinary antimicrobial consumption. ema/224954/2016: defined daily doses for animals (dddvet) and defined course doses for animals (dcdvet) tamv. verordnung über die tierarzneimittel who collaborating centre for drug statistics methodology norwegian institute of public health ambv. verordnung über die bewilligungen im arzneimittelbereich approaches for quantifying antimicrobial consumption per animal species based on national sales data: a swiss example validation of the exposure assessment for veterinary medicinal products expert opinion on livestock antimicrobial usage indications and patterns in denmark, portugal and switzerland institut für veterinärpharmakologie und -toxikologie. swiss veterinary drug compendium therapieintensität beim einsatz von fütterungsarzneimitteln bei schweinen [calculation of therapeutic intensity for pigs treated using medicated feed critically important antimicrobials for human medicine -5th rev prospective study on quantitative and qualitative antimicrobial and antiinflammatory drug use in white veal calves comparing antimicrobial exposure based on sales data antimicrobial use in pigs, broilers and veal calves in belgium. vlaams diergeneeskundig tijdschrift effect of azithromycin and clarithromycin therapy on pharyngeal carriage of macrolide-resistant streptococci in healthy volunteers: a randomised, double-blind, placebo-controlled study risk factors for death and unwanted early slaughter in swiss veal calves kept at a specific animal welfare standard antimicrobial drug use and risk factors associated with treatment incidence and mortality in swiss veal calves reared under improved welfare conditions berechnung der therapieintensität bei ferkeln und mastschweinen beim einsatz von antibiotika in fütterungsarzneimitteln monitoring of antimicrobial resistance and antibiotic usage in animals in the netherlands in 2015 the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets. 2019.00318/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2019 stebler, carmo, heim, naegeli, eichler and muentener. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-308767-trwa5grl authors: costa vallés, cristina; máñez mendiluce, rafael title: transgenic organs and xenotransplants date: 2012-03-20 journal: stem cell transplantation doi: 10.1007/978-1-4614-2098-9_6 sha: doc_id: 308767 cord_uid: trwa5grl a dvances in immunosuppressive treatments reached in the last decades of the 20th century have made solid organ transplantation the treatment of choice for cases of irreversible organ failure. however, the availability of human cadaver organs is limited and the demand for transplants is still on the rise. also, there is a recognised lack of cells and human tissues for generalised use in transplantation for the treatment of diseases that are characterised by failure of specialised cells (such as pancreatic cells to cure diabetes). xenotransplantation, which is the transplant of cells, tissues or organs from other species, became the focus of attention in the nineteen-nineties as a solution to the lack of organs and tissues for transplantation. previous clinical studies using nonhuman primates produced poor outcomes (survival from days to a few months) and confirmed the difficulty of obtaining organs from these species. since then, progress in xenotransplantation has been slow and still now various immunological and non-immunological barriers need to be overcome. these barriers are reviewed in this chapter and the various approaches explored to date to overcome them, in particular those based on the genetic modification of pigs. also, cell transplant studies such as those of pancreatic islets in monkeys have led to even more hopeful results. the range of possibilities offered by this technology will be unlimited, making it possible for xenotransplantation to be a clinical reality in a not very distant future. advances in immunosuppressive treatments reached in the last decades of the 20th century have made solid organ transplantation the treatment of choice for cases of irreversible organ failure. however, the availability of human cadaver organs is limited stem cell transplantation, edited by carlos lópez-larrea, antonio lópez-vázquez and beatriz suárez-álvarez. ©2012 landes bioscience and springer science+business media. and the demand for transplants is still on the rise. in spain, the world leader in cadaver organ donation, the greatest achievement has been to avoid a year-on-year growth in waiting lists, as occurs in most countries. it has not been possible to reduce them, despite the notable increase of live donor transplantation; rather these have only served to slow the growth of waiting lists, not to mention the risks of live donor transplantation for the donor. also, there is a recognised lack of cells and human tissues for generalised use in transplantation for the treatment of diseases that are characterised by failure of specialised cells (such as pancreatic cells to cure diabetes). xenotransplantation, which is the transplant of cells, tissues or organs from other species, became the focus of attention in the nineteen-nineties as a solution to the lack of rujdqv dqg wlvvxhv iru wudqvsodqwdwlrq $w wkdw srlqw diwhu wkh surgxfwlrq ri wkh ¿uvw wudqvjhqlf pigs, this species was selected as the best source of organs and xenogeneic tissues. [1] [2] [3] previous clinical studies using nonhuman primates produced poor outcomes (xenograft survival iurp gd\v wr d ihz prqwkv dqg frq¿uphg wkh gli¿fxow\ ri rewdlqlqj rujdqv iurp wkhvh species. moreover, the risk to public health of using the organs from nonhuman primates was quite high, as vividly demonstrated by the aids epidemic. on the other hand, pigs are domesticated animals, which produce large litters, and that, apart from their interest in the food industry, have also had some medical uses (production of insulin, heart valves). finally, they combine a physiology and anatomy that is similar to primates and they can eh jhqhwlfdoo\ prgl¿hg 7kxv lw lv qrw vxusulvlqj wkdw wkh srvvlelolw\ ri ³kxpdqlvlqj´ slj organs and tissues should be talked about with a view to using them in clinical practice. 6lqfh wkhq vljql¿fdqw dgydqfhv kdyh ehhq pdgh 7kh prvw zhoo vwxglhg slj rujdqv for their potential for xenotransplantation are the kidney and heart, followed by the lung and liver. indeed, the liver is considered as the potential bridge for allotransplantation, either through transplant or by ex vivo perfusion. research is also being carried out on cell xenografts of pancreatic islets, hepatocytes, chondrocytes and neural cells, among others. despite the great impact that their clinical application would represent, progress in xenotransplantation has been slow, mainly due to technological problems. a decade later, we are still facing various barriers that prevent the clinical application of pig organs, tissues and cells. these barriers are reviewed below and the various approaches explored to date wr ryhufrph wkhp lq sduwlfxodu wkrvh edvhg rq wkh jhqhwlf prgl¿fdwlrq ri sljv duh ghvfulehg the main obstacle for xenotransplantation to be used in clinical practice is the strong immune response caused by the pig organ in the recipient. 3 cell, tissue and organ xenografts are subject to a variety of rejection mechanisms that include humoral and cellular immune responses. the cellular immune response seems to play a key role in the rejection of cell grafts, such as hepatocytes and pancreatic islets, while rejection of vascularised organs mainly involves humoral immunological mediators. various types of rejection have been described in solid organ xenotransplantation, depending on the time elapsed following the transplant and the immune elements involved: hyperacute, acute humoral and acute cellular rejection. [1] [2] [3] +\shudfxwh uhmhfwlrq +$5 lv wkh ¿uvww\sh wr rffxu ehwzhhq plqxwhv dqg krxuv after the xenotransplant. har is triggered by a humoral immune response in which xenoreactive natural antibodies (xna) (already present in the recipient) are deposited rq wkh hqgrwkholxp ri wkh [hqrjudiw dfwlydwlqj wkh frpsohphqw v\vwhp dqg fdxvlqj àxlg extravasation from the intravascular space into the interstitium (oedema). [1] [2] [3] this process also causes the activation of the coagulation cascade and thus thrombosis, ischemia and necrosis of the xenograft in a short period of time following the transplant. the main xenoepitope recognised by xna is the disaccharide galactose-alpha-1,3-galactose (gal), widely expressed in pig tissues and synthesised by the alpha 1,3-galactosyltransferase (_1,3-gt) enzyme. humans and old world primates lack functional _1,3-gt and have high titre of anti-gal antibodies. acute humoral xenograft rejection (ahxr), also known as acute vascular rejection or delayed xenograft rejection occurs in a period of days to months after the transplant. it has not been possible to prevent it using currently available immunosuppression regimens. 1-4 ahxr shows notable similarity to har since it involves a strong humoral immune response with the participation of antibodies, the deposition of complement proteins and thrombosis in xenografts. however, in other respects ahxr differs vljql¿fdqwo\ iurp +$5 hvshfldoo\ lq wkh ruljlq ri wkh dqwlerglhv uhvsrqvleoh iru uhmhfwlrq in this case, it involves a response in which anti-gal antibodies participate, but it also includes antibodies targeting other epitopes. further, ahxr is characterised by the suhvhqfh ri lqqdwh lppxqlw\ fhoo lq¿owudwhv 1. fhoov dqg pdfurskdjhv dqg dq dfwlydwlrq ri hqgrwkholdo fhoov wkdw surprwhv lqwudydvfxodu wkurpervlv dqg ¿eulq ghsrvlwlrq acute cellular rejection occurs some days after the transplant and is a response mediated mainly by t cells against the donor antigens. the activation of these cells during xenotransplant rejection is mediated by a primary signal through the t receptor and secondary costimulatory signals conserved across species. 1 currently, it is thought that it is possible to control the cellular immune response against the xenotransplant using the various immunosuppressive protocols currently available, since no failure of pig xenograft caused by this type of rejection has been demonstrated. however, this point cannot be properly judged until ahxr can be prevented in an effective and systematic way. avascular tissue and xenogeneic cells are also subject to rejection of the graft, similar to the process that occurs in solid organ rejection, but without the vascular component and with special features for each cell and tissue. 5 pancreatic islets have drawn great attention due to their potential clinical application for the treatment of diabetes. first, adult pig beta cells do not express the gal antigen, thus reducing the humoral component of rejection. potent immunosuppressive protocols focussed on averting the activation of t cells have led to long-term survival of pig pancreatic islets in diabetic monkeys (140 days). 6 the problem for the clinical application of cellular xenotransplantation lies in the fact that, while they present fewer immunological barriers than solid organs, they vwloo uhtxluh vwurqj lppxqrvxssuhvvlrq ri wkh uhflslhqw wkdw fdqqrw eh vxvwdlqhg lqgh¿qlwho\ in any case, the strategies developed for this type of transplant may be very useful for their application in other organs of tissues of interest, especially regarding genetic prgl¿fdwlrq ri wkh grqru slj wkdw fdq lqyroyh d zlgh ydulhw\ ri fhoo dqg wlvvxh w\shv therefore any progress made in this area has an impact that goes beyond xenografts, be it to cells, tissues or organs. the various techniques have been developed to prevent har can be summarised in two groups: those focused on modifying the xenograft, and those that use systemic wuhdwphqwv wr dowhu wkh lppxqh uhvsrqvh ri wkh uhflslhqw 7kh prvw uh¿qhg phwkrgv duh edvhg rq wkh jhqhwlf prgl¿fdwlrq ri sljv xvhg dv wkh vrxufh iru rujdqv vlqfh wkh\ duh less harmful for patients and their objective is to decrease the need for immunosuppression or other conditioning treatments. however, these are complex techniques and involve slow and expensive procedures. systemic treatments, on the other hand, may help us to identify molecules or key rejection processes and to speed up the clinical application of [hqrwudqvsodqwv 6shfl¿fdoo\ wkh nh\ urohv ri ;1$ dqg frpsohphqw lq +$5 zhuh frq¿uphg e\ wkh hiihfwlyhqhvv ri dqwlerg\ devruswlrq e\ sodvpdskhuhvlv dqg v\vwhplf complement inhibition to prevent this type of rejection. 7kh ¿uvw dssurdfk wr qhxwudolvh +$5 e\ jhqhwlf hqjlqhhulqj zdv wkh lqklelwlrq ri complement activation through the expression of human complement regulatory proteins in transgenic pigs. 7, 8 the reason for this was the notable restriction of the function of the complement regulatory proteins between species, which led to the suggestion that pig molecules were unable to control the activation of the human complement. subsequent studies showed that pig complement regulatory proteins were also able to regulate, at least in part, the activity of the human complement. 9 7klv vxjjhvwv wkdw wkh ehqh¿w ri wkh h[suhvvlrq ri wkh kxpdq frpsohphqw surwhlqv lq slj fhoov lv qrw rqo\ gxh wr vshfl¿flw\ but also to the increase in expression of these proteins. initially in vitro studies demonstrated that the expression of human cd59 (hcd59) or cd55 (hdaf) in pig cells notably protects these cells from cytolysis triggered by the human serum. ex-vivo perfusion experiments with human blood also demonstrated that kidneys and hearts from pigs transgenic for human cd59 functioned for longer than control animals. later it also demonstrated the almost systematic prevention of har and longer survival rates of transgenic pig organs that expressed human complement inhibitors in transgenic pig-to-primate models. 2,10 however the use of organs from these transgenic pigs did not achieve full protection against humoral xenograft rejection, since all the organs were subject to ahxr sooner or later, the various immunosuppressive protocols being unable to modify this response (tables 1, 2 and 3). this led research to focus on wkh surgxfwlrq ri sljv zlwk jhqhwlf prgl¿fdwlrqv wkdw zrxog uhgxfh wkh uhdfwlylw\ ri wkh organs to xenoantibodies present in human serum. before homologous recombination and "knockout" techniques were available in pigs, one of the most widely investigated strategies was based on the transgenic expression of human alpha 1,2-fucosyltransferase (h transferase, ht). 11 ht produced fucosyl residues (h antigen of the o blood group) that are universally tolerated (fig. +7 zdv vkrzq wr frpshwh hi¿flhqwo\ zlwk doskd*7 iru wkh vdph vxevwudwh n-acetyl-lactosamine, preventing the transfer of the terminal galactose, the residue which gives rise to the production of the gal antigen. the reduction in the expression of the gal epitope in ht transgenic pig cells leads to a decrease in their reactivity with human antibodies and in cytolysis caused by human sera. 11 further, the hearts of ht-transgenic mice have been shown to have higher survival rates after being perfused with human serum or transplanted to 1,3gt knockout mice, which produce anti-gal antibodies. as ht and other competitive enzymes are not able to completely inhibit the expression of gal epitopes, this technique was combined with the expression of human complement inhibitors. thus, peripheral blood mononuclear cells and aortic endothelial cells of double transgenic pigs that co-express ht and hcd59 are better protected from lysis by human serum than controls or single transgenic cells for each of the genes. 12 in addition, double transgenic cells maintained their resistance to xna dqg frpsohphqw diwhu ehlqj wuhdwhg iru krxuv zlwk sruflqh surlqàdppdwru\ cytokines. 12 despite these advances, these pigs were developed to be used in cellular xenotransplantation and the organs have not been transplanted to nonhuman primates, so it has not been possible to study their effectiveness in this experimental model in frpsdulvrq wr slj rujdqv fduu\lqj rwkhu w\shv ri jhqhwlf prgl¿fdwlrqv the description of the process of nuclear transfer of a somatic cell into a germinal cell by the team led by ian wilmut in 1996 opened the door to the generation of "knockout" pigs. from that moment, several groups started the race to produce the ¿uvw doskd*7 gh¿flhqw sljv 7khuh zdv d frqfhuq wkdw sljv surgxfhg e\ wklv whfkqltxh zrxog qrw eh yldeoh exw wkh ¿uvw olwwhuv ri doskd *7 ³nqrfnrxw´ sljv zhuh khdowk\ and developed well. 13 subsequent studies showed survival times of up to 11 days for kidneys of alpha1,3-gt knockout pigs transplanted in baboons, meaning that the organs of these transgenic pigs were also protected against har, 14 as occurred in animals transgenic for human complement regulatory proteins. despite this progress, the majority of human sera show reactivity towards pig cells that lack for alpha1,3-gt gene, suggesting the existence of antibodies that recognise other antigens apart from the gal epitope. 15 therefore, the introduction of a human complement inhibitor is still necessary to completely block human serum-mediated cytotoxicity. another carbohydrate in pigs of interest to prevent har in organ xenotransplantation is the antigen of the type a blood group. as in the human, these antigens are present in some pigs and can be recognised by human antibodies directed against this blood-type. the reactivity of human serum igm against this epitope was discovered in a kidney from a type a group pig whose kidneys were extracorporeally connected to a volunteer dialysis patient. nevertheless, these antibodies should not be an obstacle to clinical xenotransplantation, since blood groups can be selected for the animals used as source of organs, in the same way as with allotransplantation. the methods described above, alone or in combination, have successfully and routinely manage to prevent har. however the same strategies have not proved successful to avoid ahxr. the studies carried out to date suggest that this type of rejection is not caused by a single immunological element, but a collection of them. we will go on to describe research carried out so far into the control of humoral as well as cellular xenograft uhmhfwlrq ,w lv gli¿fxow wr vhsdudwh wkhvh wzr surfhvvhv dv wkh\ ryhuods lq wkh wlph ri progression and possibly also in the immunological response mechanisms. however, it should be highlighted that cellular rejection has not been an insurmountable obstacle and that the various immunosuppressive protocols investigated have managed to prevent this type of rejection systematically. most of the information currently available on preclinical xenotransplantation comes from studies using hdaf transgenic pigs as a source of organs and cynomolgus monkeys or baboons as recipients. results of these studies are summarised in the tables 1 to 3, irfxvlqj rq ruwkrwrslf wudqvsodqwv ri nlgqh\ dqg khduw iurp jhqhwlfdoo\ prgl¿hg sljv given their particular preclinical importance. the transgenic expression of hdaf, alone or in combination with hcd59, protects against har and confers some protection against haxr. 16, 17 one aspect that is worth highlighting is that the species of recipient primate dovr vhhpv wr lqàxhqfh wkh vxuylydo ri wkh [hqrjudiw vxuylydo udwhv ehlqj kljkhu lq cynomolgus monkeys than in baboons, perhaps due to the fact that the latter being a model that is closer to humans (tables 1 and 2 ). without immunosuppression ahxr occurs 4 or 5 days after the transplant of hdaf transgenic organs in baboons, 18 there being no similar results from cynomolgus monkeys. the use of an immunosuppressive regimen that includes cyclophosphamide (cyp), cyclosporin (csa) and corticosteroids (cs) increases the mean survival of the renal transplant recipient to 7.5 days in baboons and approximately 12 days in cynomolgus monkeys. 2, 10 parallel studies of orthotopic heart xenotransplants in baboons show mean survival rates of 12-15 days in similar conditions of immunosuppression. the addition of other immunosuppressants, such as rapamycin, mycophenolate mofetil or methotrexate, to the aforementioned protocol and performing splenectomy during the transplantation procedure, increased the mean survival rates for renal xenografts to up to 25-35 days in cynomolgus monkeys and to 9-20 days in baboons 16, 17 (tables 1 and 2 ). the temporary use of systemic complement inhibitors such as c1 inhibitor or soluble complement receptor 1 (scr1, tp10), was also found to be effective, both in prolonging kidney xenograft survival and to revert ahxr, once diagnosed. 19 however, the toxicity of these products in the form of increased susceptibility to infections does not allow a long-term treatment with complement inhibitors. initially it was thought that anti-gal antibodies also have an important role in ahxr, which led to the development of a series of polymers containing many gal epitopes, following the failure of repeated immunoadsorption for neutralising these antibodies. 18 the most widely tested was gas914, a trisaccharide composed of _1, 3 gal with a molecular weight of 500 kda. 20 using injections of gas914 it is possible to continuously neutralise anti-gal antibodies (fig. 2) . this also reduces the intensity the arrows indicate the days on which gas914 was injected, with black corresponding to doses of 5 mg/kg and grey to 1 mg/kg. the levels of anti-gal (circles), anti-gal igg (triangles) and porcine haemolytic (squares) antibodies are shown compared to a standard human serum, from a pool of 50 different human sera, which has been arbitrarily assigned a value of 1. of the ahxr, although without improving xenograft survival even with the addition of potent immunosuppressive treatments. 21 the presence of antibodies in ahxr despite continuous depletion of anti-gal antibodies in the blood suggested that the rejection was caused by antibodies directed against other pig epitopes. these results have later ehhq frq¿uphg zlwk wkh surgxfwlrq ri _1,3-gt "knockout" pigs, that do not express wkh dqwljhq *do . lgqh\v wudqvsodqwhg iurp wkhvh jdogh¿flhqw sljv lqwr ederrqv kdyh reached mean survival rates of 10 days under mild immunosupression, and using higher-dose immunosuppression a mean of 29 days and a maximum of 34 days. 15, 22 however these organs also suffer from ahxr mediated by antibodies other than anti-gal that cannot be avoided with immunsuppressive treatments. the best renal xenotransplantation results in baboons have been obtained using kidneys of _1,3-gt knockout pigs, in combination with protocols that use chimerism to promote graft tolerance. 22 mean survival rates of 44.8 days, with a maximum of 83 days, have been attained by transplanting the pig kidney together with vascularised thymic lobe of the same animal, previously grafted under the renal capsule, with a conditioning regimen that included the temporary depletion of complement and t cells and maintenance with anti-cd154 monoclonal antibodies, mycophenolate mofetil and corticosteroids. the results represent an improvement compared to the mean and maximum survival rates of 24.4 and 30 days, respectively, achieved with similar experiments using kidneys from hdaf-transgenic pigs. this suggested that _1,3-gt knockout pigs offer advantages with respect to those that are transgenic for complement regulatory proteins in the ahxr, possibly due to the fact that the immune response is a feature that has arisen in all the preclinical studies carried out to date is that treatments that inhibit the production of antibodies, such as high doses of cyclophosphamide or anti-cd154 monoclonal antibodies, do decrease or prevent ahxr. however, they all are very aggressive and lead to excessive immunosuppression, accompanied by severe side effects (gastrointestinal lesions, anaemia, infections, etc.) which in themselves jeopardise the life of the recipient. for this reason, the experiments carried out by the group led by dr david cooper are of great importance. these researchers performed a heterotopic heart xenograft transplantation in baboons from _1,3-gt knockout pigs, using immunosuppressive levels that were acceptable for clinical practice. 23 most of these hearts were rejected with signs of ahxr or thrombotic microangiopathy. non-etheless, they reached average and maximum survival times of 78 days and 6 months respectively, the longest survivals of porcine xenografts in nonhuman primates described to date. 23 the thrombotic microangiopathy described in these experiments is considered to be another manifestation of ahxr, closely linked to the antibody-mediated response. thus, recipients dying after long survival times due to causes not associated with rejection duh irxqg wr kdyh [hqrjudiwv zlwk plqlpdo ru qrqh[lvwhqw sdwkrorjlfdo ¿qglqjv )ru this reason, although it is not possible to completely rule out an effect of clotting incompatibilities between pigs and humans, as we will see in the following section that looks at the physiological barriers between these species, the control of the response mediated by anti-nongal antibodies has become the biggest challenge for clinical xenotransplantation. our group has preliminary data suggesting that these antibodies are not directed against porcine proteins, but rather the targets, though not the gal antigen, are also carbohydrates. although it would be reasonable to expect that the antibodies are directed against many epitopes, their characterization would open the possibility of developing new treatments to prevent or treat ahxr. given that one of the principal advantages of xenotransplantation is the possibility of modifying the donor organ, the solution for ahxr would come from genetically engineering the donor animal, to avoid the expression of those elements that cause a uncontrollable production of xenoantibodies. together with these measures intended to decrease the reactivity of anti-nongal [hqrdqwlerglhv dqg srwhqwldo forwwlqj lqfrpsdwlelolwlhv rwkhu jhqhwlf prgl¿fdwlrqv pljkw decrease the immunogenicity of pig organs. key potential targets are the immunological responses induced by the porcine cells, such as those mediated by the cd80-cd86/cd28 sdwkzd\ zklfk lv frqvhuyhg dfurvv wkh vzlqhwrkxpdq vshflhv eduulhu 6shfl¿fdoo\ cd86 expressed in porcine aortic endothelial cells provides strong costimulatory signals to human t and nk cells. 1, 24 porcine cd86, in contrast to its human counterpart, is expressed in a wide variety of cells and tissues, and the signal mediated by cd28 is resistant to immunosuppression by calcineurin inhibitors. other elements which have a potential role in therapeutics are cytokines such as tnf_ and `. the use of strategies that block the tnf may be useful in the development of xenografts resistant to ahxr, as has been demonstrated in rondent xenotransplantation models. 25 in general, the objective would be for these techniques to allow clinical xenotransplantation using the minimum possible level of immunosuppression in the recipient. it is well established that xenogeneic proteins such as porcine insulin can work correctly in humans. however, it is not clear whether xenografts are able to perform their functions in an environment other than that for which they have been genetically programmed, and, if so they are, for how long this functioning can be sustained. pig kidneys have maintained the life of nonhuman primates for several months, 16, 22 xenograft function apparently failing due to rejection rather than to the existence of physiological incompatibilities between the species. recipients of _1,3-gt knockout kidneys required continuous treatment with human albumin to maintain protein levels within the normal range, 22 as a consequence of the proteinuria produced after the transplant and that continued throughout the three months that the xenograft survived. the clinical symptoms of the proteinuria (oedema) were different depending on the immunosuppression protocol used, suggesting that proteinuria was the consequence of the xenograft rejection, and not of a mutual physiological incompatibility. furthermore, nonhuman primates transplanted with _1,3-gt knockout kidneys did not suffer from the anaemia previously described zlwk wkh lppxqrvxssuhvvlyh surwrfrov wkdw lqfoxghg f\forskrvskdplgh frq¿uplqj wkdw it had been a consequence of the treatment toxicity rather than due to the inability of porcine erythropoietin to maintain erythropoiesis. ,w kdv ehhq frq¿uphg wkdw sruflqh khduwv dqg nlgqh\v duh deoh ri pdlqwdlqlqj d vlplodu sk\vlrorj\ wr kxpdq iru orqj shulrgv ri wlph dqg wkh\ duh fdqglgdwhv iru wkh ¿uvw solid organ clinical xenotransplants. in contrast, xenografts of other organs such as the lung and liver have not survived more than a few days, though even this has demonstrated that they can maintain the life of the recipient for short periods of time. nevertheless, vrph vljql¿fdqw sk\vlrorjlfdo gliihuhqfhv fdq eh h[shfwhg ehwzhhq wkh grqru dqg wkh recipient, especially in the case of the liver due to its complex metabolic system. in the case of porcine heart and kidney, the existence of some long-term minor incompatibilities cannot be ruled out. an example of this is the clotting abnormalities described in ahxr. currently it seems that these changes may be dependent of the deposit of xenoantibodies in the xenograft, but the existence of some kind of physiological incompatibility cannot be completely ruled out. in vitro, porcine cells have an inherent tendency to clot spontaneously in human plasma, an effect that seems to be dependent on some molecular incompatibilities between porcine and human blood-clotting regulators. 26 6shfl¿fdoo\ sruflqh wkurperprgxolq d nh\ dqwlfrdjxodqw h[suhvvhg e\ endotelial cells) hardly works in the human system. 26 if clotting abnormalities after [hqrwudqvsodqwdwlrq rffxuuhg lqghshqghqwo\ ri dqwlerglhv wkh gli¿fxowlhv irxqg lq preventing and treating ahxr in procine xenotransplants to nonhuman primates would be explained. nevertheless, accumulated experience suggests that phyisology should not be an insurmountable obstacle to clinical xenotransplantation of porcine organs. also some methods are currently under investigation to solve the potential clotting incompatibilities between pigs and humans, including the genetic engineering of pig organs. if these problems were to persist once the problem of ahxr has been overcome, it is very likely that they could be also treated by the introduction of suitable human genes/proteins to the donor pig. the success of organ xenotransplantation depends on the balance between the immunosuppressive treatment necessary to avoid rejection and the risk of opportunistic infections or cancer such treatment may cause. in the case of xenotransplantation, experimental data available to date suggest that more immunosuppression is required to prevent ahxr, and, therefore, that the theoretical risk of opportunistic infections is greater in xenotransplantation than in allotransplantation. furthermore, the use of nonhuman cells, tissue or organs will increase the spectrum of opportunistic infections, since it will include diseases from the animal species used as a source of organs. the possibility that a new pathogen agent could be transferred to the recipient through xenotransplantation, and the possible passing of this to the general population, as happened with aids, is cause of concern among scientists and those responsible for public health. the terms of "xenosis" and "xenozoonosis" have been proposed to describe infections produced by microorgansims of other animal species, that do not cause infection in humans in normal circumstances, but that might be transferred from a xenograft. 27 the suredelolw\ ri d vshfl¿f plfurrujdqlvp ri dq dqlpdo vshflhv fdxvlqj d glvhdvh lq kxpdqv is unknown. in theory it may be fairly high in the cases of microorganisms that are zoonotic under normal conditions (for example, toxoplasma gondii), similar to others that cause infections in allotransplantation (such as cytomegalovirus, cmv), and capable of infecting a wide spectrum of species (such as pneumocystis carinii), as well as those microorganisms that can replicate in vitro in human cells. however, it is likely that xenosis, also known as xenozoonosis, caused by bacteria, fungi and parasites, arising iurp erwk frpprq dqg vshflhvvshfl¿f sdwkrjhqv gr qrw lpso\ d sduwlfxodu ulvn iru wkh recipient of the xenograft and even less for public health. the reason for this assertion is that this type of infections should be prevented in the animal that is to be the source of organs. 7kh surgxfwlrq ri dqlpdov lq frq¿qhg dqg lvrodwhg duhdv iurp zklfk rqo\ dqlpdov that are negative to all known pathogens are prospectively selected, can minimise the number of infections that these animals carry. accordingly, it is important that strict protocols are established for clinical and microbiological assessment, using both immunocompetent and immunodepressed animals, to enable animals with any traces of infection to be detected. this applies, be they carriers of latent microorganisms, similar to those that cause infection in allotransplants, or of pathogens of other species. would rule out any animal as potential source for a lung xenograft, or the virus coxasckie for heart xenografts. the ease of obtaining spf pigs is another of the advantages of using wklv vshflhv dv d vrxufh ri rujdqv d surfhvv wkdw orjlvwlfdoo\ zrxog eh yhu\ gli¿fxow wr carry it out with nonhuman primates. the risk of transmission of the xenosis may be further reduced by the production of pigs totally free of germs (gnotobiotic). although there are currently no facilities that allow the production of mammals in this state, their construction is totally feasible, with wkh kljk frvw ehlqj wkh prvw vljql¿fdqw glvdgydqwdjh +rzhyhu jqrwrelrwlf dqlpdov duh less robust than those which have been subject to normal microbiological colonisation, and at present they do not seem to offer any advantages over spf animals in terms of minimising the risk of transmission of xenosis. therefore, the production of gnotobiotic pigs has been put on hold until clinical experimentation demonstrates its necessity. with the production of spf animals, the risk of transmission of diseases would be limited to some pathogens capable of producing latent infections in the source animal and caused by vertically transmitted viruses that cannot be prevented in the source animal by early weaning and/or caesarean birth. all species have viruses that persist within the host cells in a latent state. while the best known are the herpes viruses and retroviruses, these also include the hepatitis viruses, adenoviruses, rabies and pseudorabies viruses, reoviruses, and papovaviruses among others. it is still not known whether nonhuman latent viruses represent an infection and disease risk for humans. in vitro, it has been possible to infect human neurons with the porcine pseudorabies virus and to transmit various viruses between species in experimental models. nevertheless, it is hard to believe that in the case of a species such as the pig, with which humans have been in contact for many thousands of years, there are many infections with capacity of being transferred from person to person that have not yet manifested themselves. on the other hand, the infection caused by nipah virus in pig slaughterhouse workers in asia, after contact with infected animals, and the recent epidemic of severe acute respiratory syndrome (sars), caused by a coronavirus, transmitted through the consumption of exotic animals in china, are good examples of the ability these zoonoses to spread. the potential re-activation of latent herpes virus infections after xenotransplantation of a porcine organ has been subject of particular attention in preclinical research on [hqrwudqvsodqwdwlrq 7kuhh khushv yluxvhv kdyh ehhq lghqwl¿hg lq vzlqh 3ruflqh cytomegalovirus (pcmv), and porcine lymphotropic herpes virus 1-and 2-(plhv-1, -2) which, in pigs that have been subject to bone marrow transplantation, are associated with a lymphoid proliferative syndrome similar to post-transplant proliferative disease (ptpd) vhhq lq doorwudqvsodqwdwlrq +hushv yluxvhv duh vshflhvvshfl¿f vr li lqihfwlrq rffxuv diwhu the xenotransplant, it should be restricted to the xenograft. the replication of pcmv has been described in porcine xenografts in nonhuman primates, causing an infection that damages porcine endothelial cells and tissues. 28 the elimination of pcmv from swine litters has been possible through early weaning of newborn animals, and the absence of pcmv in xenografts has been associated with the reduction of clotting disorders and improvement of survival rates of pig xenotransplants in nonhuman primates. activation of plhv1 has not yet been demonstrated in any solid organ xenotransplant. however, unlike pcmv, this virus cannot be eliminated from source animals by early weaning of newborns, so remains a potential pathogen in porcine organ xenotransplantation. other infections that cannot be prevented using spf pigs are those caused by vertically transmitted viruses. these include porcine endogenous retroviruses (perv) which are viruses that have been permanently integrated into the genome of the host during the evolution of mammals, and are transmitted vertically from mother to offspring. although they are not pathogens in the host, these retroviruses can be xenotropic, that is, capable of infecting other species. two perv have are known to have the capacity to infect human cells in vitro, which leads to us consider the possibility of recombination or complementation of xenograft endogenous retroviruses with viruses present in human wlvvxhv dqg wkh srwhqwldo ulvn ri lqgxfwlrq ri wxprxuv dqg lppxqrgh¿flhqflhv fdxvhg by viruses. research undertaken to date, on humans in contact with living pig tissue, workers in pig slaughterhouses, human patients who have received transplants in contact with pigs and nonhuman primates who have received pig organs and severe immunosuppressive treatment, has not shown the existence of perv replication in humans or nonhuman primates. 29, 30 however work is ongoing to characterise perv, to optimising systems for their detection, and to develop approaches to avoid or minimise the associated risks. on the one hand, certain families of miniature swine, "mini pigs", that do not transmit perv wr kxpdq fhoov kdyh ehhq lghqwl¿hg uhfhqwo\ 2q wkh rwkhu kdqg wudqvjhqlf whfkqltxhv such as sirna expression technique are being applied to inhibit the expression of perv, 31 raising the prospect that this infection may be avoided by manipulating the animals to be used as sources for organs. given all this, on the basis of the currently available information, the risk of xenosis, also known as xenozoonosis, should not be an obstacle for clinical xenotransplantation of pig organs. in contrast to the situation existing a few years ago, risks have now been lghqwl¿hg lqyhvwljdwhg dqg whvwv kdyh ehhq ghyhorshg wkdw hqdeoh wkhp wr eh dvvhvvhg lq preclinical and clinical studies. although the possibility of an infection arising from the [hqrjudiw wkdw zrxog diihfw sxeolf khdowk fdqqrw eh uxohg rxw wkh ulvn vhhpv lqvljql¿fdqw and avoidable by close supervision of the source animals and recipients of xenografts. xenotransplantation has great potential to solve the problem of the lack of human tissues and organs for transplantation and continues to be a possible alternative to allotransplantation. progress has been slow in research in this area due to technological sureohpv vxfk dv wkh gli¿fxow\ ri surgxflqj ³nqrfnrxw´ sljv dqg wkh hydoxdwlrq ri [hqr]rrqrvlv ulvnv +rzhyhu wkh nh\ wrrov kdyh qrz ehhq hvwdeolvkhg dqg vr wkh ¿hog can now develop much faster. the main barrier to its clinical application is immune rejection, especially the humoral uhvsrqvh wuljjhuhg e\ ydvfxodulvhg [hqrjudiwv 7kh lghqwl¿fdwlrq ri wkh nh\ v\vwhpv dqg molecules that are involved in the process of rejection, and the development of strategies to overcome them is just a matter of time. the use of porcine organs that have been vxemhfw wr ydulrxv jhqhwlf pdqlsxodwlrqv kdv douhdg\ vkrzq vljql¿fdqw lpsuryhphqwv lq the xenotransplantation of organs to nonhuman primates. cell transplant studies such as those of pancreatic islets in monkeys have led to even more hopeful results. then the range of possibilities offered by this technology will be unlimited, making it possible for xenotransplantation to be a clinical reality in a not very distant future. discordant organ xenotransplantation in primates life-supporting pig-to-primate xenotransplantation using genetically prgl¿hg grqruv 7udqvsodqwdwlrq xenotransplantation and the future of renal replacement xenotransplantation-how far have we come? delayed rejection of porcine cartilage is averted by transgenic expression of alpha1,2-fucosyltransferase prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection human complement regulatory proteins protect swine-to primate cardiac xenografts from humoral injury role and regulation of pig cd59 and membrane cofactor protein/ cd46 expressed on pig aortic endothelial cells life-supporting pig-to-primate xenotransplantation using genetically prgl¿hg grqruv 7udqvsodqwdwlrq expression by _ixfrv\owudqvihudvh lq wudqvjhqlf sljv prgl¿hv wkh cell surface carbohydrate phenotype and confers resistance to human serum-mediated cytolysis transgenic pigs designed to express human cd59 and h-transferase to avoid humoral xenograft rejection production of _-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning acute rejection is associated with antibodies to nongal antigens in baboons using gal-knockout pig kidneys allosensitized humans are at no greater risk of humoral rejection of gt-ko pig organs than other humans long-term survival of nonhuman primates receiving life-supporting transgenic porcine kidney xenografts pig kidney transplantation in baboons: anti-gal(alpha)1-3gal igm alone is associated with acute humoral xenograft rejection and disseminated intravascular coagulation failure to deplete anti-gal_1-3 gal antibodies after pig-to-baboon rujdq [hqrwudqvsodqwdwlrq e\ lppxqrdi¿qlw\ froxpqv frqwdlqlqj pxowlsoh *do_1-3gal oligosaccharides the effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hdaf-transgenic pig-to-primate life-supporting kidney xenografts removal of anti-galalpha 1,3 gal xenoantibodies with an injectable polymer improvement in human decay accelerating factor transgenic porcine kidney xenograft rejection with intravenous administration of gas914, a polymeric form of alphagal marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience human nk cell-mediated cytotoxicity triggered by cd86 and gal_1,3-gal lv lqklelwhg lq jhqhwlfdoo\ prgl¿hg sruflqh fhoov use of porcine tumor necrosis factor receptor 1-ig fusion protein to prolong xenograft survival disordered regulation of coagulation and platelet activation in xenotransplantation xenotransplantation and xenogeneic infections porcine cytomegalovirus and coagulopathy in pig-to-primate xenotransplantation lack of cross-species transmission of porcine endogenous retrovirus (perv) in pig-to-baboon xenotransplantation with sustained depletion of anti-_ gal antibodies lack of cross-species transmission of porcine endogenous retrovirus (perv) to transplant recipients and abattoir workers in contact with pigs production of transgenic pigs that express porcine endogenous retrovirus small interfering rnas key: cord-296441-682uop9z authors: montoya, maría; foni, emanuela; solórzano, alicia; razzuoli, elisabetta; baratelli, massimiliano; bilato, dania; córdoba, lorena; del burgo, maria angeles martín; martinez, jorge; martinez-orellana, pamela; chiapponi, chiara; perlin, david s.; del real, gustavo; amadori, massimo title: expression dynamics of innate immunity in influenza virus-infected swine date: 2017-04-21 journal: front vet sci doi: 10.3389/fvets.2017.00048 sha: doc_id: 296441 cord_uid: 682uop9z the current circulating swine influenza virus (iv) subtypes in europe (h1n1, h1n2, and h3n2) are associated with clinical outbreaks of disease. however, we showed that pigs could be susceptible to other iv strains that are able to cross the species barrier. in this work, we extended our investigations into whether different iv strains able to cross the species barrier might give rise to different innate immune responses that could be associated with pathological lesions. for this purpose, we used the same samples collected in a previous study of ours, in which healthy pigs had been infected with a h3n2 swine iv and four different h3n8 iv strains circulating in different animal species. pigs had been clinically inspected and four subjects/group were sacrificed at 3, 6, and 21 days post infection. in the present study, all groups but mock exhibited antibody responses to iv nucleoprotein protein. pulmonary lesions and high-titered viral replication were observed in pigs infected with the swine-adapted virus. interestingly, pigs infected with avian and seal h3n8 strains also showed moderate lesions and viral replication, whereas equine and canine ivs did not cause overt pathological signs, and replication was barely detectable. swine iv infection induced interferon (ifn)-alpha and interleukin-6 responses in bronchoalveolar fluids (balf) at day 3 post infection, as opposed to the other non-swine-adapted virus strains. however, ifn-alpha responses to the swine-adapted virus were not associated with an increase of the local, constitutive expression of ifn-alpha genes. remarkably, the equine strain gave rise to a serum amyloid a response in balf despite little if any replication. each virus strain could be associated with expression of cytokine genes and/or proteins after infection. these responses were observed well beyond the period of virus replication, suggesting a prolonged homeostatic imbalance of the innate immune system. the current circulating swine influenza virus (iv) subtypes in europe (h1n1, h1n2, and h3n2) are associated with clinical outbreaks of disease. however, we showed that pigs could be susceptible to other iv strains that are able to cross the species barrier. in this work, we extended our investigations into whether different iv strains able to cross the species barrier might give rise to different innate immune responses that could be associated with pathological lesions. for this purpose, we used the same samples collected in a previous study of ours, in which healthy pigs had been infected with a h3n2 swine iv and four different h3n8 iv strains circulating in different animal species. pigs had been clinically inspected and four subjects/group were sacrificed at 3, 6, and 21 days post infection. in the present study, all groups but mock exhibited antibody responses to iv nucleoprotein protein. pulmonary lesions and high-titered viral replication were observed in pigs infected with the swine-adapted virus. interestingly, pigs infected with avian and seal h3n8 strains also showed moderate lesions and viral replication, whereas equine and canine ivs did not cause overt pathological signs, and replication was barely detectable. swine iv infection induced interferon (ifn)-alpha and interleukin-6 responses in bronchoalveolar fluids (balf) at day 3 post infection, as opposed to the other nonswine-adapted virus strains. however, ifn-alpha responses to the swine-adapted virus were not associated with an increase of the local, constitutive expression of ifn-alpha genes. remarkably, the equine strain gave rise to a serum amyloid a response in balf despite little if any replication. each virus strain could be associated with expression of cytokine genes and/or proteins after infection. these responses were observed well beyond the period of virus replication, suggesting a prolonged homeostatic imbalance of the innate immune system. keywords introduction the first line of defense after viral infections is based on innate immunity, which has the capacity to respond to pathogens by sensing pathogen-associated molecular patterns (pamps) through germline-encoded pattern recognition receptors. this process leads to the production of several cytokines such as type i (α and β) interferons (ifns). these bind to the type i ifn receptor to signal the induction of hundreds of interferonstimulated genes in the local uninfected and virus-infected cells, thus creating an antiviral state that suppresses virus infection and serves to promote the onset of adaptive immunity (1, 2) . innate immune responses are crucial not only for blunting viral replication in the first instance but also for orchestrating effective adaptive immune responses. therefore, a transient induction of cytokines and chemokines is required for efficient antiviral responses. however, overreacting and prolonged immune responses may lead to undesired secondary effects, contributing to immunopathology. as for influenza virus (iv), the innate immune response may vary between mild and severe infections, and include widely different soluble innate immune inhibitors: sialic acid-based inhibitors, ca-dependent lectin inhibitors, anti-microbial peptides, complement, natural igm, ifns, to name a few (3). toll-like receptors 3 and 7 (tlr-3 and tlr-7) have been found to play an important role in influenza a virus recognition and initiation of the immune response in human respiratory epithelial cells and plasmacytoid dendritic cells (pdcs) (4). activation of both tlrs and cytoplasmic receptors leads to a potent type i ifn release and simultaneous pro-inflammatory cytokine expression. type i ifns, ifn-γ, and pro-inflammatory cytokines, such as interleukin (il)-1, il-6, il-8, il-12, and tumor necrosis factor-alpha (tnf-α), have been shown to be upregulated in lung tissue and lung lavage after experimental infection of pigs with swine iv (5-7). three iv subtypes (h1n1, h1n2, and h3n2) are currently circulating in swine herds in europe (8) (9) (10) and have been associated with disease occurrence and gross lesions in swine (11) . also, pigs are susceptible to infection with low pathogenic and highly pathogenic avian ivs (lpaiv and hpaiv, respectively) (12) . most lpaiv subtypes diagnosed in field samples possess a h1 or h3 hemagglutinin usually restricted to birds. hpaiv are able to infect pigs under natural and experimental conditions (12) . besides, it is well known that some ivs are able to infect humans and pigs, as it was the case in the last h1n1 2009 pandemic infection (13, 14) . in vitro, different iv strains can interact with porcine dendritic cells (dcs) and induce sequential waves of cytokine production that are dependent on time and virus strain (15) . this effect could account for the different immune responses generated by iv strains. on the other hand, we have previously examined the potential of h3n8 iv from canine, equine, avian, and seal origin to productively infect pigs. our results demonstrated that avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation (16) . however, no studies in vivo have addressed whether innate immune responses of pigs to swine-adapted and non-adapted iv strains might be different. the above findings outline the main working hypothesis of our study, which implies that swine iv strains might give rise to peculiar innate immune responses and time courses thereof in pigs, clearly different from those triggered by other iv strains. in order to answer these questions, samples from pigs infected with a swine (h3n2) and four different non-swine-adapted h3n8 iv strains circulating in different animal species (dogs, horses, wild aquatic birds, and seals) from our previous study (16) were analyzed and innate immune responses in the respiratory tract were thoroughly investigated. in line with the above operational framework, six groups of pigs (7-8 weeks old, landrace × pietrain, free from common pathogens) were housed in separate isolation rooms and adapted to the new environment and stockmen over a 1-week period under veterinary supervision. the animals used in our study were seronegative at that time to influenza a viruses by competition elisa (id screen ® influenza a antibody competition elisa, id-vet, france). also, they had been always healthy and thrifty before the present study. the experimental infection of pigs with different iv strains was described in our previous paper (16) . pigs were infected with the following iv strains: at day 0, each pig in groups 2, 3, 4, 5, and 6 was intratracheally infected with 1 ml of virus suspension in pbs, containing 2 × 10 5 chicken embryo infectious doses 50% (eid50) of the corresponding virus strain. animals were clinically inspected on a daily basis. rectal temperature and weight were measured at the same times. four pigs of each virus-infected group were euthanized at day 3, 6, and 21 post infection (p.i.), respectively. in the mock-infected group, two pigs were euthanized at each of the same times. blood serum samples in vacuum tubes were collected from jugular veins at day 0 and just before sacrifice at the aforementioned days. after sacrifice, bronchoalveolar fluids (balf) were collected by lung lavage with pbs, as described by busquets et al. (14) . briefly, the right lung of sacrificed pigs was used to perform a bronchoalveolar lavage (bal) using around 100 ml of pbs, and the left one was sampled for histopathological and virological studies. a complete necropsy was performed. lung lesions were classified depending on the extent of pneumonia. mild lesions were recorded when affecting small areas (<2 cm 2 ) of cranial or medial lung lobes, moderate lesions when affecting extended areas (2-5 cm 2 ) of cranial or medial lobes, and severe lesions when affecting large areas (>5 cm 2 ) of cranial, medial, diaphragmatic, and accessory lobes. histopathology samples from nasal turbinates, trachea, and lungs were collected, fixed in 10% buffered formalin and processed for histopathology, i.e., they were dehydrated through graded alcohols and embedded in paraffin. the 3-µm thick sections were cut, stained with hematoxylin-eosin and examined in a "blind-fashion" manner. in particular, we aimed to evaluate bronchiolar epithelial changes and peribronchiolar inflammation in large, medium, and small or terminal bronchioles, as well as inflammatory changes in alveoli. in the lung, bronchointerstitial pneumonia intensity was assessed by means of a semi-quantitative lesion score. the pathological scores for tracheal and pulmonary tissues were as follows: 0: no lesions, 1: mild lesions, 2: moderate lesions, 3: severe lesions. in order to check iv replication in the lower respiratory tract of pigs, balf samples collected from pigs killed at day 3, 6, and 21 were tested for gene m of the influenza a virus using a quantitative real-time pcr procedure (17) . the sensitivity of the test was equal to 10 2 tissue culture infectious doses 50% (tcid50)/25 microliters. anti-influenza a virus nucleoprotein (np) antibody levels were investigated in serum using the id screen ® influenza a antibody competition elisa (id-vet, france) following the manufacturer's instructions at days 0, 6, and 21 p.i. as previously described (14) . the threshold level was set at <50% of the od level observed in the control wells without any swine serum. the kit detects antibodies against all influenza a subtypes and antigenic variants thanks to the use of a monoclonal antibody against a highly conserved epitope of the influenza a virus np and it was used with pig serum as previously described (13, 14) to avoid background. acute phase proteins (app) of pigs (haptoglobin and serum amyloid a, saa) were investigated in balf samples by commercial colorimetric kits (haptoglobin kit, tridelta development, code tp801. multispecies saa elisa kit, tridelta development, code tp802), according to the manufacturers' directions. interferon-alpha was measured in balf samples by two different assays. first, a sandwich elisa with monoclonal antibodies (mab) f17 and k9 to porcine ifn-alpha1 was used as previously described (18) . ifn-alpha was also measured by a cytopathic effect inhibition assay on mdbk cells with vesicular stomatitis virus (19) . the test was calibrated with a preparation of porcine recombinant ifn-α1 (pbl biomedical laboratories, cat. 17100-1). the units of this preparation are determined with respect to the international reference standard for human leukocyte ifn (ga-902-530) provided by national institutes of health (bethesda, md, usa). this assay for porcine ifn-α in serum had been also validated in a previous study (20) . identification of the cytokine was performed by a neutralization assay on mdbk cells of ifn α-positive balf samples (21) , using monoclonal antibody (mab) g16 to porcine ifn-α1 (serotec, cat. mca1935z). the assay was calibrated with porcine recombinant ifn-α1. interleukin-6 and tnf-α were determined in balf samples by bioassays on 7td1 and wehi 164 cells, respectively, as previously described (22, 23) . cytokine concentrations were determined from a standard curve created with reference preparations of human recombinant il-6 and tnf-α (pierce endogen, rockford, il, usa). immunophenotyping of balf cells was carried out by flow cytometry. this was performed using indirect labeling of cells by hybridoma supernatants for cd3 (clone ppt3), cd172a (swc3) (clone 74-22-15a), and swine mhc (sla) ii (clone 1f12) (24) . the antibody against γδ tcr (clone pgbl22a) was purchased from euroveterinaria s.a. and the one against cd4 (clone 74-12-4) was purchased from serotec s.a., both conjugated with r-phycoerythrin (pe). as for the hybridoma supernatants, the concentration of antibody was empirically calculated after titration of each preparation and the secondary antibodies were either pe or apc-conjugated goat anti-mouse igg (jackson immunoresearch, suffolk, uk) used following the manufacturer's instructions. each primary antibody was compared with its relevant isotype control in each sample using the same concentrations. briefly, 2.5 × 10 5 cells/50 μl/well were labeled for 1 h at 4°c with 50 µl of each monoclonal antibody (relevant and irrelevant) at a pre-established, optimal dilution. after 1-h incubation at 4°c, cells were washed with cold pbs with 2% fcs by centrifugation at 450 × g, 4°c, 5 min. then, the secondary antibody conjugated to r-phycoerythrin diluted 1:200 was added. cells were incubated for a further 1 h at 4°c, then washed as before and resuspended in pbs with 2% fcs. stained cells were acquired using facsaria i (becton dickinson ® ) and analyzed by software facsdiva v.6.1.2. a gating strategy was applied to living cells using their forward and side scatter (fs/ss) characteristics. staining with isotype controls did not exceed 1% in all the samples. total rna was extracted from balf cells using rneasy mini kit (qiagen, milan, italy) by the qiacube system (qiagen, milan, italy) in accordance with the manufacturer's instructions. the protocol included a dnase treatment for eliminating genomic dna. rna concentration was evaluated by uv absorbance (biophotometer, eppendorf, milan). three µl of rna (10 ng/µl) were added to the reaction mix for cdna synthesis. this was performed in the presence of random hexamers as previously described (25) . then, the expression of porcine ifn-α, il-8, il-6, il-1β, and tnf-α genes was determined using the primer sets shown in table 1 . the choice of the porcine ifn-α subtypes shown in table 1 was dictated by their important role in constitutive expression of type i ifns (26) and, therefore, by their possible expression before iv infection. porcine beta2-microglobulin was used as housekeeping control gene ( table 1) to normalize the test results at different sampling times. evagreen real-time pcr amplification was performed over 40 cycles in a cfx96 real-time system (bio-rad, milan, italy) as described by razzuoli et al. (27) . in each sample, the relative expression of the selected genes was calculated using the formula δct = ct (target gene)-cycle threshold (ct) (housekeeping), where ct (cycle of threshold) values were the mean of three test replicates ±1 sd, as previously described (28) . negative samples were given a ct 40 fictitious value for further statistical examination. gene expression data (δct values) obtained on balf cells were checked for normality by the kolmogorov-smirnov test. the data sets passing the normality test were checked for significant differences between time points (days 3, 6, 21 p.i.) by one-way anova for unpaired data, whereas the others were evaluated by the non-parametric kruskal-wallis procedure (graphpad prism 5.03, graphpadsoftware, san diego, ca, usa). the threshold for significance was set at p < 0.05. statistical analyses on flow cytometry data were carried out using the sas system v.9.1.3 (sas institute inc., cary, nc, usa). for all analyses, the individual pig was used as the experimental unit. shapiro wilk's and levene tests were used to evaluate the normality of the distribution of the continuous variable and the homogeneity of variances, respectively. a statistical analysis was performed to study the association between the different variables with the different experimental groups at the different time points (0, 3, and 21 days post-infection). to analyze the association between continuous, normally distributed variables and the different experimental groups, an anova test was used at 0, 3, and 21 days post-infection. finally, a wilcoxon rank-sum test was used for the continuous, non-normally distributed variables at the different time points. no clinical signs were shown during the experimental procedure, such as fever, weight loss, anorexia, depression, nasal discharge, or altered breathing. temperature was not higher than 40.5°c in any animal at any time point. there were no traumatic lesions as a result of intratracheal virus inoculation. gross lesions were mostly shown by animals infected with the swine iv, but some lesions were also detected in lungs from pigs infected with other iv strains. pulmonary lesions ( table 2) were consistent with bronchointerstitial pneumonia at different grades of severity (0-3). the pigs infected with swine iv presented the highest level of severity followed by those infected with seal iv, avian iv, and either equine or canine iv in descending order. histopathology results are shown in figure 1 . nasal turbinates sections did not differ between iv and mock-infected animals. tracheal lesions of iv-infected pigs consisted of variable grades (0-3) of lymphoplasmacytic infiltration in the lamina propria. in the most severe cases, focal ulceration of the mucosa with fibrin deposition and neutrophilic infiltration was observed. a lymphoplasmacytic infiltration was observed in the bronchioli with extension to surrounding alveolar septa. in the most severe cases, at day 3 p.i., necrosis of the bronchiolar epithelium was observed with accumulation of necrotic cells in the bronchiolar lumen and alveolar spaces. in agreement with the gross lesion scores shown in table 2 , histopathological lesions were much more severe in pigs of the swine group at day 3 p.i. followed by the animals infected with seal or avian iv. the pigs infected with either equine or canine iv showed the least severe lesions ( table 3) . to confirm the virological data (iv titration in embryonated chicken eggs) shown in our previous paper (16) , viral replication was evaluated by quantitative real-time pcr on balf samples of animals sacrificed at days 3, 6, and 21 p.i. (figure 2) . the swine-adapted iv actively replicated in the pig respiratory tract at both day 3 and 6 p.i., as shown by ct values ranging from 21 to 33. instead, we could detect only one iv-positive balf sample in the equine group at day 3 p.i. with a ct value of 35 and none in the canine group, indicating very little replication of those viruses. on the contrary, the avian and seal virus strains showed a moderate replication at day 3 in most pigs, with ct values ranging from 33 to 37. one animal from the seal group was still iv-positive at day 6 p.i. with a ct value of 37. the above pcr data are in agreement with iv titrations in embryonated chicken eggs, reported in our previous paper (16) . total anti-np antibody levels were measured by a competition elisa kit in serum samples from all animals at the beginning of the assay (day 0), days 6, and 21 p.i. all animals were seronegative at the beginning of the experiment (day 0, figure 3) . also, no antibody response was observed in mock-infected animals. on the other hand, all iv-infected animals seroconverted (od < the threshold) by day 21 p.i. most of them seroconverted by day 6 p.i. in groups infected with swine, avian, and seal viruses, whereas the response was delayed in groups infected with the equine and canine viruses (figure 3 ). the above findings confirmed a different invasiveness in pigs of swine-adapted and non-adapted iv strains in terms of virus replication and pathological lesions. on this basis, we investigated innate immune responses during iv infection in the respiratory tract that might differ between pathogenic and non-pathogenic iv strains in pigs. protein cytokine levels were measured in all animals from all groups by different assays. firstly, levels of ifn-α in balf were analyzed by elisa. all samples were negative except those collected from animals infected with swine iv at day 3 p.i. (figure 4a) . these findings were confirmed by a bioassay for type i ifn on mdbk cells, and the specificity of the response was confirmed by an ifn-neutralization assay based on a mab to porcine ifn-alpha 1 (data not shown). there was only one pig (sw-2) with a slight tnf-α response in balf at day 3 p.i. in the swine iv-infected group. balf samples were always tnf α-negative in the other groups (data not shown). on the other hand, il-6 was clearly detected at day 3 p.i. in the balf samples of swine iv-infected pigs. in the other groups, there was no significant difference with respect to mock animals, with the exception of pig se-4 in the seal group (day 3 p.i.) ( figure 4b) . the high il-6 response in balf of the swine group at day 3 p.i. led us to investigate haptoglobin and saa in balf samples of the mock, swine, and equine groups at day 3 p.i. all the samples were haptoglobin-negative (<0.01 mg/ml). on the contrary, all the equine iv-infected pigs were saa-positive (range: 2.9-5.8 mg/ml), as also was one sample of the swine group (4.7 mg/ml). all the other samples were saa-negative (<0.01 mg/ml). the expression of some cytokine genes was investigated by quantitative real-time pcr on balf cells. results are shown in figure 5 and detailed hereunder. mock group: there was no significant difference between the different sampling times for each cytokine gene under study. swine group: there was no significant difference of gene expression at the three time points. only tendencies (p < 0.10) were shown for il-8, il-10, and ifn-gamma genes. equine group: significant differences were shown for ifna9, ifn-gamma, and il-10 genes. in particular, at day 21 p.i., an increase was observed of ifn-gamma and il-10 and a decrease of ifna9 gene expression. canine group: significant differences were shown for ifna5/6, ifna7/11, and ifn-gamma genes. at day 6 p.i., there was a downregulation of ifna5/6 and 7/11, as opposed to a significant increase of ifngamma gene expression at day 21 p.i. no other significant change of expression was detected. avian group: there were significant differences for ifna5/6, ifna13, ifna16, and ifn-gamma gene expression and tendencies for ifna7/11 and ifna9 genes. in particular, a downregulation of these genes occurred at day 6 p.i. seal group: ifna5/6 was downregulated at day 21 p.i., whereas ifn-gamma and il-10 genes were upregulated at the same time. based on the fact that local responses in balf from swine iv-infected animals were different from those of the other iv-infected pigs, we investigated whether there was any difference in cells infiltrating the lungs of infected pigs (figure 6) . therefore, cells from balf were analyzed by flow cytometry to detect changes among animals of each group. swine iv-infected animals had significantly more t cells (cd3+) at day 3 and 6 p.i. than the rest of the infected groups (p < 0.05), and the highest concentrations also corresponded to the appearance of a low concentration of the γδ t cell receptor phenotype. at day 3 p.i., γδ t cell percentages were significantly different from those of the other infected pigs (p < 0.05). also, only swine iv-infected animals showed a clear increment of pdcs (defined as cd172a + cd4 + cells) at day 6 p.i., with a statistical tendency (p = 0.06). no major changes were detected concerning the surface expression of sla-ii or cd172 in balf cells (data not shown). in this study, the pathogenicity of iv strains with different host specificity was characterized in swine in terms of clinical symptoms and tissue lesions. also, we aimed to define a possible association between innate immune responses to iv strains, and clinical and postmortem findings. the main findings of our study are outlined in table 4 . despite wide variations among individual pigs, the iv strains under study could be discriminated in terms of pathogenicity. our results confirmed a widely different replication of iv strains in the lower respiratory tract of swine: the swine-adapted virus replicated to a large extent, as opposed to the other iv strains under study. these pcr results were fully in agreement with virus titration results in our previous study (16) . interestingly, the avian and seal h3n8 strains replicated more than the equine and canine strains. replication of the canine and equine strains could have transiently taken place at days 1-2 post infection, before the first sampling. this is consistent with low-grade postmortem lesions and histopathological analyses on samples at day 3 p. i., as reported in tables 2 and 3 . these findings highlight different invasiveness of swine, seal and avian, equine, and canine viruses, in descending order (figure 2) . the replication to a higher level of swine, avian, and seal iv was associated with an earlier antibody response compared with groups equine and canine (figure 3) , where a possible low-titered replication could account for the late antibody response to np. also, we cannot rule out that defective or partially assembled particles in the viral inoculum could also have induced antibodies to np, and that different iv strains could elicit antibodies with different reactivity in the assay. please notice, however, that amino acid identity of nps with respect to swine-adapted iv was very high, ranging between 92% (canine strain) and 97% (avian and seal strains). therefore, the combination of an ab response to iv np with macro and microscopic lesions and the clear discrimination from mock-infected animals indicate that pigs can be productively infected by h3n8 viruses, which is also in agreement with the replication of both seal and avian h3n8 strain in tracheal explants (16) . most importantly, the pathogenic role of swine-adapted iv strain was confirmed by both gross and histological lesions in lungs, not induced by the other viruses under study. in turn, the presence of lesions could be temporarily associated with local innate immune responses in the lower respiratory tract (balf samples): ifn-α and il-6 ( figure 4) . these findings are in agreement with those of other experimental infections of pigs with ivs (5, 6) . moreover, previous results in our group showed that ifnα secretion was only detected in vitro when swine iv-infected myeloid porcine dcs but not when other ivs from human or avian origin were used (15) . interestingly, the ifn-α titers in bal fluids of swine iv-infected pigs were not associated with the expression of ifn-α genes, in agreement with our previous data in vitro (25) and another study in vivo (29) . but for one animal, the il-6 local response in balf samples of the swine group at day 3 p.i. was not in agreement with the presence of app. unexpectedly, saa was clearly expressed in balf samples of the equine group at day 3 p.i. this indicates that saa responses can be induced despite poor virus replication in the lower respiratory tract. also, the equine strain widely affected the expression of cytokine genes (see figure 5 ). this result suggests that innate immune responses can be triggered by some iv strains regardless of their replication efficiency. also, our findings confirm the possible extrahepatic expression of app observed in other disease models (30) . the swine iv strain also caused detectable changes of the balf-infiltrating leukocyte populations. in particular, some cd4+cd172a+ cells were observed in balf from swine iv-infected animals but not in balf from the other iv groups. these are considered pdcs in swine (31) , and their main function is the release of high levels of type i ifns. this is in agreement with the fact that ifn-α was only observed in balf samples of the swine iv-infected group (figure 4a) . ifn-α was detected at day 3 p.i., whereas the increased prevalence of pdcs was detected at day 6 p.i. this apparent discrepancy could be explained by our detection limit for pdcs in balf, as a certain minimum concentration would probably be required for detection by flow cytometry. also, we do not rule out a distinct or overlapping release of ifn-α by alveolar macrophages or other cells (e.g., epithelial cells) at an early time point after infection. despite the lesions caused by the swine iv strain, no disease signs were observed. this result is in contrast with an experimental infection of pigs with a danish h1n2 iv strain, which led to the development of typical clinical signs of influenza (29) . the absence of clinical signs was probably associated in our study with the reduced infectious challenge (2 × 10 5 eid50), which was necessary to standardize the challenge infection with iv strains showing high or poor replication in embryonated chicken eggs (16) . yet, our data are in agreement with the fact that many pigs become infected with one or more iv subtypes without showing clinical signs (32) . the gene expression data point at a crucial neglected issue: iv strains seem to cause a long-term regulation of the innate immune response, by far beyond the actual period of virus replication and persistence in organs and tissues. instead, the modulation of cytokine genes was more related to tissue damages, as indicated by the gross and hystopathological lesions persisting in some pigs until day 21 p.i. retrospectively, earlier sampling of lung tissues and/or balf samples could have provided a more clear picture of cytokine gene expression levels, which were probably in a descending phase at day 3 p.i. after a possible major peak at day 1 (29) . this is particularly true of ifn-β, which is highly up-regulated for a short time after iv infection (29) . indeed, our previous results actually showed that ifn-β gene upregulation was only detected 8 h after swine iv infection of myeloid porcine dcs in vitro (15) . this is also in agreement with its role as immediate-early type i ifn gene in non-lymphoid cells (33) . some innate immune responses were triggered by the swineadapted iv strain, only. diverse iv strain-specific factors could account for these responses, such as hemagglutinin receptor specificity and affinity, polymerase efficiency and activity of iv anti-ifn proteins, which may show additive or synergistic interactions with figure 5 | rt real-time pcr for some cytokine genes was carried out on bronchoalveolar fluids cells collected at the indicated times after infection. for each sample, the relative expression of the selected genes was calculated using the formula δct = ct (target gene)-cycle threshold (ct) (housekeeping), where ct values were the mean of three test replicates. results are shown as n-fold change in gene expression (2 −δct ). the same superscripts (a, b, c) on the bar indicate significant differences (p < 0.05) in one-way anova or kruskal-wallis test. samples from day 3 p.i. are shown as gray bars, from day 6 p.i. as striped bars, and from day 21 p.i. as black bars. the host's immune system. on the basis of our previous results on tracheal explants of pigs (16) , components other than affinity for sialic acid receptors of swine iv strains are likely to account for high innate immune responses. in particular, gross and histological lesions are probably induced following recognition of pamps of the swine-adapted iv strains and activation of the nlrp3 inflammasome (cryopyrin) (34) . such a response is probably less intense after infection with other iv strains, which can be accounted for by different causes: (a) amino acid changes of some viral pamps. (b) suppressive regulations by odns of non-swine iv strains [as shown, e.g., for porcine circovirus 2 (35) , and/or by ifn-α subtypes with anti-inflammatory control activity (36) ]. (c) third, the innate immune response to the swine-adapted iv could be simply due to the much higher and/or quicker replication of this strain. in this respect, viral replication measured by quantitative real-time pcr correlated with the degree of lesions. beyond the above hypotheses, a biological explanation of the observed results could be obtained by reverse genetics studies: changes or disruptions should be engineered in the viral genome, and effects of such alterations should be checked in vitro or in vivo. on the whole, the emerging picture outlines a host/virus relationship characterized by a strict control of the innate immune responses of pigs to non-adapted iv strains. the swine-adapted ones can circumvent these controls, in the framework of a rapid, effective, non ab-dependent containment of the primary virus infection in the lower respiratory tract. interestingly, the uncomplicated exposure to the swine iv strain did not induce clinical signs in our study. this finding points at a crucial role of the infectious pressure caused by common pathogens on farm for disease onset. this feature probably exerts additive and/or synergistic effects with common environmental stressors (animal density, seasonal changes, hierarchy fights after commingling) and genetic traits. as author contributions mm supervised the entire study. er performed real-time pcr assays for cytokine genes and data analysis thereof. mb and pm-o performed flow cytometry experiments and data analysis thereof. db, lc, and jm supervised sample collection, storage and distribution, as well as cytokine, clinical immunology, histopathology, and antibody assays. ef and cc performed realtime pcr assays for ivs and data analysis thereof. as, dp, mab, and gr provided scientific support. ma supervised clinical immunology assays, cytokine protein assays, and the manuscript writing. acknowledgments regulation of adaptive immunity by the innate immune system type i interferons in host defense the amazing innate immune response to influenza a virus infection innate antiviral responses by means of tlr7-mediated recognition of single-stranded rna correlations between lung proinflammatory cytokine levels, virus replication, and disease after swine influenza virus challenge of vaccination-immune pigs cytokines and acute phase proteins associated with acute swine influenza infection in pigs swine influenza h1n1 virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human h1n1 influenza virus seroprevalence of h1n1, h3n2 and h1n2 influenza viruses in pigs in seven european evidence of the concurrent circulation of h1n2, h1n1 and h3n2 influenza a viruses in densely populated pig areas in spain genetic characterization of influenza a viruses circulating in pigs and isolated in north-east spain during the period influenza a virus infections in swine: pathogenesis and diagnosis avian and swine influenza viruses: our current understanding of the zoonotic risk pathogenesis and transmission of the novel swine-origin influenza virus a/ h1n1 after experimental infection of pigs experimental infection with h1n1 european swine influenza virus protects pigs from an infection with the 2009 pandemic h1n1 human influenza virus swine, human or avian influenza viruses differentially activates porcine dendritic cells cytokine profile crossspecies infectivity of h3n8 influenza virus in an experimental infection in swine real time reverse transcription (rrt)-polymerase chain reaction (pcr) methods for detection of pandemic (h1n1) 2009 influenza virus and european swine influenza a virus infections in pigs a sensitive immunoassay for porcine interferon-alpha quantification of interferons by anti-viral assays and their standardization appearance of interferon-alpha in serum and signs of reduced immune function in pigs after transport and installation in a fattening farm antibodies against interferons: characterization of interferons and immunoassays isolation and characterization of biologically active murine interleukin-6 produced in escherichia coli increased levels of tumor necrosis factor and interleukin 1 in bronchoalveolar lavage fluids from pigs infected with mycoplasma hyopneumoniae characterization of five monoclonal antibodies specific for swine class ii major histocompatibility antigens and crossreactivity studies with leukocytes of domestic animals characterization of the interferon-alpha response of pigs to the weaning stress reverse transcription real-time pcr for detection of porcine interferon alpha and beta genes ipec-j2 cells as reporter system of the anti-inflammatory control actions of interferon-alpha analysis of relative gene expression data using realtime quantitative pcr and the 2(-delta delta c(t)) method expression of innate immune genes, proteins and micrornas in lung tissue of pigs infected experimentally with influenza virus (h1n2) rapid and widely disseminated acute phase protein response after experimental bacterial infection of pigs ) for the invaluable help in measuring some clinical immunology parameters, dr. l. fraile (udl, spain) for assistance in statistical analysis dendritic cells in innate and adaptive immune responses against influenza virus porcine innate and adaptative immune responses to influenza and coronavirus infections differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7 caspase-1: the inflammasome and beyond identification of a sequence from the genome of porcine circovirus type 2 with an inhibitory effect on ifn-alpha production by porcine pbmcs differential biological activities of swine interferon-alpha subtypes we would like to thank dr. jaime maldonado the research leading to these results has received funding from: the european community's seventh framework programme (fp7, 2007 (fp7, -2013 , research infrastructures action, under the grant agreement no. fp7-228393 (nadir project), and from the project agl2010-22200-c02-01 of spanish ministry of science and innovation. key: cord-332049-geh9aaf5 authors: wagner, judith; kneucker, annette; liebler-tenorio, elisabeth; fachinger, vicky; glaser, melanie; pesch, stefan; murtaugh, michael p.; reinhold, petra title: respiratory function and pulmonary lesions in pigs infected with porcine reproductive and respiratory syndrome virus date: 2010-01-20 journal: vet j doi: 10.1016/j.tvjl.2009.12.022 sha: doc_id: 332049 cord_uid: geh9aaf5 pulmonary dysfunction was evaluated in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv, isolate vr-2332) and compared to clinical and pathological findings. infected pigs developed fever, reduced appetite, respiratory distress and dullness at 9 days post-inoculation (dpi). non-invasive pulmonary function tests using impulse oscillometry and rebreathing of test gases (he, co) revealed peripheral airway obstruction, reduced lung compliance and reduced lung co-transfer factor. prrsv-induced pulmonary dysfunction was most marked at 9–18 dpi and was accompanied by a significantly increased respiratory rate and decreased tidal volume. expiration was affected more than inspiration. on histopathological examination, multifocal areas of interstitial pneumonia (more severe and extensive at 10 dpi than 21 dpi) were identified as a possible structural basis for reduced lung compliance and gas exchange disturbances. porcine reproductive and respiratory syndrome virus (prrsv), a member of the family arteriviridae, genus arterivirus, causes porcine reproductive and respiratory syndrome (prrs), an important cause of production losses in pigs (rossow, 1998) . the consequences of prrsv infection have been well documented for the reproductive system and for systemic infection (christianson et al., 1992; botner et al., 1994; mengeling et al., 1996; lager et al., 1997; kranker et al., 1998) . numerous studies have evaluated the clinical, pathological and immunological features of the respiratory form of prrs (labarque et al., 2000; samsom et al., 2000; opriessnig et al., 2002) , but the effects on pulmonary function have not been investigated, even though respiratory dysfunction may have a significant impact on the clinical outcome of the disease. typical clinical signs of respiratory infection with prrsv are tachypnoea or dyspnoea that can be accompanied by an increased respiratory effort, lethargy, fever and occasionally coughing, sneezing and chemosis (rossow et al., 1994; opriessnig et al., 2002) . gross lung lesions include failure of the lungs to collapse, as well as moderately well demarcated, mottled, brown areas of pneumonia (opriessnig et al., 2002) . microscopic lung lesions are charac-terised by type ii pneumocytic hypertrophy and hyperplasia, septal infiltration with mononuclear cells and alveolar exudates (opriessnig et al., 2002) . no data are currently available on the pathophysiological features and derangements of pulmonary function induced by prrsv. since the lungs are important for oxygen supply to all tissues, pulmonary dysfunction might have significant clinical and subclinical systemic consequences. pulmonary function testing has been applied to pigs with bacterial respiratory infections by our group (reinhold et al., , 2008 . a combination of impulse oscillometry and rebreathing of test gases can be used to evaluate lung ventilation, respiratory mechanics and pulmonary gas exchange in spontaneously breathing pigs. since both methods are non-invasive and applicable to conscious animals, changes in pulmonary function can be evaluated over time. in this study, pulmonary dysfunction was characterised in relation to clinical signs and pathological changes in pigs with experimental prrsv infection. twenty-four german hybrid pigs from a closed specific-pathogen-free herd known to be free of prrsv were transported to our institute at 24-27 days of age and were enrolled in the study after a quarantine period of 21 days. pigs were housed according to the guidelines for animal welfare and fed twice daily with a 1090-0233/$ -see front matter ó 2009 elsevier ltd. all rights reserved. doi:10.1016/j.tvjl.2009.12.022 commercial grower diet without antibiotics. water was supplied ad libitum. pigs were housed in two groups of 12 animals each, with pens of six pigs being separated from each other. virus vr-2332, the prototype type ii prrsv isolated in 1989 in minnesota, usa, has been shown to be virulent for sows and piglets (rossow et al., 1994; opriessnig et al., 2002; nielsen et al., 2003) . sixth passage vr-2332, propagated and titrated on ma-104 cells, was prepared at a concentration of 593 log 10 50% tissue culture infectious doses/ml for use as the inoculum. the study had a randomised, negatively controlled design (table 1 ) and was performed at biosafety level 2. ethical approval was obtained from the commission for the protection of animals of the state of thuringia, germany (registration number 04-001/07). twelve pigs were exposed to prrsv and 12 pigs served as controls. on the day of challenge, pigs exposed to prrsv had an age of 62.2 ± 1.0 days (mean ± standard deviation, sd) and a body weight of 22 ± 2 kg, while controls were 61.2 ± 0.7 days old and weighed 22 ± 2 kg. pigs were inoculated intranasally (1 ml per nostril) and intramuscularly (1 ml per pig) with either prrsv vr-2332 or 0.9% saline (controls). for intranasal inoculation, a 10 ml syringe was connected to a tube (40 cm â 1.7 mm inner diameter feeding tube, rüsch sterile, ref 224000, size no. 3. willy rüsch gmbh) which had been inserted approximately 3 cm into the nose. one millilitre of virus solution, along with 9 ml air, per syringe was administered into each nostril with manual pressure, followed by im injection of 1 ml into the gluteal muscle. clinical observations were recorded twice daily and included general behaviour, feed intake, appetite, rectal temperature, respiratory rate (rr) and the presence or absence of clinical signs of respiratory disease or diarrhoea. to monitor for the presence of prrsv by pcr and for seroconversion, blood samples were collected three times before challenge (à11, à5 days and à1 h) and six times after challenge (3, 7, 10, 14, 17 and 21 days post-inoculation, dpi). blood samples, nasal swabs, rectal swabs and tracheal swabs were also collected to exclude concurrent infections (table 1) . pulmonary function tests (pfts) were performed twice before challenge (à7 and à3 days) and seven times after challenge (2, 4, 6, 9, 12, 15, 18 and 21 dpi) in eight pigs exposed to prrsv and in eight controls (table 1) . body weight was deter-mined 1 day before each pft and at postmortem examination. four pigs per group (without pft) were sacrificed at 10 dpi and eight pigs per group (with pft) were sacrificed at 21 dpi for gross pathological, histopathological and immunohistochemical examination (table 1) . to quantify the prrsv load in blood, serum samples were analysed by real-time pcr for prrsv strain vr-2332 and its attenuated form, ingelvac mlv. rna was extracted using qiaamp viral rna mini kit (qiagen). reverse transcription was carried out using the multiscribe rt enzyme kit (applied biosystems). reverse transcription conditions were 25°c for 10 min, 37°c for 120 min and 85°c for 5 s. amplification was carried out in triplicate using the taqman universal pcr master kit (applied biosystems) and the prrs vr-2332-specific primers mlv f1 (5 0 -gcagctcccatctacagctgatt-3 0 ) and mlv r1 (5 0 -agacaatgtgagtca aaacgggaaagat-3 0 ). the probe was tet-5 0 -ttggctagctaacaaatttgattgggc agtggagagttt-3 0 -tamra. thermal cycling conditions were 50°c for 2 min, 95°c for 10 min, then 50 cycles of 94°c for 15 s and 60°c for 1 min. prrsv cdna quantification was achieved by comparison of the unknown sample with a standard curve derived from known amounts of plasmid dna. a commercial prrsv elisa (herdcheck prrs elisa, idexx) was used to detect anti-prrs antibodies in the blood serum of pigs before and after challenge. samples with sample-to-positive (s/p) ratio p 0.4 were considered to be positive for antibodies against prrsv, as recommended by the manufacturer. routine bacteriological culture was performed on nasal, tracheal and faecal swabs for bordetella spp., pasteurella spp., haemophilus spp., actinobacillus pleuropneumoniae (app) and salmonella spp. (table 1 ). samples were tested for mycoplasma spp. by indirect immunofluorescence and for chlamydia spp. by pcr. paired serum samples from each pig (blood collected at the beginning of the study and at postmortem examination) were used for serology (table 1) . commercial elisa test kits were used to detect antibodies against mycoplasma hyopneumoniae (dako m. hyo elisa, oxoid), app (cypress diagnostics), swine influenza virus (siv; idexx), transmissible gastroenteritis virus (tgev; svanova) and porcine table 1 study design for prrsv monitoring, additional microbiological analysis, pulmonary function testing and postmortem examination. m. hyopneumoniae, mycoplasma hyopneumoniae; app, actinobacillus pleuropneumoniae; pcv-2, porcine circovirus type 2; prcv, porcine respiratory coronavirus; siv, swine influenza virus; tgev, transmissible gastroenteritis virus; pft, pulmonary function tests (8 pigs per group examined postmortem at 21 dpi). a n = 12 pigs per group. b n = 8 pigs per group (with pft). c n = 4 pigs per group (without pft). respiratory coronavirus (prcv; svanova). testing for antibodies against porcine circovirus type 2 (pcv-2) was performed by an indirect fluorescent antibody test (fachinger et al., 2008) . approximately 15 min prior to pft, each pig was sedated with diazepam (1.5-2.0 mg/kg body weight im; faustan, weimer pharma). the sedated animal was restrained in a canvas sling with openings for the limbs and wore a tightly fitting face mask, allowing spontaneous breathing. after an adaptation period of approximately 5 min, two non-invasive lung function techniques were applied consecutively: (1) impulse oscillometry system (ios; masterscreen ios; jaeger); and (2) rebreathing system (masterscreen diffusion; jaeger). both systems were originally produced for human medicine and have been successfully applied to pigs previously (reinhold et al., , 2008 . ios was used to measure variables of respiratory mechanics based on a forced oscillation technique, as previously validated for pigs (klein and reinhold, 2001; klein et al., 2003) . externally generated test impulses given by a loudspeaker were superimposed on spontaneous airflow of the breathing animal. variations in pressure and flow signals were analysed using fast fourier transformation to calculate the complex respiratory impedance, which consists of both respiratory resistance (rrs) and respiratory reactance (xrs) (smith et al., 2005) . airflow was registered during spontaneous breathing using a lilly-type pneumotachograph with a mesh resistance of 36 pa/(l/s) and used to calculate spirometric variables of spontaneous breathing (rr and tidal volume, vt). each test per day and animal consisted of three consecutive ios measurements free of any artefacts (e.g. coughing or irregular breathing pattern). each measurement lasted for 60 s. three impulses were generated per second, leading to 180 independent results per min. the sampling rate was set at 200 hz (period between two sampling points of 5 ms), selecting 32 sampling points after each impulse. results of the three measures per pig and per time point were averaged for statistical analysis. the following variables of ventilation and respiratory mechanics were analysed: (1) rr; (2) vt; (3) volume of minute ventilation (mv); (4) both vt and mv related to body weight (vt/kg, mv/kg); (5) rrs; (6) xrs, each at 3, 5, 10 and 15 hz, and separated for inspiration and expiration at each frequency (rrs,in 3hz . . . rrs,in 15hz ; rrs,ex 3hz . . . rrs,ex 15hz ; xrs,in 3hz . . . xrs,in 15hz ; xrs,ex 3hz . . . xrs,ex 15hz ); (7) resistance of proximal airways (r prox ); and resistance of distal airways (r dist ). using different test gases and a multiple breath approach (i.e. steady state method), the rebreathing system permitted the simultaneous measurement of two additional pulmonary function variables. the functional residual capacity (frc) of the lungs was measured by the helium (he) dilution technique (washin). the transfer factor of the lungs for carbon monoxide (tl co) was determined in order to evaluate the transfer of oxygen from the lungs into the blood. for assessing both frc and tl co, each pig inhaled the test gas mixture (9% he, 0.25% co in synthetic air) from a reservoir bag. since animals were growing, the volume filled in the reservoir bag was 4 l in the pre-challenge period and was adjusted for the increasing lung volume with growth to 5 l from 2 to 18 dpi. the time to perform this test (i.e. rebreathing time) increased from 91 ± 17 s (mean ± sd) 1 week before challenge (body weight of 17 ± 1 kg, mean ± sd) to 116 ± 16 s at 18 dpi (body weight of 32 ± 2 kg) due to a significant correlation with increasing body weight (r = 0.25; r 2 = 6.28%; p = 0.003) or increasing frc (r = 0.29; r 2 = 8.26%; p = 0.0005), respectively. frc was calculated per kg body weight (frc/kg) to avoid body weight being a confounding factor. data for tl co were corrected for the individual concentration of haemoglobin in the blood (tl co hb ), as determined before each pft using a haemoxymeter that allows species-specific analysis of haemoglobin fractions for pigs (osm3, radiometer). to correct for body weight and metabolism, tl co hb was calculated per kg body weight (tl co hb /kg), as well as per kg metabolic body weight (tl co hb /kg 0.75 ). pigs were euthanased by intravenous injection of pentobarbital sodium (release, wdt) at p600 mg/10 kg body weight. the trachea was exposed, severed distal to the larynx and the lungs were instilled with neutral buffered formalin (nbf) at a pressure of 300 mm water column. after 15 min of intrathoracic fixation, the trachea was closed and the lungs were removed from the thoracic cavity and placed in nbf for 24 h. samples collected from proximal and distal parts of the cranial and caudal pulmonary lobes and from one area of the accessory lobe were embedded in low-melting paraffin (50-52°c). paraffin sections were stained with haematoxylin and eosin for histological examination. immunohistochemistry prrsv antigen was detected by immunohistochemistry using the alkaline phosphatase anti-alkaline phosphatase (apaap) method. paraffin sections collected on charged slides were pretreated with proteinase k (0.05% in phosphate buffered saline, ph 7.4) for 10 min. the monoclonal antibody sr30-a (rural technologies) was used as the primary antibody and rabbit anti-mouse ig (dianova) was used as the secondary antibody to bind the apaap complex (dako). alkaline phosphatase was visualised using neufuchsin. slides were counterstained with methylene green. positive control sections (bioscreen evdmc) were included with each reaction. statistical analyses were performed to determine the relationship between prrsv infection and clinical signs, body weight or lung function variables. for statistical evaluation of clinical signs, the study period from 6 days before inoculation until 21 dpi was separated into nine sub-periods, each of three consecutive days. within each sub-period, data obtained per day and per animal were averaged. for calculating baseline data obtained by pft, all measures per pig before inoculation were averaged. numeric data are presented as medians, minima and maxima. the mann-whitney-wilcoxon-test (w-test; comparison of medians) was used to determine significant differences between groups at p 6 0.05. for regression analyses a linear model was used (y = a + bx) and both coefficient of linear correlation (r) and coefficient of determination (r 2 ) were calculated. pcr for quantification of viral load and elisa for antibody detection demonstrated that all pigs were negative for prrsv before challenge. controls remained free of prrsv infection throughout the trial. in pigs exposed to prrsv, infection was confirmed by increasing viral loads in serum. viraemia was detected in all pigs at 3 dpi and reached a maximum 10 dpi (fig. 1a) . antibody titres against prrsv were first observed at 7 dpi in one pig and at 14 dpi in all other pigs, reaching a maximum at 17 dpi (fig. 1b) . none of the control pigs exhibited any clinical signs of respiratory disease during the study. pigs exposed to prrsv developed clinical signs initially observed at 1-3 dpi, including reduced appetite, dullness and fever. body temperature was significantly increased in challenged pigs compared to controls from 1 to 15 dpi and this increase showed a biphasic character, with maxima at 1-3 dpi and 7-9 dpi ( fig. 2a) . most prrsv challenged pigs remained dull until the end of the trial, whereas reduced appetite and/or diarrhoea was identified only sporadically from 7 to 18 dpi. the daily weight gain was 568 ± 61 g (mean ± sd) in controls and 573 ± 67 g in pigs exposed to prrsv; there was no significant difference between groups. coughing, dyspnoea and ocular discharge were detected in pigs exposed to prrsv. coughing was present from 1 to 21 dpi in 9/12 pigs and was most prominent from 13 to 15 dpi. rr increased significantly in prrsv challenged pigs compared to controls starting in the period 1 to 3 dpi and reached maximal values at 13-15 dpi (fig. 2b) . dyspnoea developed in 8/12 pigs in the period 10-21 dpi, with a maximal intensity at 16-18 dpi. one prrsv challenged pig exhibited ocular discharge in the period 7-12 dpi. interestingly, no nasal discharge and no vomiting was observed. compared to controls, vt/kg was significantly reduced in pigs exposed to prrsv after challenge (fig. 3a) . this decrease was most prominent in the period 9-18 dpi. in contrast, the volume of mv in relation to body weight (mv/kg) increased significantly in prrsv challenged pigs. compared to controls, the most marked increase in mv was seen between 6 and 15 dpi (fig. 3b) . in pigs experimentally exposed to prrsv, marked changes in respiratory impedance occurred at 9-18 dpi; the changes were more prominent during expiration than during inspiration. spectral curves of rrs and xrs within the frequency range of 3-15 hz at 12 dpi are shown in fig. 4 . rrs at 3 and 5 hz measured during expiration was significantly elevated in comparison with controls, leading to a significantly larger difference between inspiratory and expiratory resistance values. this increase in rrs,ex was statistically significant on days 12 and 18 after challenge for both rrs,ex 3hz and rrs,ex 5hz , as well as for rrs,ex 3hz alone on days 4 and 9 post-challenge (supplementary table 1 ). in contrast, rrs at 3 and 5 hz measured during inspiration was not significantly different between groups (fig. 4, supplementary table 1 ). both rrs,ex and rrs,in assessed at 10 and 15 hz were lower in pigs exposed to prrsv compared to controls (supplementary table 1 ). due to significantly increased expiratory resistance data at low frequencies (3 and 5 hz) and significantly lower rrs,ex data at 15 hz, the resistance curves during expiration became inversely frequency dependent after prrsv challenge, i.e. resistance decreased with increasing frequency (fig. 4) . xrs at all frequencies (3-15 hz) was significantly lower in prrsv infected pigs compared to controls (fig. 4) . this observed negativity of the total xrs curves was more prominent during expiration compared to inspiration, mainly from 12 to 18 days after exposure (supplementary table 2 ). proximal and distal airway resistances are shown in table 2 . after challenge, pigs exposed to prrsv had significantly higher distal airway resistance compared to controls. in contrast, r prox was significantly lower in prrsv challenged pigs than in control pigs. as shown in table 3 , volumes of frc increased in growing pigs of both groups during the study (linear correlation between body body temperature (a) and respiratory rate (b) of pigs exposed to prrsv and controls. box-and-whisker plot represents median value, 25% and 75% percentiles (box), range, outlier values (o) and extreme values (ã). days à6 to 9: n = 12 per group; days 10-21: n = 8 per group. # indicates significant differences between prrsv challenged pigs and controls (w-test, p 6 0.05). weight and frc: control pigs: r = 0.69; r 2 = 47.96%; p 6 0.00001; prrsv infected pigs: r = 0.44; r 2 = 19.05%; p 6 0.00001). neither absolute volumes of frc nor frc/kg body weight (data not shown) differed significantly between groups at any time point. in controls, tl co hb ranged from 0.43 mmol/min/kpa (median; minimum-maximum: 0.33-0.95) 7 days before challenge to 1.23 mmol/min/kpa (minimum-maximum: 0.97-3.2) at 18 dpi. due to the increase in body weight of pigs during the trial (r = 0.51; r 2 = 25.56%; p 6 0.0001) and because gas exchange in the lung physiologically adapts to metabolism, groups were compared on a basis of tl co hb data corrected for either body weight (fig. 5a ) or metabolic body weight (fig. 5b ). compared to controls, reduced gas transfer became evident in pigs challenged with prrsv from 9 dpi until the end of the study. this decrease was statistically significant for tl co hb /kg at 12 and 18 dpi and for tl co hb /kg 0.75 at 12 dpi. there were no gross changes in the lungs of prrsv infected or control pigs. systemic enlargement of lymph nodes (including the tracheobronchial lymph nodes) was evident at 10 and 21 dpi in the prrsv inoculated group. histopathological examination of the lungs of inoculated pigs revealed mild to moderate multifocal interstitial pneumonia characterised by thickening of interalveolar septa, septal infiltration with mononuclear cells, hyperplasia and hypertrophy of type ii pneumocytes and alveolar exudates of macrophages, necrotic debris and occasionally multinucleated cells (fig. 6a) . pulmonary lesions were more frequent and more severe in cranial lobes compared to caudal lobes and in the proximal part of lobes compared to the distal part. at 10 dpi, mild to moderate interstitial pneumonia was evident in 3/4 pigs. at 21 dpi, lesions were milder, but all eight pigs were affected. hyperplasia of the tracheobronchial lymph nodes was evident in 7/8 pigs at 21 dpi. in the lungs of control pigs, there were mild to moderate peribronchiolar and perivascular lymphocytic infiltrates, along with mild lymphocytic infiltrates in interalveolar septa and mild multifocal clusters of alveolar macrophages in alveolar spaces. there was no thickening of interalveolar septa (fig. 6b ). prrsv antigen was detected only in pigs inoculated with prrsv. there was a close correlation between pulmonary lesions and the presence of viral antigen. prrsv antigen was present in the cytoplasm of alveolar macrophages and occasionally in multinucleate cells within alveoli (fig. 6c) . it was also seen in mononuclear cells within thickened interalveolar septa and in type ii alveolar epithelial cells. prrsv antigen was also detected in one pig which did not develop interstitial pneumonia. at 10 dpi, the highest amount of viral antigen was found in two pigs with moderate pulmonary lesions. there was less prrsv antigen present in the lungs at 21 dpi compared to 10 dpi. all pigs were free of app, bordetella spp., mycoplasma spp., pasteurella spp., siv and tgev. chlamydia spp. were detected in rectal swabs of all pigs. all animals had antibodies against pcv-2 and prcv. haemophilus parasuis was detected in a nasal swab from 1/ 12 pigs in the control group before challenge. salmonella spp. were detected sporadically in rectal swabs before and after challenge in both groups. this study characterised respiratory dysfunction induced by the well-characterised prototypical na-type prrsv isolate vr-2332 (rossow et al., 1994; opriessnig et al., 2002; nielsen et al., 2003) . moderate clinical disease was induced that allowed consecutive pulmonary function testing until 3 weeks after challenge without any spontaneous deaths before the end of the study. this is the first simultaneous within-subject study of clinical, functional and morphological changes in pigs experimentally infected with prrsv. in the absence of other major infectious causes of respiratory disease, changes in clinical status, pulmonary dysfunction and pathology in pigs in this study can be attributed to prrsv. prrsv viraemia and seroconversion were evident in all challenged pigs. fig. 3 . tidal volume per kg body weight (vt/kg; a) and minute ventilation per kg body weight (mv/kg; b) of pigs exposed to prrsv (n = 8) and controls (n = 8) as measured with the impulse oscillometry system. box-and-whisker plot represents median value, 25% and 75% percentiles (box), range, outlier values (o) and extreme values (ã). a.i. represents data measured prior to inoculation (averaged per pig). # indicates significant differences between prrsv challenged pigs and controls (wtest, p 6 0.05). challenge with vr-2332 induced reproducible and representative respiratory disease consistent with previous studies using the same strain (rossow et al., 1994; opriessnig et al., 2002) . prrsv viraemia was detected at 3 dpi and coincided with an increase in rectal temperature. an increase in rr was evident at 7 dpi and lasted until the end of the study, i.e. until at least 21 days after infection. other clinical signs included dyspnoea and coughing and were most prominent from 15 to 18 dpi. pulmonary dysfuncfig. 4 . medians of spectral respiratory impedance within the frequency range of 3-15 hz separated for respiratory impedance during inspiration (a) and respiratory impedance during expiration (b) of pigs exposed to prrsv (n = 8) and controls (n = 8) 12 dpi. rrs, respiratory resistance; xrs, respiratory reactance, both during expiration (rrs,ex, xrs,ex, respectively) and inspiration (rrs,in, xrs,in, respectively) . # indicates significant differences between prrsv challenged pigs and controls (w-test, p 6 0.05). distal airway resistance (r dist ) and proximal airway resistance (r prox ) in pigs exposed to prrsv (n = 8) and controls (n = 8). control prrsv a.i. represents data measured prior to inoculation with prrsv (averaged per pig). # indicates significant difference between pigs exposed to prrsv and controls (w-test, p 6 0.05). functional residual capacity (frc) in pigs exposed to prrsv (n = 8) and controls (n = 8). tion was evident earlier (4 dpi) and was most prominent at 12 dpi. microscopic lung lesions were more severe at 10 dpi than at 21 dpi which is in accordance with opriessnig et al. (2002) . some authors have reported coughing as a clinical sign of prrs (done et al., 1996; beyer et al., 2000) , whereas others have attributed coughing to secondary infections (e.g. m. hyopneumoniae, b. bronchiseptica, pcv-2) (thacker et al., 1999; brockmeier et al., 2000; thacker and thanawongnuwech, 2002) . we observed a spontaneous dry cough in most of the pigs exposed to prrsv, but not in controls. due to the absence of other major respiratory pathogens in the present study and because there was no evidence of tracheitis, bronchiolitis or airway mucus on pathological examination, we interpret the observed cough as being induced by bronchospasms as a component of the respiratory illness caused by prrsv. this interpretation is supported by the presence of peripheral airway obstruction, as evaluated by pulmonary function testing. the pattern of ventilation, as indicated by spirometric data determined during spontaneous breathing, includes rr, vt and mv. in our study, rr increased significantly in pigs exposed to prrsv, with maximal values at 13-15 dpi (216% compared to baseline data). increased rr may be a response to hyperthermia, as well as an attempt to compensate for reduced vt and/or arterial oxygen deficiency caused by reduced gas transfer from the lungs into the blood. relative vt (vt/kg) was significantly reduced (<10 ml/kg) in pigs exposed to prrsv, while vt/kg was always >10 ml/kg in healthy controls. reduced vts indicate changes in the pattern of breathing caused by obstructive or restrictive disorders and/or by disorders in gas exchange. pulmonary function tests in prrsv challenged pigs indicate both obstructive and restrictive disorders (confirmed by increased rrs at frequencies 65 hz and decreased xrs), as well as disorders in gas exchange (confirmed by decreased tl co hb ). the latter correlates with the pathological findings of thickened interalveolar septa and the presence of alveolar exudates. the observed increases in mv in prrsv challenged pigs were caused by increases in rr and indicate compensation for reduced vts. pigs infected with prrsv had shorter breathing cycles and shallower inspiration. this pattern of breathing, however, is pathophysiologically linked to a higher percentage of dead space venti-lation compared to alveolar ventilation and consequently carries the risk of alveolar hypoventilation. in addition, the energy requirement for breathing increases due to increased effort of respiratory muscles. impulse oscillometry measures complex respiratory impedance, which consists of rrs and xrs (smith et al., 2005) . while rrs reflects mainly airway resistance, xrs is determined by inertive and capacitive components of the respiratory system. pressure impulses generated by a loudspeaker in a frequency range of 0-100 hz are used as test signals. low frequencies (3-5 hz) penetrate deep into the respiratory tract and provide a representation of the peripheral airway system, whereas high frequencies (10-15 hz) provide a representation of the upper and central airway systems. results at frequencies >15 hz were disregarded, since they are mainly influenced by mechanical properties of the face mask, which acts as a confounding factor for respiratory impedance measurements in animals (reinhold et al., 1998; klein et al., 2003) . in addition to the spectral curves of rrs and xrs, two model-derived resistances (i.e. r dist and r prox ) were analysed to differentiate between the effects of prrsv on either proximal airways (nasal cavities, larynx and pharynx) or distal airways (lung periphery), respectively. all variables of respiratory mechanics were significantly different in pigs challenged with prrsv compared to controls. rrs at 3-5 hz, r dist and xrs are important variables with respect to the lung periphery, including peripheral airways. rrs at frequencies 65 hz was significantly elevated in pigs exposed to prrsv and this increase was only evident during expiration. this phenomenon indicates airflow limitation predominantly in the peripheral airway system, since peripheral airway obstruction affects expiration much more than inspiration. the presence of peripheral airway obstruction is emphasised by significant increases in model-derived resistance of distal airways (r dist ). due to the absence of mucus and bronchial wall oedema, peripheral airway obstruction was most likely to have been caused by bronchospasms. morphological correlates for bronchospasms, however, are lacking, since contractions or spasms of airway muscles do not persist after euthanasia and the fixation method used in this study (intratracheal instillation with formalin) opens airways and fills alveolar regions, irrespective of the ventilation status . transfer factor of carbon monoxide corrected for haemoglobin in relation to body weight (tl co hb /kg; a) and metabolic body weight (tl co hb /kg 0.75 ; b) in pigs exposed to prrsv (n = 8) and controls (n = 8). box-and-whisker plot represents median value, 25% and 75% percentiles (box), range, outlier values (o) and extreme values (ã). a.i. represents data measured prior to inoculation (averaged per pig). # indicates significant differences between prrsv challenged pigs and controls (w-test, p 6 0.05). during normal breathing. decreases in xrs indicate reduced compliance of the lung. this might be caused by restrictive disorders due to both stiffness of obstructed airways and pulmonary tissue components. the lungs of pigs challenged with prrsv had thickened interalveolar septa and septal infiltration with mononuclear cells, which may be responsible for reduced compliance. findings with respect to central and/or upper airways are reflected by both rrs at 10-15 hz and r prox . after prrsv challenge, proximal airway resistance in exposed pigs was significantly lower compared to controls. this phenomenon might be interpreted as 'upper airway opening', an attempt to compensate for peripheral airway obstruction by increasing the diameters of upper airways (nasal cavities, larynx and pharynx). due to the functional character of this phenomenon, no morphological equivalent is visible at postmortem examination. in humans, enlargement of the aperture of the glottis is recognised during a high frequency, low vt breathing pattern (stȃnescu et al., 1972) . a further approach to enlarge the upper airway diameter is reduction of mucosal thickness by changes in blood flow. in rats, cervical sympathetic nerve stimulation induces a reduction in upper airway resistance, which is most likely induced by a-adren-ergic vasoconstriction of the upper airway mucosal vasculature, resulting in reduced mucosal thickness (o'halloran et al., 1998) . stimulation of cervical sympathetic nerves, however, can be caused by systemic hypoxia and hypercapnia (matsumoto et al., 1987) . prrsv challenged pigs showed a significantly increased partial pressure of co 2 compared to controls in venous blood gas analysis (data not shown). other factors contributing mainly to proximal airway resistance are anatomical peculiarities in the upper airways and head position. while performing pfts, head position was standardised as recommended for pigs (klein et al., 2003) . the anatomical structure of the nasal cavities, however, might contribute to different upper airway resistances between individuals and could explain significantly different rrs data p10 hz between groups even before exposure. influence of prrsv on functional residual capacity frc represents the volume present in the lung at the end of spontaneous expiration. therefore, it might be an indicator of 'trapped air', hyperinflation or emphysema. in our trial, no significant changes in frc could be identified, indicating that airway obstruction was not severe enough to induce 'air trapping' or obstructive emphysema. this is in accordance with a lack of emphysema on pathological examination in our study, as well as studies of others (pol et al., 1991; halbur et al., 1995; rossow, 1998) . in the present study, both tl co hb in relation to body weight (tl co hb /kg) and metabolic body weight (tl co hb /kg 0.75 ) revealed significant decreases in the diffusion of oxygen from the lungs into the blood in pigs exposed to prrsv compared to controls. limiting oxygen diffusion from the alveoli to the blood can be explained by increased diffusion distance due to infiltration of alveolar walls by mononuclear cells. in addition, hyperplasia and hypertrophy of the alveolar epithelial cells, as well as the presence of alveolar exudates, decreases the amount of gas within alveoli. alveolar hypoventilation due to a rapid and superficial pattern of breathing may also contribute to decreased oxygen exchange. in the pig, which has no collateral airways (mclaughlin et al., 1961) , each airway obstruction results in regional heterogeneity of alveolar ventilation (robinson, 1982) . consequently, obstruction of ventilation in pigs exposed to prrsv most likely resulted in imbalances between ventilation and perfusion, leading to reduced oxygen transfer from the lungs into the blood. this would result in respiratory acidosis characterised by reduced ph and increased partial co 2 pressure in venous blood, indicating reduced alveolar ventilation. this is the first study evaluating disorders of lung function caused by prrsv in pigs. significant imbalances in respiratory mechanics, lung ventilation and pulmonary gas exchange were correlated with clinical signs and pathological findings. prrsv isolate vr-2332 alone was capable of inducing respiratory distress for at least 3 weeks after exposure. studies using other prrsv strains would be useful to identify differences in the pathogenesis of infections caused by different prrsv isolates. evaluation of therapeutic and vaccination strategies will be supported by combining the functional approach of this study with knowledge generated from animal studies focussing on inflammatory markers and the immune response. none of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. porcine reproductive and respiratory syndrome virus (prrsv): kinetics of infection in lymphatic organs and lung isolation of porcine reproductive and respiratory syndrome (prrs) virus in a danish swine herd and experimental infection of pregnant gilts with the virus effects of intranasal inoculation of porcine reproductive and respiratory syndrome virus, bordetella bronchiseptica, or a combination of both organisms in pigs experimental reproduction of swine infertility and respiratory syndrome in pregnant sows porcine reproductive and respiratory syndrome (prrs): a review, with emphasis on pathological, virological and diagnostic aspects the effect of vaccination against porcine circovirus type 2 in pigs suffering from porcine respiratory disease complex comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus analysis of respiratory mechanics by impulse oscillometry in non-sedated and diazepam-sedated swine respiratory mechanics in conscious swine: effects of face mask, head position and bronchoconstriction evaluated by impulse oscillometry experimental inoculation of swine at various stages of gestation with a danish isolate of porcine reproductive and respiratory syndrome virus (prrsv) effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs duration of homologous porcine reproductive and respiratory syndrome virus immunity in pregnant swine a study of the subgross pulmonary anatomy in various mammals cervical preganglionic sympathetic nerve activity and chemoreflexes in the cat comparison among strains of porcine reproductive and respiratory syndrome virus for their ability to cause reproductive failure generation of an infectious clone of vr-2332, a highly virulent north american-type isolate of porcine reproductive and respiratory syndrome virus influence of cervical sympathetic nerves on ventilation and upper airway resistance in the rat comparison of molecular and biological characteristics of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine (ingelvac prrs mlv), the parent strain of the vaccine (atcc vr-2332), atcc vr2385, and two recent field isolates of prrsv pathological, ultrastructural and immunohistochemical changes by lelystad virus in experimentally induced infections of mystery swine disease (synonym: porcine epidemic abortion and respiratory syndrome (pears)) validation of impulse oscillometry in friesian and blue belgian calves with respect to changes in extrathoracic upper airway resistance evaluation of lung function in pigs either experimentally or naturally infected with chlamydiaceae an experimentally induced chlamydia suis infection in pigs results in severe lung function disorders and pulmonary inflammation some functional consequences of species differences in lung anatomy experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and 10-week-old pigs porcine reproductive and respiratory syndrome changes of leukocyte phenotype and function in the broncho-alveolar lavage fluid of pigs infected with porcine reproductive and respiratory syndrome virus: a role for cd8(+) cells forced oscillation technique and impulse oscillometry glottis opening and airway resistance mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia porcine respiratory disease complex (prdc) the authors are very grateful to annelie langenberg, sylke stahlberg, ines lemser and to all colleagues working in the team of the animal house (fli jena, germany) for their skilful assistance during the study. furthermore, the authors are thankful to colleagues of the institute of bacterial infections and zoonoses (ibiz) in the 'friedrich-loeffler-institut' (jena, germany), namely dr. ulrich methner and his team for salmonella spp. diagnosis and dr. astrid raßbach and co-workers for performing bacterial screening for pasteurella spp., bordetella spp., haemophilus spp. and app. in addition, the authors thank dr. konrad sachse and staff of the oie reference laboratory for chlamydiosis for chlamydia spp. testing and renate haß for assaying mycoplasma spp. this work was funded by the non-profit organisation 'akademie für tiergesundheit e.v.' (germany), which provided a scholarship for judith wagner. supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tvjl.2009.12.022. key: cord-002272-c7f1l13s authors: sauter, kristin a.; waddell, lindsey a.; lisowski, zofia m.; young, rachel; lefevre, lucas; davis, gemma m.; clohisey, sara m.; mcculloch, mary; magowan, elizabeth; mabbott, neil a.; summers, kim m.; hume, david a. title: macrophage colony-stimulating factor (csf1) controls monocyte production and maturation and the steady-state size of the liver in pigs date: 2016-07-21 journal: am j physiol gastrointest liver physiol doi: 10.1152/ajpgi.00116.2016 sha: doc_id: 2272 cord_uid: c7f1l13s macrophage colony-stimulating factor (csf1) is an essential growth and differentiation factor for cells of the macrophage lineage. to explore the role of csf1 in steady-state control of monocyte production and differentiation and tissue repair, we previously developed a bioactive protein with a longer half-life in circulation by fusing pig csf1 with the fc region of pig igg1a. csf1-fc administration to pigs expanded progenitor pools in the marrow and selectively increased monocyte numbers and their expression of the maturation marker cd163. there was a rapid increase in the size of the liver, and extensive proliferation of hepatocytes associated with increased macrophage infiltration. despite the large influx of macrophages, there was no evidence of liver injury and no increase in circulating liver enzymes. microarray expression profiling of livers identified increased expression of macrophage markers, i.e., cytokines such as tnf, il1, and il6 known to influence hepatocyte proliferation, alongside cell cycle genes. the analysis also revealed selective enrichment of genes associated with portal, as opposed to centrilobular regions, as seen in hepatic regeneration. combined with earlier data from the mouse, this study supports the existence of a csf1-dependent feedback loop, linking macrophages of the liver with bone marrow and blood monocytes, to mediate homeostatic control of the size of the liver. the results also provide evidence of safety and efficacy for possible clinical applications of csf1-fc. lations in mice (24) , there were a number of clinical trials of applications in cancer and other indications (23) . the interest in csf1 as a therapeutic agent has been reinvigorated by evidence of the requirement for macrophages in tissue regeneration in multiple organs (7) and the finding that macrophages generated in response to csf1 have trophic roles (9, 43) . csf1 treatment has been shown to promote regeneration and repair in injury models in the kidney (1), brain (5, 37) , and bone (6) . the pleiotropic impacts of csf1 mutations in mice (12) suggest that repair in most tissues is reliant on macrophages that depend on this growth factor. applications of csf1 therapy were constrained by the very short half-life of the 150-amino acid active form of csf1. we developed a bioactive protein with a longer half-life in the circulation. we fused pig csf1, which is equally active in all mammalian species tested (20) , with the fc region of pig igg1a (csf1-fc) (21) . the expected increase in half-life was confirmed, and csf1-fc administration to mice produced substantial increases in circulating monocyte and tissue macrophage numbers, at much lower doses than the native protein. an unexpected effect of the treatment was a substantial increase in the size of the liver, associated with extensive hepatocyte proliferation (21) . this observation was consistent with previous data implicating csf1-dependent macrophages in hepatic regeneration. acute liver failure in human patients, for example due to paracetamol toxicity, is associated with the loss of clearance functions that protect the body against the contents of the portal blood. circulating csf1 levels in human patients upon admission to hospital with paracetamol poisoning were found to be predictive of subsequent prognosis (46) . administration of csf1-fc in mouse models produced both accelerated regeneration of the liver and, perhaps more importantly, very rapid restoration of clearance functions (21, 44, 46) . pigs have been used increasingly as models of human disease (17) . the gene expression profiles of stimulated mouse and human macrophages differ greatly, and those of pig macrophages are much more human-like (29) . aside from the possible applications as human disease models, pigs are a major livestock species. early weaning in pigs, when the mucosal barrier and innate immune systems are immature, is associated with susceptibility to a very wide range of mucosal and systemic bacterial and viral infections that produce significant losses (3) . the results obtained in mice cannot necessarily be extrapolated to large animals, including humans, in which the profiles of macrophage activation by both csf1 and microbial stimuli are very different (29) . our results confirm that, as in the mouse, csf1-fc can drive hepatocyte proliferation and modulate the size of the liver in a large animal model. the data support applications of csf1-fc in liver regeneration and provide further evidence for the role of csf1 in monocyte/ macrophage maturation. in combination with earlier data, they reinforce the conclusion that circulating csf1 is a central contributor to the homeostatic control of the size of the liver. animals. approval was obtained from protocols and ethics committees of roslin institute or agri-food and biosciences institute (afbi) for the trials. the experiments were carried out under the authority of a uk home office project license, under the regulations of animals (scientific procedures) act 1986. csf1-fc was made as previously described (21) and provided by zoetis. large white pigs ϳ8.5 wk of age from one litter were used. four days prior to the first injection each pig was weighed and had blood collected into an edta tube. pigs were injected subcutaneously once a day for a total of 3 days with the appropriate volume of csf1-fc (0.75 mg/kg; n ϭ 6) or pbs vehicle (n ϭ 5). pbs injection was used to control for the possible impact of stress and restraint associated with treatment. in experiments on weaners, large white ϫ landrace pigs ϳ4 wk of age from three litters were used. six days prior to the first injection each pig was weighed and an estimated weight was extrapolated for each for the first injection day. pigs were injected intramuscularly once a day for 2 days with the appropriate volume of csf1-fc (0.75 mg/kg; n ϭ 12) or pbs (n ϭ 12). on the second injection day the pigs were weaned. all pigs were sedated with ketamine and azaperone before being euthanized by captive bolt. neither subcutaneous nor intramuscular injection produced any side effects. isolation of pbmc and bmc. blood was collected into blood collection bags containing acid citrate dextrose (acd) (sarstedt) or into beakers containing acd (sigma). the buffy coat was layered onto lymphoprep (axis-shield) and centrifuged for 25 min at 1,200 g with no brake. peripheral blood mononuclear cells (pbmc) were retrieved and red cells were removed with cell lysis buffer (biolegend). pig bone marrow cells (bmc) were obtained by flushing the bone marrow from ribs with rpmi/5 mm edta followed by removal of red cells with cell lysis buffer. all isolated cells were suspended in pbs prior to counting and cryopreservation. flow cytometry analysis. cells were washed, pelleted, resuspended in blocking buffer (pbs/2% heat inactivated fcs), transferred to a 96-well plate (v-bottom), and incubated on ice for 15-20 min. the plate was centrifuged for 4 min at 400 g followed by removal of supernatant. cells were resuspended in 100 l of pbs containing the appropriate antibody or isotype control (table 1) . samples were incubated at 4°c in the dark for 30 min before being washed two times with 200 l pbs. cells were resuspended in 600 l pbs with 0.1% sytox blue (invitrogen) immediately prior to analysis using a bd fortessa lsr flow cytometer (becton dickinson). analysis was performed using flowjo software (flowjo). complete blood count analysis. an aliquot of blood from acd blood collection bags was analyzed for complete blood cell counts. total white blood cell (wbc) was measured on the siemens advia 2120 analyzer. wbc differential counts were performed by making a blood smear counterstained with giemsa stain prior to cells of each cell type being counted. the absolute value for each wbc type was determined by using the total wbc and % leukocytes. manual platelets counts were carried out using a hemocytometer slide (by the r(d)svs clinical pathology laboratory, university of edinburgh). plasma analysis. plasma was analyzed by r(d)svs clinical pathology laboratory for cortisol (performed on siemens immulite analyzer) as well as a large animal liver damage profile (performed on the il650 analyzer from instrumentation laboratories). tissue processing. tissues were dissected, weighed (liver, spleen, and kidney), and placed in 10% neutral buffered formalin or rnalater (ambion). for histology, tissues were processed overnight using an excelsior tissue processor (thermo fisher scientific). sections were embedded in paraffin wax prior to 4-m sections cut and mounted onto slides (superfrost plus, thermo fisher scientific). slides were dried overnight at 37°c before 60°c for 25 min. sections were stained with h&e or immunohistochemistry was performed by r(d)svs pathology department. immunohistochemistry for cd163. antigen retrieval was performed with proteinase k (dako s302030) for 10 min. nonspecific protein binding was blocked using 2.5% goat serum (vector laboratories) for 20 min. endogenous peroxidase activity was blocked using dako real peroxidase blocker (dako s202386) for 10 min. sections were incubated for 60 min using mouse anti-pig cd163 (serotec mca2311ga) diluted 1/30. visualization using secondary reagent dako envision mouse hrp (dako k4007) for 40 min followed by dab (newmarket scientific monosan dab substrate kit cat. no. mon-app177) for 10 min and dab enhancer for 3 min (newmarket scientific dab concentrate cat. no. co7-25) was performed by the r(d)svs pathology department. the staining was analyzed using image j (fiji). immunohistochemistry for ki67 and pcna. antigen retrieval was performed by boiling in 10 mm sodium citrate buffer. nonspecific protein binding was blocked using 2.5% goat serum (vector laboratories) for 20 min. endogenous peroxidase activity was blocked using dako real peroxidase blocker (dako s202386) for 10 min. sections were incubated for 60 min using rabbit anti-human ki67 (abcam ab15580) diluted 1/10,000. visualization was performed with secondary reagent immpress hrp anti-rabbit igg (peroxidase polymer; vectormp-7401) for 30 min followed by dab (newmarket scientific monosan dab substrate kit cat. no. mon-app177) for 10 min and dab enhancer for 3 min (newmarket scientific dab concentrate cat. no. co7-25). statistical analysis. data were analyzed by t-tests. results are presented as treatment group means ϯ se. all analyses were performed using graphpad prism 5.0 (graphpad software). a p value ͻ 0.05 was considered statistically significant. microarray. total rna was prepared from liver samples using trizol, prepared for hybridization using the ambion wt expression kit (life technologies), following the manufacturer's instructions, except for the input amount of rna (500 ng input instead of 100 ng) and hybridized in a random order to the affymetrix porcine gene 1.1 st array (performed by edinburgh genomics, university of edinburgh). statistical analysis of the array data utilized partek genomic suite (partek). for network analysis, the normalized array data were uploaded to the software biolayout express 3d (http://www.biolayout.org/) as described previously (18, 30) . the data from the microarray are available at gene expression omnibus ncbi (http://www.ncbi.nlm.nih.gov/geo/) accession code gse78837. we first examined 8-wk-old pigs of both sexes from the same litter, with control and treated groups weight matched in pairs. based on mouse data (21) we used a dose of 0.75 mg/kg for three daily treatments followed by cull 24 h after final injection. in previous studies, we have compared csf1-fc with the native, non-fc conjugate, form of csf1 as a control protein. native csf1 has a much shorter half-life and at the same dose had no effect on monocyte-macrophage numbers (21) . we have not compared csf1-fc directly with an irrelevant fusion protein, or with an isotype control for the fc component. however, an equivalent dose of igg1 (10 -20 mg depending on the size of the pig) would have no impact on the plasma igg concentration (15-30 mg/ml). csf1-fc treatment of macrophages in vitro did not induce proinflammatory cytokines (21) and, in keeping with the lack of intrinsic proinflammatory activity, there was no evidence of any reaction at the sites of injection in any treated animals. the most obvious effect of csf1-fc administration was hepatosplenomegaly. csf1-fc doubled the spleen/body weight ratio and increased the liver/body weight ratio by 40% after only 4 days (fig. 1a ). the total wbc count was significantly increased, mainly due to lymphocytosis in addition to the expected monocytosis (fig. 1b) . csf1-fc accelerates the maturation of macrophage populations in peripheral blood monocytes. csf1 has been implicated in the maturation of blood monocytes in both mice and humans, driving the formation of the nonclassical (cd14 low , cd16 high ) subset in humans (ly6c low in mice) (31, 33) . pig blood monocytes can also be separated into subsets based on expression of surface markers, although the distinctions are not as clear as in other species (16) . expression of various markers by peripheral blood monocytes was assessed by flow cytometry staining (fig. 2 ). in addition to the increase in total wbc seen in fig. 1 , the proportion of monocytes, detected by cd172a (sirpa) was increased around twofold (fig. 2b ). the proportion of cells expressing cd16 was also increased ( fig. 2a) . no increase was seen in the percentage of cd3 ϩ lymphocytes ( fig. 2c ) the best-characterized monocyte maturation marker in pigs is the haptoglobin receptor, cd163 (16) , which has also been implicated as a receptor for the major pig viral pathogen, porcine reproductive and respiratory syndrome (prrsv) (14) , and which varies inversely with cd14 expression. double staining with the two markers indicated that csf1-fc shifted the profiles of both markers, favoring expansion of the cd163 ϩ cells (fig. 2d ). the results are consistent with an impact of csf1-fc to promote both monocyte production/ release and maturation. impact of csf1-fc treatment on the bone marrow. we next investigated whether the ability of csf1-fc to promote monocytosis was associated with expansion of progenitor pools in the marrow (fig. 3) . figure 3 , a and b, demonstrates a substantial increase in large cd14 ϩ monocytes, and even greater increase in cd163 ϩ cells in the marrow of treated animals, consistent with the pattern seen in blood. aside from monocyte progenitors, a key population of macrophages in bone marrow forms the center of hemopoietic islands. in mice, these cells express sialoadhesin (cd169, which provides a receptor for immature erythrocytes) and are critical for suc-cessful engraftment in bone marrow transplantation (10) . as shown in fig. 3c , the csf1-fc treatment produced a substantial increase in the cd169 ϩ population in pigs. the csf1-fc treatment did not expand the small percentage of cells that express cd117 (kit), a marker of the stem cell population (fig. 3d) , suggesting that csf1-fc acts primarily to promote proliferation/expansion of committed progenitors. in bone marrow of pig, neither cd172a nor cd16 provides a useful marker of monocyte lineage cells, being detected on the large majority of the cells (fig. 4, a and b) and only marginally increased by csf1-fc. we also examined the expression of the csf1r (cd115) using either a recently described monoclonal antibody (39) or labeled csf1-fc. there was some evidence of expansion of the positive cell populations in each case, but the levels of labeling were very low (fig. 4, c and d) . we suggest that the receptor may be downregulated by csf1-fc. counts. pigs (8-wk-old males and females) were injected with pbs or 0.75 mg/kg csf1-fc for 3 days prior to euthanasia on day 4. blood was collected into edta tubes postmortem and complete blood count assessment was performed. graphs show means ϯ se. **p ͻ 0.01, ***p ͻ 0.001, ****p ͻ 0.0001 by t-test; n ϭ 5-6 pigs per treatment. a: liver weight/body weight ratio, spleen weight/body weight ratio, and kidney weight/body weight ratio. b: total wbc count, lymphocyte number, neutrophil number, monocyte number, and platelet number. origin of the increase in liver and spleen weight. csf1-fc treatment caused a substantial increase in macrophage numbers in both organs, detectable by immunolocalization of cd163. in immunostained sections of liver csf1-fc treatment increased cd163 ϩ area (quantified with imagej) from an average of less than 0.5% to an average of over 9% (fig. 5a) . in spleen csf1-fc treatment had an even greater effect, causing an increase of cd163 ϩ area from an average from ϳ1% to 16% (fig. 5b ). as in mice (21) , in the spleen the majority of the increase in size could be attributed to increased red pulp macrophages and also to expansion of the marginal zones. by contrast, the increase in the area apparently occupied by macrophages is not sufficient to explain the substantial increase in the size of the liver. sections of liver were stained for the figure 6 shows images of the liver from two control and two csf1-fc-treated pigs. the pigs are relatively young, and still growing, and accordingly there is significant ongoing proliferation evident from ki67 staining. the vast majority of ki67 ϩ nuclei in both control and csf-1-fc-treated pig livers were large and round, consistent with identity as hepatocytes. macrophage nuclei are more difficult to visualize in histological sections, because the cells and nuclei are much smaller and ramified in the sinusoids. very occasional smaller ki67 ϩ nuclei visible in the sinusoids suggested that some infiltrating monocyte-macrophages were also proliferative, as shown directly in the mouse system (46) . the images in fig. 6 also show an obvious increase in cellularity in response to csf1-fc. we counted the total nuclei and the proportion stained with anti-ki67 in representative large fields from each animal. as shown in fig. 6 , csf1-fc treatment almost doubled the total number of nuclei in each field and produced a threefold increase in the percentage of those nuclei stained with anti-ki67. essentially the same findings were made with staining for proliferating cell nuclear antigen (pcna) (not shown). the sections in fig. 6 show no evidence of pathology in the liver; notably, there are no pyknotic nuclei and granulocytes are absent. granulocyte infiltration is the hallmark of tissue injury, including injury to the liver (27) . changes in the liver might occur secondary to alterations in the gut. csf1 has been attributed indirect functions in control of proliferation and differentiation of gastrointestinal epithelium (26, 45) . treatment with csf1-fc in pigs produced a small but significant increase in the mean villus length of the mid jejunum but had no detectable effect in the ileum, cecal base, or ascending colon (fig. 7) . there was also no significant change in goblet cell number. effect of csf1-fc on liver function. a panel of biochemical tests to measure serum enzymes, bile acid, bilirubin, and protein concentrations was performed to assess hepatic function. the only change was a small increase in bile acids and bilirubin in serum from csf1-fc-treated pigs (fig. 8) , only marginally outside the normal range (50) . since standard enzymic indicators of liver injury (alkaline phosphatase, alanine aminotransferase, ␥-glutamyl transpeptidase) were unchanged, the increase in bile acids probably reflects the increased size of the liver. to examine the impact of csf1-fc on liver function in more detail, we profiled the transcriptome. the expression results were filtered to remove genes detected below an arbitrary relative intensity threshold and also genes that did not differ by more than 1.5-fold between the highest and lowest value in the nine samples. the second criterion removed around 30% of probes on the microarray, including many hepatocyte-specific gene products. figure 9c shows that the relative abundance of representative examples of these known hepatocyte gene products, albumin (alb), cd14, fetuin, and transferrin (tf), within the total liver rna pool was unchanged in response to csf1-fc. in other words, the infiltration of the liver by macrophages was insufficient to dilute the contribution of hepatocyte mrna to the total mrna pool. that finding is consistent with the histological observation above, that even in the csf1-fc-stimulated state the infiltrating macrophages appear to make up no more than 10% of the total area of the liver. hence, the 40% increase in total liver weight can be attributed primarily to an increase in using imagej software the total cd163 ϩ area was calculated from 2 representative images per pig per organ. graphs show means ϯ se. *p ͻ 0.05, **p ͻ 0.01 by t-test; n ϭ 4 -5 pigs per treatment. g539 csf1 controls the steady-state size of the liver hepatocytes, consistent with the extensive proliferation and increased cellularity shown in fig. 6 . we clustered the included probe sets based on expression pattern and displayed them using biolayout express 3d . the advantage of using the clustering approach is that genes that might appear regulated, but in only a subset of animals, appeared in separate smaller clusters. these may reflect the interanimal variation in macrophage-inducible gene expression that we documented previously in a study of pig breeds (30) . the gene lists of specific clusters are provided in supplemental table s1 . (supplemental material for this article is available online at the journal website.) functional annotations of the two large clusters were tested using david (supplemental table s2 ). cluster 1, the set of genes elevated in all csf1-fctreated pigs, was clearly enriched for genes involved in the cell cycle and innate immunity, whereas cluster 2, the set that was reduced in the csf1-fc-treated pigs, was enriched in genes involved in metabolism. importantly, there is no evidence among the induced genes of expression of apoptosis-associated genes, no induction of acute phase genes, and no appearance of classical granulocyte marker genes such as s100a8/s100a9 or mpo. figure 9 shows the expression profiles of a number of genes that highlight the biological processes involved. cluster 1 (fig. 9a ) includes many genes that were shown previously (18) to be restricted to macrophages, such as the transcription factor spi1; surface markers sirpa, emr1, and itgam; known csf1-inducible genes plau, mmp9, chi3l1, and c1q; endocytic receptors marco, msr1, and fcgr1a; and pattern recognition receptors tlr1, 2, 4, 6, 7, 8, and 9 . on average, the relative contribution of cluster 1, macrophagespecific genes, to the liver total rna increased by three-to fivefold in response to csf1-fc, again consistent with the fig. 5 . in mice, the recruited monocytes express the chemokine receptor ccr2 and apparently respond to ccl2 (49) . by contrast, ccr1 was enriched in the liver mrna of treated pigs, alongside three of its known ligands, ccl8, ccl14, and ccl3l1. as previously observed in mice (46) , monocytes recruited to the treated livers apparently responded to proinflammatory signals, since cluster 1 contained numerous known lps-inducible genes (29) such as inflammatory cytokines tnf, il1a, and il1b; interferon targets ido1; multiple type 1 interferon targets irf1, irf5, ifitm2, and ifit3; tgfb1; and costimulators of t cell activation cd40, cd80, and cd86. cluster 1 also contains numerous cell cycle-associated genes, including pcna; the key transcription factors foxm1, e2f4, e2f7, and e2f8; and several cyclin genes ccna2, ccna3, ccnb2, ccnb3, ccnd2, and ccnd3. the increased expression of enzymes of glycolysis hk1, hk2, hk3 pfkp, pgk1, pgd, pkm, gpi, gapdh, and ldha also reflects the requirement for aerobic glycolysis in proliferating cells (25) . cluster 2 (fig. 9b) , the set of genes reduced in the csf1-fc-treated pigs, most likely reflects the functional zonation of the liver between periportal and perivenous regions of liver lobules (8, 19, 48) and the selective proliferation of cells derived from portal progenitors that has been observed in regenerating liver (15, 34, 36) . it includes genes involved in xenobiotic metabolism and detoxification, notably p450 family (e.g., cyp1a1, cyp2e1), glutathione s-transferases (e.g., gstaa2) and aldo-ketoreductases (e.g., akr1c1), and the gluconeogenic enzyme pck2, that are known to be enriched in perivenous locations. the cluster contains the gene for the regulator of hepatocyte stem cells, sox9 (2), indicating that these cells are not expanded in the csf1-fc-treated livers. unexpected members of this cluster are genes for the growth hormone receptor (ghr) and the target, igf1, and both estrogen (esr1) and androgen (ar) receptors. also unexpected is the inclusion of the receptor for hepatocyte growth factor, met, which is implicated in regeneration (36), but this might reflect autoregulation in response to its ligand (52) . although models of acute liver failure in pigs have been described (32, 41) , and may be one path to clinical development of csf1-fc as a treatment, it is challenging to perform sufficient replicates to test a clinical intervention. we therefore considered an alternative in production pigs. commercial pigs are normally weaned at 4 wk, when the gut is immature. diarrhea and disseminated infections with organisms such as escherichia coli and streptococcus suis are relatively common (38) . in this respect, the pig has been studied as a model of early-life stress (42) . the biology of early weaning in pigs may also be relevant to intestinal failure-associated liver disease in neonates and children (40) . weaner pigs were treated with a higher dose of 1.0 mg/kg csf1-fc for two daily injections, immediately prior to weaning and on the day of weaning followed by euthanasia 24 h following the final injection. at this early time point, there was already a significant increase in the spleen/body weight ratio and a trend toward increased liver/body weight ratio (fig. 10a ). the number of cd163 ϩ cells was more than tripled in the bone marrow, from ϳ10% to over 30% (fig. 10b) , and increased numbers of cd163 ϩ macrophages were confirmed by immunostaining in liver and spleen (fig. 10c ). at this time point, there was not a significant monocytosis, indicating that both marrow expansion and tissue macrophage proliferation precede monocyte expansion and are likely direct effects of csf1-fc. we repeated the treatment in a larger cohort of weaned pigs. this study was conducted in a high health status research unit, which reflected commercial practice. we explicitly removed zinc from the feed, which is usually added to reduce weaningassociated infections. given the production of inflammatory cytokines and reduction in igf-1 in the liver of treated pigs, we measured weight gain daily in all animals. csf1-fc (0.75 mg/kg) was administered to pigs for two daily intramuscular injections on the day before and the day of weaning, and pigs were killed 5 days after the second injection. although some pigs showed evidence of mild postweaning diarrhea, all the animals in both groups continued to gain weight rapidly (fig. 11a) . the treated pigs, like the treated mice left for longer after the final injection, demonstrated hepatosplenomegaly (fig. 11b) , and the increased numbers of cd163 ϩ macrophages in the liver remained evident after 5 days (fig. 11c ). in this study we have extended previous studies in mice (21) to examine the impact of a sustained increase in csf1 activity on monocyte-macrophage homeostasis. all of the impacts we have observed are consistent with the known biological activity of csf1. in mice, the same impacts on monocyte-macrophage numbers and maturation can be generated by injection of very much higher doses of native csf1 (21) or injection of a much larger native form of human csf1 (24) . the doses of native csf1 required are prohibitive in a large animal. although we cannot entirely eliminate other functional contributions of the fc component, the increase in circulating half-life is the most obvious explanation for the increased efficacy compared with native csf1. the nature of the so-called hepatostat, which determines that the liver returns to a size that is strictly proportional to body size, has continued to be something of a mystery (34, 35) . although there are many candidates, including growth factors and inhibitors, extracellular matrix proteins and metabolites, and circulating hormones that can regulate hepatic regeneration, it is unclear how any of them functions as a sensor. in a previous study, we made the striking observation that csf1 treatment of mice (using an fc conjugate with an increased circulating half-life) was able to increase the size of the liver as well as the number of kupffer cells. this ability is quite unique. in the present study, we have extended the finding to the domestic pig, an animal that is considerably more humanlike in size and vascular biology. the data in fig. 6 show that a major impact of csf1-fc treatment in pigs is to increase the number of hepatocytes through extensive proliferation, so that the total cellularity of the liver is increased even more than the increase in total liver weight. hepatocyte proliferation, as opposed to hypertrophy, is also a feature of liver regeneration in response to partial hepatectomy (34 -36) . we have made the reciprocal observation in mice; namely, that prolonged depletion of kupffer cells with anti-csf1r treatment leads to a reduction in the size of the liver (45) . others have shown that liver regeneration is greatly impaired in csf1-deficient or anti-csf1r-treated mice (46) and in mice depleted of blood monocytes (15) and have promoted liver repair by infusing csf1-stimulated macrophages into the portal vein (47) . the impact of csf1-fc on hepatic growth in mice was dependent on monocyte recruitment, as evident from the impact of knockout of ccr2 (46) . the role of monocyte-macrophage products, including the inflammatory cytokines tnf, il1, and il6, in hepatocyte proliferation has been well recognized (22, 46) . csf1-fc action in mice was partly dependent on il6 (46) , which was also induced in all of the csf1-fc-treated pigs. the effect of csf1-fc treatment demonstrates that csf1-dependent monocyte recruitment is both necessary and sufficient to drive hepatic proliferation and can drive it beyond the homeostatic limits even in a large animal. the effect of csf1 treatment supports other evidence of the existence of a homeostatic feedback loop. macrophages, notably those of the liver (4) and blood monocytes (51) , together regulate the level of circulating csf1 via receptor-mediated endocytosis. this mechanism is evident from the massive increase in circulating csf1 seen in animals treated with anti-csf1r (33), and the importance of the liver is evident in patients following partial resection (46) . the csf1-fc treatment reveals that elevated csf1 can provoke expansion of the committed monocyte pool in the marrow (fig. 3) , maturation of the monocytes toward a resident phenotype (fig. 2) , and infiltration of the liver (fig. 5) . hence, the physiological hepatostat (34 -36) may actually be a "macrostat." of course, the increased macrophage numbers in the liver elicited by csf1 produce secondary impacts, not only removing potential toxins from the portal blood, but altering the balance of metabolism in the liver between portal and venous-associated functions. interestingly, resident kupffer cells are selectively located toward the portal vein in mouse liver lobules (13) , which might also serve to localize macrophage-derived hepatocyte proliferative signals. the expression profiling of the livers of the csf1-fc-treated pigs (fig. 9 ) closely parallels results obtained previously in the mouse system (21) . the newly recruited monocytes clearly respond to tlr-mediated and other signals to express the large majority of transcripts seen when csf1-stimulated bone marfig. 10 . effect of a short course of csf1-fc in weaning pigs. pigs (4-wk-old males and females) were injected with pbs or 1 mg/kg csf1-fc for 2 days prior to euthanasia on day 3. graphs show means ϯ se. *p ͻ 0.05, **p ͻ 0.01, ***p ͻ 0.001 by t-test; n ϭ 5 pigs per treatment. a: blood was collected into edta tubes postmortem and complete blood count assessment was performed. graphs show liver weight/body weight ratio, spleen weight/body weight ratio, and monocyte number. b: bm from ribs was collected as described in materials and methods. bm cells were analyzed via flow cytometry for expression of cd163 with exclusion of dead cells using sytox blue. representative flow cytometry plots are shown. c: formalin-fixed liver and spleen tissue was prepared and stained for cd163. representative images are shown. fig. 11 . long-lasting effect of csf1-fc in weaning pigs. pigs (4-wk-old males and females) were injected with pbs or 0.75 mg/kg csf1-fc for 2 days prior to euthanasia 5 days after the final injection; n ϭ 12 pigs per treatment. a: body weight was recorded at each of the time points shown and total body weight change over the duration of the experiment was graphed for pbs treated pigs (black) and csf1-fc-treated pigs (red). b: bar graphs show means ϯ se. ***p ͻ 0.001, ****p ͻ 0.0001 by t-test. graphs show liver weight/body weight ratio, spleen weight/body weight ratio, and kidney weight/body weight ratio. c: formalin-fixed liver tissue was prepared and stained for cd163. representative images are shown. scale bar ϭ 10 m. csf1 controls the steady-state size of the liver row-derived macrophages respond to lps (29) . however, classical neutrophil chemoattractants such as il8 were not detected; we found no evidence of neutrophil infiltration, no induction of classical acute phase response or apoptosis genes, and no evidence of damage to liver cells. furthermore, the pigs showed no adverse impacts, and in weaners the treatment did not impair their rapid growth (fig. 11) . this is consistent with earlier data, in which recombinant csf1 has previously been administered in phase 1 clinical trials by continuous infusion to humans and was well-tolerated (11, 28) , and indicates that the fc fusion protein does not produce any additional toxicity. the lack of severe consequences may be attributed in part to induction of the anti-inflammatory cytokines il10 and tgfb1 in the liver of the treated animals. whatever the mechanism, the outcome suggests that csf1-fc specifically promotes a proregenerative cellular response in liver, as it does in other organs (23) . in the final set of experiments (fig. 11) , we progressed toward the clinical application of csf1-fc in a pig model. the model may also reflect a practical application in pig production to bolster the innate immune system at weaning. we showed that only two doses, administered intramuscularly, were sufficient to produce a sustained increase in monocyte count, liver size, and liver macrophage numbers and produced no adverse local reaction. we propose that csf1-fc could provide protection against disseminated infections arising from the immature gut of early-weaned animals. similarly, clearance functions of the macrophages of the liver are crucial to prevent sepsis in acute liver failure in humans, and csf1-fc rapidly promoted clearance functions in mouse disease models (46) . the fact that administration of csf1-fc to pigs increased the size of the liver and the liver macrophage population (i.e., the clearance capacity, noting also the increased expression of clearance receptors in the array profiles) has an obvious relevance to human acute liver failure. accordingly, we suggest that the fundamental understanding of the central role of csf1 in liver homeostasis can potentially translate into both human clinical and veterinary applications. colony-stimulating factor-1 promotes kidney growth and repair via alteration of macrophage responses hepatocyte turnover and regeneration: virtually a virtuoso performance the development of the mucosal immune system pre-and post-weaning: balancing regulatory and effector function macrophages specifically regulate the concentration of their own growth factor in the circulation powerful beneficial effects of macrophage colony-stimulating factor on beta-amyloid deposition and cognitive impairment in alzheimer's disease m-csf neutralization and egr-1 deficiency prevent ovariectomy-induced bone loss macrophages: supportive cells for tissue repair and regeneration lack of ccr2 results in increased mortality and impaired leukocyte activation and trafficking following infection of the central nervous system with a neurotropic coronavirus colony-stimulating factor-1 in immunity and inflammation cd169(ϩ) macrophages provide a niche promoting erythropoiesis under homeostasis and stress phase i trial of recombinant human macrophage colonystimulating factor administered by continuous intravenous infusion in patients with metastatic cancer targeted disruption of the mouse colonystimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects immunohistochemical analysis of the involvement of f4/80 and ia-positive macrophages in mouse liver infected with lymphocytic choriomeningitis virus effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) kupffer cell-monocyte communication is essential for initiating murine liver progenitor cell-mediated liver regeneration comparative analysis of monocyte subsets in the pig the mononuclear phagocyte system of the pig as a model for understanding human innate immunity and disease a gene expression atlas of the domestic pig t-cell factor 4 and beta-catenin chromatin occupancies pattern zonal liver metabolism in mice cloning and expression of porcine colony stimulating factor-1 (csf-1) and colony stimulating factor-1 receptor (csf-1r) and analysis of the species specificity of stimulation by csf-1 and interleukin 34 characterisation of a novel fc conjugate of macrophage colony-stimulating factor immunology in the liver -from homeostasis to disease therapeutic applications of macrophage colony-stimulating factor-1 (csf-1) and antagonists of csf-1 receptor (csf-1r) signaling the effect of human recombinant macrophage colony-stimulating factor (csf-1) on the murine mononuclear phagocyte system in vivo role and regulation of glucose metabolism in proliferating cells csf-1 receptor-dependent colon development, homeostasis and inflammatory stress response mechanisms of liver injury. ii. mechanisms of neutrophilinduced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions phase i study of continuous-infusion recombinant macrophage colony-stimulating factor in patients with metastatic melanoma pig bone marrow-derived macrophages resemble human macrophages in their response to bacterial lipopolysaccharide the impact of breed and tissue compartment on the response of pig macrophages to lipopolysaccharide monoclonal antibodies against macrophage colony-stimulating factor diminish the number of circulating intermediate and nonclassical (cd14(ϩϩ)cd16(ϩ)/cd14(ϩ)cd16(ϩϩ)) monocytes in rheumatoid arthritis patient a reproducible, clinically relevant, intensively managed, pig model of acute liver failure for testing of therapies aimed to prolong survival an antibody against the colony-stimulating factor 1 receptor depletes the resident subset of monocytes and tissue-and tumor-associated macrophages but does not inhibit inflammation advances in liver regeneration liver regeneration after partial hepatectomy: critical analysis of mechanistic dilemmas principles of liver regeneration and growth homeostasis macrophage colony stimulating factor promotes phagocytosis by murine microglia stress signaling pathways activated by weaning mediate intestinal dysfunction in the pig development and characterisation of monoclonal antibodies reactive with porcine csf1r (cd115) the prevention and treatment of intestinal failure-associated liver disease in neonates and children development of an invasively monitored porcine model of acetaminophen-induced acute liver failure early-life stress origins of gastrointestinal disease: animal models, intestinal pathophysiology, and translational implications trophic macrophages in development and disease the macblue binary transgene (csf1r-gal4vp16/uas-ecfp) provides a novel marker for visualisation of subsets of monocytes, macrophages and dendritic cells and responsiveness to csf1 administration pleiotropic effects of extended blockade of csf1r signaling in adult mice csf1 restores innate immunity after liver injury in mice and serum levels indicate outcomes of patients with acute liver failure macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function molecular determinants of liver zonation critical roles for ccr2 and mcp-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites serum total bile acids monitoring after experimental orthotopic liver transplantation fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis regulation of the met oncogene: molecular mechanisms we thank the staff at the biological resource facility and dryden farm (at the roslin institute) and staff at the afbi-ni for their assistance in planning and executing the animal studies. we thank colleagues at zoetis for providing the csf1-fc, specifically the authors listed in our previous publication (21) . we also show our gratitude to iveta gazova, anna raper, and jun hu for helping to process samples and deliver them to their appropriate locations.present address for g. m. davis: the university of manchester, oxford road, manchester m13 9pl, united kingdom. key: cord-351834-9pclxek0 authors: cohen, liza miriam; grøntvedt, carl andreas; klem, thea b.; gulliksen, stine margrethe; ranheim, birgit; nielsen, jens peter; valheim, mette; kielland, camilla title: a descriptive study of acute outbreaks of respiratory disease in norwegian fattening pig herds date: 2020-06-24 journal: acta vet scand doi: 10.1186/s13028-020-00529-z sha: doc_id: 351834 cord_uid: 9pclxek0 background: respiratory diseases are major health concerns in the pig production sector worldwide, contributing adversely to morbidity and mortality. over the past years there was a rise in reported incidents of respiratory disease in pigs in norway, despite population wide freedom from aujeszky´s disease, porcine reproductive and respiratory syndrome, porcine respiratory corona virus and enzootic pneumonia. the main objective of this study was to investigate acute outbreaks of respiratory disease in conventional norwegian fattening pig herds. the study included 14 herds. in seven herds with reported outbreaks of acute respiratory disease, data on clinical signs was recorded and samples for laboratory examination were collected. diagnostic protocols were compared by parallel analysis of clinically healthy pigs from seven non-outbreak herds. results: the most commonly reported clinical signs were sudden deaths and dyspnea. an average compartment morbidity of 60%, mortality of 4% and case fatality of 9% was recorded in the outbreak herds. post-mortem examinations revealed acute lesions resembling porcine pleuropneumonia in all 28 pigs investigated from the outbreak herds and in 2 of the 24 (8%) pigs from the non-outbreak herds. chronic lesions were recorded in another 2 pigs (8%) from the non-outbreak herds. actinobacillus pleuropneumoniae serovar 8 was isolated from lungs and/or pleura from all tested pigs (n = 28) in the outbreak herds, and from 2 out of 24 pigs (8%) in the non-outbreak herds, one pig with an acute and another pig with a chronic infection. no other significant bacterial findings were made. seroconversion to a. pleuropneumoniae antibodies was detectable in all outbreak herds analyzed and in six out of seven non-outbreak herds, but the risk ratio for seroconversion of individual pigs was higher (risk ratio 2.3 [1.503.43 95% ci; p < 0.001]) in the outbreak herds. all herds tested positive for porcine circovirus type 2 and negative for influenza a viruses on oral fluid rt-qpcr. conclusion: the main etiological pathogen found during acute outbreaks of respiratory disease was a. pleuropneumoniae serovar 8. all pigs from outbreak herds had typical lesions of acute porcine pleuropneumonia, and only a. pleuropneumoniae serovar 8 was identified. co-infections were not found to impact disease development. are however hard to determine in field conditions. studies show that coinfections with different respiratory agents are common in pigs [4, 5] . viral infections often predispose for secondary bacterial infections. this has been studied under experimental conditions, i.e. coinfections of porcine reproductive and respiratory syndrome virus (prrsv) and mycoplasma hyopneumoniae [6] , prrsv and actinobacillus pleuropneumoniae [7] , swine influenza virus (siv) and bordetella bronchiseptica [8] . moderate to marked fever, lethargy, coughing, sneezing and dyspnea are common clinical signs during disease outbreaks [9, 10] . the presence of multiple pathogens often increases the severity of disease and occurrence of lesions in the respiratory tract [8, 11, 12] . there are differences in occurrence and distribution of pathogens between countries, regions and herds [13, 14] that contribute to the complexity of respiratory disease. due to strict import regulations in norway, there is negligible import of live pigs to the commercial pig population [15] . the national yearly yield was approximately 1.6 million slaughtered pigs in 2018, originating mainly from 2580 registered fattener pig herds with a concession limit of maximum 2100 slaughtered pigs per year [16, 17] . the norwegian pig production is also characterized by stringent regulation of antimicrobial drug use and a tradition of eradicating diseases from animal populations [18, 19] . the commercial pig population in norway has documented freedom from several important respiratory pathogens including aujeszky's disease virus, prrsv, siv (apart from influenza a [h1n1]pdm09) [20] and m. hyopneumoniae [18] . after the pandemic in 2009/2010, antibodies to siv (h1n1)pdm09 have been detected regularly from 25 to 50% of examined herds in norway [21] , but siv (h1n1)pdm09 infections in the norwegian pig population has been considered to have limited clinical impact [22] . in cases of respiratory disease in norwegian herds, a. pleuropneumoniae has regularly been isolated from lungs of carcasses submitted for routine diagnostics [23] . several studies from other countries conclude that a. pleuropneumoniae is normally present in most conventional pig herds, having a main reservoir in the tonsils of carrier pigs [24, 25] . accordingly, outbreaks in conventional herds are most often triggered by factors related to animal housing, management and environment rather than an introduction of the bacteria in a naïve herd [26] . preceding infection with a primary viral pathogen is also a possible triggering factor [4] . in the years between 2010 and 2014 there was an increase in reported acute cases of respiratory disease requiring veterinary treatment in norway [27] . a systematic investigation of porcine respiratory disease outbreaks in norway has not recently been performed, and updated knowledge is needed for appropriate disease prevention and intervention. the main objective of this study was to investigate clinical outbreaks of acute respiratory disease in norwegian fattening pig herds, using a group of non-outbreak herds to compare diagnostic procedures. the source population was the conventional fattening pig herds located in central and southern parts of norway in the period between september 2017 and october 2018. the conventional herds are not part of the norwegian specific pathogen free (spf) sub-population, in which herds are free from e.g. toxin producing pasteurella multocida and all serotypes of a. pleuropneumoniae. seven conventional fattening pig herds with acute outbreaks of respiratory disease (outbreak herds) and seven pig herds without respiratory disease outbreaks (nonoutbreak herds) were included in this study. the inclusion criteria for outbreak herds were; three or more pigs displaying acute signs of respiratory disease including fever and coughing and/or dyspnea, and/ or otherwise reduced general condition e.g. lethargy or inappetence. non-outbreak herds inclusion criteria were; absence of acute clinical signs of respiratory disease at the time of sampling, situated in the same geographical area as the outbreak herds. the non-outbreak herds were not matched to the outbreak herds by means of other parameters. herds were included only if there were more than three weeks until planned slaughter, due to follow-up sampling per protocol. two herds were excluded, due to treatment with antimicrobial drugs before sampling could be carried out, and insufficient time from outbreak to planned slaughter, respectively. descriptive herd data are listed in table 1 . a network of veterinary practitioners was established to collect samples and herd data. the practitioners were contacted through emails, letters, meetings and announcements in relevant journals and national newspapers. the veterinarians contacted the project group immediately upon being called out to examine pigs with symptoms of acute respiratory disease. outbreak herds were recruited for participation by the veterinary practitioners after meeting the inclusion criteria. non-outbreak herds were then recruited by the veterinary practitioners contacting herd owners meeting the matching criteria, asking their participation and arranging a visit. complete kits containing materials and detailed instructions for sample collection, preservation and transport were pre-distributed to designated pick up points at abattoirs and veterinary practice offices and sent to veterinarians across the country upon request. each outbreak herd was visited on three occasions ( fig. 1, green boxes) ; the first visit was conducted as soon as possible during the reported outbreak for initial sampling. the second visit was performed 2 to 5 days later to conduct interviews and register herd demographic data. during the third and final visit two to four weeks after the first, follow-up samples were collected, as described in fig. 1 . non-outbreak herds were visited on two occasions, once for initial sampling, farmer interviews and herd registrations, and secondly for follow up sampling. details about the diagnostic sampling are shown in fig. 1 . diagnostic sampling in outbreak herds was performed the day the veterinarian was notified about the disease. the veterinary practitioner reported observed clinical signs on a standardized submission form. in these herds, three to five pigs were selected for organ collection, 28 pigs were sampled in total. the selection was made from pigs with clinical signs of respiratory disease prior to death or euthanasia by captive bolt and exsanguination. short time from death to sampling was considered, no additional criteria for sampling were applied. in non-outbreak herds three to five pigs were haphazardly selected, 24 pigs were sampled in total. lungs and mediastinum (including pericardium, excluding the heart) and trachea caudal to the thoracic inlet were collected. within each herd, care was given not to sample pigs treated with any antimicrobial drugs up to 14 days prior to the sampling. blood sampling was performed on a total of 10 pigs per herd by haphazard selection from as many pens in the compartment as possible, up to 10 pens. a total of 141 pigs were sampled. the pigs were selected regardless of clinical presentation and restrained by snaring the upper jaw. during restraint the pigs were ear-tagged for individual identification at follow-up sampling during the final visit. rectal temperature was measured, and blood samples were collected (details in fig. 1 ). pooled oral fluid (of) samples were collected from two haphazardly selected pens (n = 14 pooled of samples from 28 pens) using chewing rope as described by prickett et al. [28] . care was given to keep the stress of the animals during sampling to a minimum. demographic data sampling was obtained by interviewing the farmers using a purpose-built questionnaire, see details in fig. 1 . relevant information regarding the disease outbreaks including information about the first 5 days after noticing the first clinical signs was registered in table 1 overview of descriptive data in both outbreak and non-outbreak herds (n = 14) a herd type: 5 finishers, 2 farrow-to-finish. 6 herds: one compartment affected and tested. 1 herd: two compartments affected and tested, compartment average presented b herd type: 6 finishers, 1 farrow-to-finish. one compartment tested per herd fig. 1 overview of the timeline and procedure of the study outbreak herds. the following data was registered: dates of the pigs' arrival to compartment, a description of earliest observed clinical signs, onset of disease, time to veterinary contact, time from the first clinical signs to the initial sampling, numbers of pigs displaying clinical signs, applied antibiotic treatment, and number of sick and dead pigs from the start of the outbreak until the time of the interview. during the final visit, second blood samples were collected from individually ear tagged pigs, and rectal temperature measured in the same pigs. procedures for sample handling are presented in fig. 1 . organs from 52 pigs were subject to post-mortem examination. the pericardium, pleura, trachea, bronchi, lung parenchyma and tracheobronchial lymph nodes from 28 to 24 pigs from outbreak herds and non-outbreak herds respectively, were examined at the norwegian veterinary institute (nvi) according to a standardized protocol (additional file 1). tissue samples from the lungs, pleura and lymph nodes were fixed, processed, sectioned and stained for histological examination (additional file 2). in total, 112 histological sections from the outbreak herds and 36 sections from 3 non-outbreak herds were examined following a standardized protocol (additional file 2). sampling (on charcoal transport swabs) for bacterial cultivation was performed during postmortem examination of 52 lungs and pleurae, see details in table 3 . the lung surface was flamed and aseptically incised before swabbing of lung tissue. the swabs were cultivated as a part of the routine diagnostics at nvi (additional file 3). serovar identification of cultured a. pleuropneumoniae (n = 31 isolates) was performed on sequence data, generated through whole genome sequencing of the a. pleuropneumoniae isolates at statens serum institut (ssi), copenhagen, denmark. the serovar was determined based on the presence of the serovar specific cps operons [29, 30] . details regarding the method are described in additional file 3. the serum samples (n = 282) were analyzed using commercial diagnostic kits for antibodies to a. pleuropneumoniae, influenza a virus, prrsv, prcv and m. hyopneumoniae. the analyses were performed as described by the manufacturers; details are given in additional file 3. interpretation of the test results were categorical, based on the cut-off values recommended by the test manufacturers. presence of antibodies to prrsv, porcine respiratory corona virus (prcv) and m. hyopneumoniae were tested in the second serum sample (n = 141). serum elisa was conducted on paired serum samples (n = 282) from individual pigs for antibodies to influenza a virus and a. pleuropneumoniae. the presence of influenza a virus and porcine circovirus type 2 (pcv2) nucleic acids in pooled oral fluids (n = 14) were analyzed with real time polymerase chain reaction (pcr) by in-house procedures (additional file 3). a cycle threshold (ct) value for influenza virus below 37 was considered positive. pcv2 quantitative pcr (qpcr) is a quantitative test where results are given as measured nucleotide copies in 200 µl sample, calculated from repeated measures at different ct values and results are reported as low (< 10 4 copies), moderate (10 4 -10 7 copies) or high (> 10 7 copies). our sample size of 10 serum samples per herd was chosen based on an estimate of at least one positive animal if the prevalence of our disease in question is around 25% at a 95% confidence level. the same sample size was used for agents not present in the population, that we did not expect to find, due to practical reasons. statistical analyses of the data were performed using the software stata (stata se/15 for windows; stata corp., college station, tx, usa). descriptive numeric results are presented as average values and the standard deviation (sd) for data with a normal distribution, or median value followed by the interquartile range (iqr) for data that was not normally distributed. rectal temperatures from the first visit and from the final visit to the herd were compared. the variable "fever" was defined as a rectal temperature above 39.5 °c. odds ratios for fever during the outbreak sampling compared to fever during follow-up visits, were calculated using a stata 15 case-control odds-ratio calculator. morbidity was measured as the proportion of pigs with clinical signs of respiratory disease of the total number of pigs in the herd (herd morbidity) and in the compartment (compartment morbidity). mortality was measured as the proportion of pigs dying during the outbreak, out of the total number of pigs in the herd (herd mortality) and in the compartment (compartment mortality). case fatality, an indicator of pathogen virulence and disease lethality, was measured as the proportion of pigs that died during the outbreak and displayed clinical signs of respiratory disease prior to their death, out of the total number of pigs displaying respiratory disease. a herd was classified as seroconverted if at least one pig shifted from negative to positive status and no pigs shifted from positive to negative status. the proportion of seroconverted pigs in each herd was calculated. samples from pigs that could not be identified by ear tags (one herd, n = 10) were excluded. when calculating the incidence proportion and risk ratios for seroconversion to a. pleuropneumoniae, pigs that were seropositive on the first serum sample were excluded from the population at risk. incidence proportion was defined as the proportion of the seronegative pigs that seroconvert during the time at risk. time at risk was defined as time between paired serum samples. the risk ratio (rr) for a pig to seroconvert in outbreak herds, compared to nonoutbreak herds, was calculated using a stata15 cohort study risk-ratio calculator the 95% confidence interval (ci). the statistical significance of the calculated association, whether it was likely that the rr was different from 1, was indicated by the reported p value. median number of days from the farmers noticed clinical signs of respiratory disease until calling the local veterinary practitioner was 1 day. onset of outbreak was 35 days (median, iqr 43) after the pigs arrived at the compartment. the severity of the clinical signs varied between outbreak herds. clinical signs reported by the veterinary practitioner were mainly sudden deaths (four herds) and dyspnea (three herds). signs such as fever, bloody froth from oronasal openings, cough and lethargy were also reported, and it was observed that sick pigs were reluctant to chew on the cotton ropes used for of sampling. in all herds, intramuscularly administrated procaine benzylpenicillin was used to treat sick pigs over 3 to 5 days. in one herd, tiamulin was additionally administered in the drinking water for 4 days. treatments were started by the veterinary practitioner during the first visit after the outbreak of disease. all herd owners reported the treatment to effectively reduce acute clinical signs and stop the further spread of disease. the average compartment morbidity during the outbreak was 60% (sd 43, range 6-100%), while herd morbidity was 25% (sd 19, range 0.9-51%) in the outbreak herds. case fatality rate during the disease outbreaks was on average 9% (sd 12, range 0-34%) over 5 days, suggestive of a low virulent agent. during the outbreaks, compartment mortality was 4% on average (sd 3, range 0-10%), while herd mortality was 2% (sd 2, range 0-5%). proportion of pigs in the outbreak herds measuring a rectal temperature above 39.5 °c was 57.6% (n = 54) and 30% at the first and final visit, respectively. for the non-outbreak herds the proportion of pigs with a rectal temperature above 39.5 °c was 42.4% (n = 50) and 10% at first and final visit, respectively. the odds for a temperature above 39.5 °c were higher (odds ratio = 2.8, 95% ci 1.17-6.70), during outbreak than during follow-up in the outbreak herds. there were no dropouts among the study animals, the number of animals tested at the visits was the same. median number of days between first and final visit was 22 days (iqr 5) in outbreak herds and 18 days (iqr 4) in non-outbreak herds. results from the pathological examinations of 52 organs are presented on herd level in table 2 . gross pathology of the lungs was detected in all pigs (n = 28) from the outbreak herds. acute pleural lesions were reported in 25 of these pigs (89%) and chronic pleural lesions, were found in one. typical lesions of acute pneumonia were found in all the pigs. the acute lesions were principally dorsally distributed in all lung lobes, but the caudal lobes were the most affected. chronic lung lesions were observed in one pig. moderate to severe enlargement of the tracheobronchial lymph nodes was a prevalent finding (n = 22, 73%) in the pigs with pneumonia. characteristic gross lung lesions are shown in fig. 2 . in the non-outbreak herds various gross lung lesions were detected in seven of the 24 pigs (29%). pleuritis was observed in two of 24 pigs (8%), where one had an acute pleuritis, and the second pig focal chronic pleuritis. pneumonia was observed in four other pigs. mild, focal, acute lesions were seen in two of them, while similar acute lesions and abscess formation was seen in another. multifocal, necrotizing, chronic pneumonia was diagnosed in the fourth pig. a single pig from a non-outbreak herd had gross lung lesions of multifocal bleeding and mottled grayish green areas indicative of larval migration by ascaris suum. diagnostic results for individual herds, including the gross findings are summarized in table 2 . histopathological changes agreed with the acute macroscopic lesions observed. histological examination revealed fibrin and neutrophil deposits on the pleura. in the lung parenchyma there was alveolar filling with necrotic leukocytes, neutrophils and fibrin. interstitial edema and hemorrhage, peribronchial and peribronchiolar leukocyte infiltration was observed. subacute to chronic, necrotic lesions of varying sizes were demarcated by macrophages, lymphocytes and plasma cells table 2 results from gross pathology, bacteriology, serology and virology from seven outbreak herds (from 1 to 7) and seven non-outbreak herds (from 8 to 14) of on the pleura or in alveolar lumen, and areas of interstitial bleeding. actinobacillus pleuropneumoniae was cultured from all sampled pigs (n = 28) from outbreak herds (n = 7). abundant growth of a. pleuropneumoniae was present in lung tissue in all 28 pigs and on pleura in 26 pigs. in samples from 20 of the lungs and 13 pleurae, a. pleuropneumoniae was the sole microbial species detected. in the remaining samples, a range of bacteria were detected in addition to a. pleuropneumoniae and the results are shown in table 3 . swabs from non-outbreak pigs' lungs produced mostly negative bacteriology. from non-outbreak herds, a. pleuropneumoniae was isolated from lung parenchyma in two out of 24 pigs. the a. pleuropneumoniae isolates originated mainly from areas with acute gross pathology ( table 2 ). in one non-outbreak pig a. pleuropneumoniae was cultured from a chronic lung lesion. serotyping of a. pleuropneumoniae on genome level revealed that all sampled isolates belonged to serovar 8. the 282 serum samples were successfully analyzed in one session. antibodies to a. pleuropneumoniae were detected in samples from six (86%) outbreak herds and four (57%) non outbreak herds. at the first serum sample, 35% (25 of 71) of the pigs in the outbreak herds were seropositive, and 37% (26 of 70) in the non-outbreak herds. at the second serum sample, 89% (63 of 71) and 60% (42 of 70) of the pigs were positive in the outbreak and non-outbreak herds respectively, details are listed in table 2 . six outbreak herds and six non-outbreak herds were considered seroconverted, indicative of an active infection in the period from the first to the second visit. seroconversion in the seventh outbreak herd could not be assessed due to missing ear tags. proportion of seroconverted pigs in each outbreak herd ranged from 30 to 100%, and from 0 to 80% in non-outbreak herds ( table 2) . incidence proportion was 0.96 (sd 0.10) in outbreak herds over the median time at risk of 22 days. incidence proportion in the non-outbreak herds was 0.44 (sd 0.36) over the median time at risk of 18 days. the risk for seroconversion was more than double compared to pigs from non-outbreak herds (rr 2.3 [1.50-3.43 95% ci; p < 0.001]). antibodies to influenza a virus were detected in one outbreak herd, where one pig seroconverted during the sampling period, and two pigs were found to have a reduced antibody titer to below cutoff. influenza a-antibodies were not detected in the remaining six outbreak herds or the non-outbreak herds. the proportion of siv seropositive herds was 7% out of the herds combined. antibodies to m. hyopneumoniae, prcv and prrsv were not detected in samples from any herds. the 14 pooled of samples from 28 pens, median number of pigs per pen was 10 (range [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] , were all negative for influenza a viruses. quantification of pcv2 by rt-qpcr turned out low or moderate in all samples, results per herd are shown in table 2 . field outbreaks of acute respiratory disease in norwegian fattening pigs were investigated and a. pleuropneumoniae serovar 8 was the main pathogen detected, with negligible presence of co-infections. clinical signs reported were in agreement with previous reports of a. pleuropneumoniae infections, which are described to have a diverse clinical presentation [31] . even with the large variation in morbidity and mortality rates, the results from this study were in line with observations from other studies, as research on outbreak characteristics of respiratory disease show that morbidity can range from 10 to 100% [26] . mortality during outbreaks of acute porcine pleuropneumonia is usually reported to be between 1 and 10% [26] . case fatality rates are not commonly included in this research literature but is a more precise measure of the lethality of a disease, especially if little information about other illnesses is available. disease that affects mortality are likely to have common risk factors [32] and the use of case fatality rate is a more robust measurement and less subjected to confounders such as that of other illnesses. even as a single infectious primary agent, a. pleuropneumoniae can cause severe clinical signs. during acute porcine pleuropneumonia, high fever is common [33, 34] . for pigs in the age range from 3 to 6 months, body temperatures normally span from 38.5 to 39.3 °c [35] , and the proportion of pigs displaying a fever can be indicative of an outbreak. in the present study, the pigs were restrained by snaring the upper jaw during clinical examination and blood collection, which is stressful for the animal [36] . the cutoff for fever at 39.3 °c + 0.2 was used in the study to compensate for this stress. higher odds for displaying fever in the herds during outbreak than at the final visit were found among the pigs in this study. this signified body temperature as a disease characteristic during outbreaks of porcine pleuropneumonia, although technical biases like personnel and thermometers used might have influenced our results. this coincided with results from a recent study from finland [37] . there are 18 acknowledged a. pleuropneumoniae serovars, of which some were recently described [38] . from the norwegian pig population, serovars 2, 6, 7, 8 and 10 have previously been reported [39] . serovar 8 has been most commonly associated with clinical disease in recent years, followed by type 6 [40] . however, these previous findings were all based on antibody agglutination tests which are prone to cross-reactions, for instance between serovars 3, 6, 8 and 15 [41] . all a. pleuropneumoniae strains in this study belonged to type 8, raising questions about the importance of serovar 6. underestimation of serovar 8 has occurred in canada [42] , england and wales [43] . serovar 8 is typically viewed as low virulent and is less often associated with clinical disease globally. in a study describing clinical presentation of different serovars in experimentally infected pigs [33] , serovars that were less commonly associated with disease were able to produce severe clinical signs, including high fever. this could perhaps be a result of absence of other respiratory agents including more virulent serovars of a. pleuropneumoniae. the macro-and histopathologic findings were typical for acute pleuropneumonia caused by a. pleuropneumoniae [44] [45] [46] , supporting that a. pleuropneumoniae was the main etiologic pathogen in these outbreaks. direct agent detection, primarily by bacteriological culturing in affected lung tissue obtained during necropsy, is considered the most adequate method for diagnosing porcine pleuropneumonia [31] . direct pcr is a method that would be expected to yield similar results but would not allow for storing of the bacterial isolates for further molecular testing, as was done in this study. we observed a low incidence of pathological lesions in non-outbreak herds, and a. pleuropneumoniae was only isolated from lesions resembling porcine pleuropneumonia. other bacteria, including p. multocida and streptococcus spp., were also detected in a few samples in this study. both are known opportunistic bacteria that colonize the upper respiratory tract of healthy pigs [4] . streptococcus suis is the most important streptococcal swine pathogen found to contribute to bronchopneumonia [47] . it is not unlikely that the bacteria could colonize areas already infected with a. pleuropneumoniae. the lesions might then be hard to distinguish from the primary pathogen, particularly if large parts of the lungs are affected. in one outbreak herd all five lungs had growth of other bacteria. they could have been contaminated during collection, transport or sampling. alternatively, these pigs were all colonized by secondary bacterial pathogens. the number of herds included in this study was too low to investigate whether the presence of these bacteria was linked to any differences in outbreak characteristics or diagnostic results. the low occurrence of common secondary invaders could have been explained by the short time span between registered disease and sampling. it has been questioned whether the actions that led to the eradication of m. hyopneumoniae from the norwegian pig population [18] also significantly reduced the occurrence of other pathogens. this has not yet been investigated. treatment with procaine benzylpenicillin was in line with the therapeutic guidelines published by the norwegian medicines agency as the drug of choice for acute porcine pleuropneumonia [48] . similar recommendations have been published in finland and sweden [49, 50] . in denmark, tilmicosin and tulathromycin have been commonly used against acute pleuropneumonia [51] partly due to the convenience of peroral administration, not due to reduced susceptibility to benzylpenicillin. national surveillance programs for antimicrobial resistance in these countries have recently reported a high proportion of a. pleuropneumoniae isolates being susceptible to benzylpenicillin [51] [52] [53] . nevertheless, there are no recently published studies on the efficacy of procaine benzylpenicillin for porcine pleuropneumonia in norway. such knowledge of causative pathogens is the fundament for correct and prudent use of antimicrobials. the details to antimicrobial resistance patterns of a. pleuropneumoniae in norway are currently being studied further. seroconversion to a. pleuropneumoniae had occurred in most of the herds, in many cases in absence of clinical disease. the risk for seroconversion to a. pleuropneumoniae for pigs in outbreak herds was more than double compared to pigs from non-outbreak herds, despite small within-herd populations at risk due to many seropositive pigs in the first serum samples. seroconversion to less virulent strains might have happened without resulting in a cross-protection to the outbreak-causing serovar. in finland, haimi-hakala et al. observed no difference in either prevalence of seroconverted herds or proportion of seroconverted pigs per herd in the outbreak case group and non-outbreak control group [37] . they discuss that neither single or paired serum sampling for the diagnosis of acute respiratory disease in field conditions is of much value due to both a lack of details concerning the initiation time of infection and a high prevalence of subclinical infections with a. pleuropneumoniae. the risk for seroconversion was not addressed in their paper. a danish study from 2004 investigated correlations in seroconversion to a. pleuropneumoniae and concluded that variation in seroconversion was mainly explained by a common batch level factor, that varies between farms and batches within a farm [54] . outbreaks of disease might be viewed as a batch level factor in this sense. in cases of all-in-all-out rearing by compartment, which is common, batches of pigs are usually housed separately. as we observed, the outbreaks were often restricted to single compartments. risk factors can be related to animal housing, management and environment [26] , and infection pressure might be increased during clinical disease and is a likely trigger for seroconversion. risk factor analyses were beyond the scope of this paper due to a lower number of herds in our study than what was expected. the seeming decrease in outbreak occurrence might have resulted from of a collective effort in the norwegian pig production system to increase the health status of herds with reoccurring problems with respiratory disease prior to our sampling. when investigating siv antibody titers we found that only one outbreak herd was seropositive. even though one pig seroconverted during the sampling period, two pigs were found to have reduced antibody titer. since a single false-positive serological reactor could not be excluded, the true status of these animals was uncertain. there being multiple false-positive reactions in one herd, which would have been the case here, was perhaps less likely. the proportion of seropositive herds in this study was less than what is found on a national level, where approximately 25% of the herds are reported positive [21] . the virology results from our study suggested that neither siv nor pcv2 contributed to the disease outbreaks in the study population. the absence of siv in all of samples supported the lack of pathological lesions and serological results indicative of siv infection. no difference was detected in pcv2 levels between the outbreak-and the non-outbreak herds. reluctancy of sick pigs to chew on the ropes could have resulted in unrepresentative pcv2 levels. since pcv2 levels was tested on pooled samples we have no information on the individual pig's contribution to the sample. the health status of the norwegian pig population is very good and have many similarities to the one of finland in the sense that they are free from m. hyopneumoniae, prrsv and until recently prcv [21] . in finland, a more diverse outbreak etiology has been observed [37] . in the finnish study, a. pleuropneumoniae was found to be the most likely cause of disease in 14 of the 20 sampled herds. in most of these herds, a. pleuropneumoniae was the only etiologic pathogen identified. similarly, 16 outbreaks of respiratory disease were studied in the netherlands [10] concluding that five of these were most likely caused by a. pleuropneumoniae, while seven were caused by siv (h1n1) and (h3n2). like in our study, they did not find any clear evidence of specific dual infections. the main etiological pathogen of acute outbreaks of respiratory disease in the included norwegian fattening pigs was a. pleuropneumoniae. all pigs from outbreak herds were found to have typical lesions of acute porcine pleuropneumonia, and only a. pleuropneumoniae serovar 8 was identified. the clinical presentation and pathology of a. pleuropneumoniae was in line with previous reports on field outbreaks internationally. co-infections did not seem to be of impact on disease development. supplementary information accompanies this paper at https ://doi. org/10.1186/s1302 8-020-00529 -z. additional file 1. protocol for postmortem sampling. a scheme for a standardized postmortem evaluation and sampling of pigs' lungs. the scheme was compiled at the pathology department at the norwegian veterinary institute to be used in the study of acute respiratory disease outbreaks. additional file 2. histology protocol. a scheme for a standardized histologic evaluation of sections from pigs' lungs, pleura and tracheobronchial lymph nodes, including a description of section preparation. the scheme was compiled by members of the project group grisefine lunger to be used in the study of acute respiratory disease outbreaks. additional file 3. details of sample handling and diagnostics. a document containing extended details of sample handling and laboratory diagnostic methods performed in the study of acute respiratory disease outbreaks. we would like to thank professor mari heinonen at the university of helsinki and tijs tobias (dvm, ph.d., msc) for their advice and contributions to the planning of this study. at the norwegian veterinary institute several people at different departments have contributed significantly to the diagnostic work that is presented in this study. we would especially like to thank pathologists øyvor kolbjørnsen and helene wisløff, bacteriologist marianne gilhuus, molecular biologist torfinn moldal. in preparation for the field trial, there had been a substantial marketing campaign for the project, reaching out to pig farmers, veterinarians and farm advisors throughout the country. we would like to thank everyone involved in the sampling and data collection for their time and attention. results presented in this article have not been previously published. authors' contributions lmc, cag, tbk, smg, br and ck planned this study, lmc, ck and br performed the outbreak investigations/collected materials. lmc, mv and ck analyzed and interpreted the data. lmc and ck prepared the tables and figures. lmc had the primary responsibility of writing and revising the manuscript. all authors contributed to revising the manuscript. all authors read and approved the final manuscript. we are grateful for the funding provided by the agricultural agreement research fund and the foundation for research levy on agricultural products, grant number nfr-267422. thanks to our collaborators who also funded the research: animalia, nortura and klf. the norwegian veterinary institute covered all costs related to administration of sampling equipment and sample analysis for this study. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. to be able to perform sampling from live animals, the norwegian food safety authority approved the study design for 'grisefine lunger' in september 2017, maintaining compliance of ethical guidelines and the three r's. fots norwegian food safety authority reference id 13185. diseases of swine effect of pneumonia on growth rate and feed efficiency of minimal disease pigs exposed to actinobacillus pleuropneumoniae and mycoplasma hyopneumoniae a tool to assess the economic impact of pleurisy in slaughter pigs porcine respiratory disease complex associations between pathogens in healthy pigs and pigs with pneumonia mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia enriched housing reduces disease susceptibility to co-infection with porcine reproductive and respiratory virus (prrsv) and actinobacillus pleuropneumoniae (a. pleuropneumoniae) in young pigs influenza virus coinfection with bordetella bronchiseptica enhances bacterial colonization and host responses exacerbating pulmonary lesions porcine respiratory disease complex (prdc): a review i etiology, epidemiology, clinical forms and pathoanatomical features survey of infectious agents involved in acute respiratory disease in finishing pigs kinetics of single and dual infection of pigs with swine influenza virus and actinobacillus pleuropneumoniae interaction between mycoplasma hyopneumoniae and swine influenza virus an investigation of the pathology and pathogens associated with porcine respiratory disease complex in denmark association of porcine circovirus 2 with porcine respiratory disease complex annual report 2018 koorimp and kif 7b27e 28ef6 bf4e8 78416 cc666 4a440 e1/koori mp-arsme lding -2018-web.pdf. accessed 12 des 2019. 16. regulation on swine and poultry production annual statistical report the successful eradication of mycoplasma hyopneumoniae from norwegian pig herds -10 years later. in: abstract book of the 11 th european symposium of porcine health management the norwegian livestock industry´s joint action plan on antimicrobial resistance influenza a(h1n1)pdm09 virus infection in norwegian swine herds 2009/10: the risk of human to swine transmission the surveillance programme for specific viral infections in swine herds in norway adverse effects of influenza a(h1n1)pdm09 virus infection on growth performance of norwegian pigs-a longitudinal study at a boar testing station the norwegian veterinary institute transmission of actinobacillus pleuropneumoniae in pigs under field-like conditions: emphasis on tonsillar colonisation and passively acquired colostral antibodies actinobacillus pleuropneumoniae infections in closed swine herds: infection patterns and serological profiles simulation study of the mechanisms underlying outbreaks of clinical disease caused by actinobacillus pleuropneumoniae in finishing pigs respiratory disease in pigs-an increasing problem? oral-fluid samples for surveillance of commercial growing pigs for porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 infections comparative sequence analysis of the capsular polysaccharide loci of actinobacillus pleuropneumoniae serovars 1-18, and development of two multiplex pcrs for comprehensive capsule typing complete genome sequence of midg2331, a genetically tractable serovar 8 clinical isolate of actinobacillus pleuropneumoniae hennig-pauka i. update on actinobacillus pleuropneumoniae-knowledge, gaps and challenges risk factors for mortality in grow-finishing pigs in belgium convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year ready to submit your research ? choose bmc and benefit from evaluation of actinobacillus pleuropneumoniae diagnostic tests using samples derived from experimentally infected pigs detection of actinobacillus pleuropneumoniae in pigs by real-time quantitative pcr for the apxiva gene herd evaluation restraint, but not frustration, induces prostaglandinmediated hyperthermia in pigs etiology of acute respiratory disease in fattening pigs in finland proposal of serovars 17 and 18 of actinobacillus pleuropneumoniae based on serological and genotypic analysis occurrence of lung lesions and antibodies to serotypes 2 and 6 of actinobacillus pleuropneumoniae and haemophilus parasuis in 5176 slaughter weight pigs from 113 elite herds in norway quantitation of serotype-specific and crossreacting group-specific antigens by coagglutination and immunodiffusion tests for differentiating actinobacillus (haemophilus) pleuropneumoniae strains belonging to cross-reacting serotypes 3, 6, and 8 the challenge of detecting herds sub-clinically infected with actinobacillus pleuropneumoniae actinobacillus pleuropneumoniae serovar 8 predominates in england and wales diseases of swine an abattoir survey of pneumonia and pleuritis in slaughter weight swine from 9 selected herds. i. prevalence and morphological description of gross lung lesions an abattoir survey of pneumonia and pleuritis in slaughter weight swine from 9 selected herds. iii. serological findings and their relationship to pathomorphological and microbiological findings diseases of swine docum ents/veter in%c3%a6rme disin /terap ianbe falin ger/ terap ianbe falin g_bruk%20av%20ant ibakt eriel lt%20mid ler%20til %20pro duks recommendations for the use of antimicrobials in the treatment of the most significant infectious and contagious diseases in animals dling srek-vet/rek%20-%20dos ering %20av%20ant ibiot ika%20til l%20gri s%5b1%5d national food institute. danmap 2018 -use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in denmark finres-vet 2018: finnish veterinary antimicrobial resistance monitoring and consumption of antimicrobial agents. finnish food authority publications swedres-svarm 2017: consumption of antibiotics and occurrence of resistance in sweden intra-unit correlations in seroconversion to actinobacillus pleuropneumoniae and mycoplasma hyopneumoniae at different levels in danish multi-site pig production facilities springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-281309-c9y7m5do authors: guo, baoqing; lager, kelly m.; henningson, jamie n.; miller, laura c.; schlink, sarah n.; kappes, matthew a.; kehrli, marcus e.; brockmeier, susan l.; nicholson, tracy l.; yang, han-chun; faaberg, kay s. title: experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus date: 2013-01-20 journal: virology doi: 10.1016/j.virol.2012.09.013 sha: doc_id: 281309 cord_uid: c9y7m5do the pathogenesis of type 2 highly pathogenic porcine reproductive and respiratory syndrome virus (hp-prrsv) in 10-week old swine in the united states was investigated. rjxwn06, rescued from an infectious clone of chinese hp-prrsv, replicated in swine with at least 100-fold increased kinetics over u.s. strain vr-2332. rjxwn06 caused significant weight loss, exacerbated disease due to bacterial sepsis and more severe histopathological lung lesions in pigs exposed to hp-prrsv than to those infected with vr-2332. novel findings include identification of bacterial species present, the degree of thymic atrophy seen, and the inclusion of contact animals that highlighted the ability of hp-prrsv to rapidly transmit between animals. furthermore, comprehensive detailed cytokine analysis of serum, bronchoalveolar lavage fluid, and tracheobronchial lymph node tissue homogenate revealed a striking elevation in levels of cytokines associated with both innate and adaptive immunity in hp-prrsv infected swine, and showed that contact swine differed in the degree of cytokine response. in 2006, investigators from several chinese provinces reported a unique syndrome in growing swine that was highlighted by the predominant clinical signs of high fever, anorexia, listlessness, red discoloration of skin, respiratory distress and very high morbidity and mortality rates (li et al., 2007; tian et al., 2007; tong et al., 2007; wu et al., 2009; zhou et al., 2008) . originally known as porcine high fever disease (phfd), this syndrome spread to vietnam in 2006 and to cambodia, laos, the philippines, bhutan, myanmar, thailand, south korea and russia in later years feng et al., 2008) . although there was concern that this syndrome may have been caused by a new disease agent, extensive diagnostic testing revealed only known pathogens. one consistent finding was the detection of porcine reproductive and respiratory syndrome virus (prrsv) with two discontinuous deletions in the replicase polyprotein known as nonstructural protein 2 (nsp2), and two single nucleotide deletions in the 5 0 and 3 0 untranslated regions (utrs) (zhou and yang, 2010) . experimental infection of chinese swine with the initial novel prrsv field isolates reproduced the clinical disease (li et al., 2007; tian et al., 2007; tong et al., 2007; wu et al., 2009; zhou et al., 2008) , providing strong evidence for its role as the causal agent of phfd. since the severity of the clinical disease was greater than expected for a typical prrsv infection, there remained the chance that an unknown agent in the prrsv isolates exacerbated the disease symptoms. this question was resolved when phfd was reproduced in chinese swine with virus derived from an infectious clone of the jx143 prrsv isolate (lv et al., 2008) . the prior studies demonstrated that prrsv isolates with a common genetic pattern had a causal role in phfd, leading to the new designation of this viral lineage as highly pathogenic prrsv (hp-prrsv). the presence of the unique nsp2 deletion motif was initially thought to contribute to the severity of disease (tian et al., 2007) . however, studies showed that the nsp2 region of hp-prrsv strain rjxwn06 containing the novel deletion could be replaced with 458 amino acids from low virulence strain hb-1/3.9 to result in a chimeric virus with only a minor delay in mortality (zhou et al., 2009) . there was also concern that there may be some unique aspect that may predispose asian pigs to a severe outcome, e.g., husbandry practices, endemic infections with other pathogens, climate, or host genetics. although a number of experiments have associated genetic changes in prrsv with an attenuation phenotype using point mutations (grebennikova et al., 2004; nielsen et al., 2001; storgaard et al., 1999) or by the construction of chimeric viruses (ellingson et al., 2010; kwon et al., 2008; wang et al., 2008; zhou et al., 2009 ), there appears to be no single locus for which mutations confer a predictable change in virulence. collectively, this area of study suggests the factors that contribute to prrsv pathogenicity are complex and viral strain-specific . as part of the single-strand positive-sense rna virus order nidovirales, family arteriviridae, prrsv genomes can vary between 15 kb to 15.5 kb, consist of at least 10 open reading frames (orfs) and replicate through a nested set of subgenomic rnas (van hemert and snijder, 2008) . members of this highly variable virus have been grouped into two genotypes, type 1 (european-like, prototype strain lelystad) or type 2 (north american, prototype strain vr-2332), which differ in nucleotide similarity by approximately 45% (nelsen et al., 1999) . both genotypes have representatives of varying pathogenicity, and intragenic nucleotide sequence variation in each can be as much as 20% (shi et al., 2010) . for this study, we imported a plasmid containing the cdna copy of the hp-prrsv strain rjxwn06 genome, a type 2 prrsv genome that is 15,321 bases in length and has 89% pairwise nucleotide identity to the prototype genome of vr-2332 (zhou et al., 2009) . to investigate the pathogenesis of hp-prrsv infection and the potential contribution of climate, host genetics, commensal bacteria, other environmental conditions and husbandry practices to the pathogenicity of hp-prrsv in u.s. swine, we compared and contrasted the pathogenicity of rescued jxwn06 (rjxwn06) virus to that of vr-2332 in 10-week old swine. we found that this hp-prrsv strain caused extreme morbidity, as was seen in asia, but novel to this study, resulted in up to 100x higher abundance of circulating virus when compared to vr-2332, caused extremely exacerbated thymic atrophy such that the thymus was often difficult to discern, and the host response was assessed in comparison to animals infected with strain vr-2332 for the first time by a swine protein array including 5 innate and 5 adaptive cytokines in serum, bronchoalveolar lavage fluid and lymph nodes. moreover, we completed bacterial speciation and loads after necropsy and specified the degree of weight loss seen. lastly, we showed that hp-prrsv readily transmitted to contact swine causing a different pattern of cytokine responses. as a result of our study, the remaining plausible contributing factors to the high virulence seen in asia were discredited, and thus hp-prrsv strains pose a serious threat to the u.s. swine industry. pigs challenged with chinese hp-prrsv strain rjxwn06 (group 2) began exhibiting clinical signs of disease within 2-3 days post exposure (dpe). as a group, the pigs developed fevers, became listless, anorexic, and began shivering and huddling together in a pile. clinical signs became more severe over the next few days, with pigs rapidly losing body weight (fig. 1) , becoming dehydrated and weak. respiratory distress, characterized by dyspnea, tachypnea and coughing was common in all pigs. erythema of the skin was present in most of the pigs, and several developed cutaneous hemorrhages and cyanotic extremities (blue ears) (fig. 2 ). on 7 dpe, one pig from group 2 was found dead. two others were euthanized due to severe weakness and moribund condition on dpe 8 and 9, and one was found dead on dpe 13. on 10 and 11 dpe, clinical signs of disease in some of the hp-prrsv challenged group began to decrease in severity. while pigs remained huddled in a pile with respiratory signs, approximately half of them were subjectively less listless, would move away when approached and began showing an interest in feed again. during the study, contact pigs displayed similar clinical signs as those challenged with hp-prrsv . pigs challenged with vr-2332 (group 4) were clinically normal until dpe 7, when they began to exhibit slightly increased respiratory rates and became a little less active than the control group. one pig from the vr-2332 group was found dead on dpe 9; however, the cause of death was attributed to gastric dilatation and volvulus, rather than to clinical disease typical of prrsv strain vr-2332 . control pigs (group 1) remained clinically normal for the duration of the study. postmortem examination revealed severe lesions in the hp-prrsv challenged pigs including: marked interstitial pneumonia, lymphadenopathy and thymic atrophy. necropsy findings in this group include: pulmonary edema, pleuritis, peritoneal and pericardial effusions, renal petechia, and fibrinous peritonitis that are delineated in table 1 . lungs and representative lesions are shown in fig. 2 . no pathologic lesions were identified in control pigs (group 1). pigs in the vr-2332 challenge group (group 4) had diffuse interstitial pneumonia, characteristic of an uncomplicated prrsv infection. although lung lesions were subjectively more severe in the hp-prrsv and contact groups, a statistically significant difference in lung scores was only found between the rjxwn06 contact group and the group inoculated with vr-2332 (groups 4 and 3, po0.003) (fig. 3a) . lymphadenopathy was present in all infected pigs (groups 2-4 ), with no statistically significant difference between hp-prrsv and vr-2332 infected groups (fig. 4a ). thymic atrophy was also a common finding in infected pigs and was significantly more severe in hp-prrsv infected and contact pigs compared to vr-2332 infected pigs (p ¼0.0003 and po0.0001; fig. 4b ). table 2 summarizes aspects of the clinical disease seen in this study. shown are adwg and associated standard errors for each of the four groups for days à 1-7 and days 7-13. sham inoculated control swine (group 1) and those challenged with vr-2332 (group 4) gained weight at an expected rate for their age group. group 2 swine challenged with hp-prrsv strain rjxwn06 and their contacts (group 3) lost body weight during the last week of the experiment. the adwg for group 2 on days à 1-7 includes only 11 pigs, and on days 7-13 includes only 8 pigs. the body weights for the contact animals were taken on the same day as pigs in the other groups. significance values are shown. microscopic lung lesions consisted of histiocytic interstitial pneumonia with increased numbers of macrophages in alveolar septa and lumina (fig. 5a , c). type ii pneumocyte hyperplasia was also present. these lesions were seen in the hp-prrsv challenge group (group 2), the hp-prrsv contact pigs (group 3) and the vr-2332 challenge group (group 4). mean interstitial pneumonia lesions were 3.33, 3.75, and 2.00 for chinese prrsv challenge group (group 2), contact chinese prrsv pigs (group 3) and vr-2332 challenge (group 4), respectively (fig. 3b ). lesions were significantly (p¼0.0002) more severe in the chinese prrsv challenge group (group 2) and contact chinese prrsv pigs (group 3) as compared to the vr-2332 challenge (group 4). two sham inoculated control pigs had mild lesions with a score of 1, while the remaining pigs had no lesions. immunohistochemistry labeling for prrsv nucleocapsid was positive in 100% of chinese prrsv challenge and contact pigs (groups 2 and 3) and 88% of vr-2332 challenge pigs (group 4), with mean ihc labeling scores of 2.08, 1.75, and 1.63, respectively (fig. 5) . positive labeling was present in alveolar macrophages and interstitial macrophages for all infected groups ( fig. 5b and c). no significant difference was present in the amount of prrsv labeling in the lung among the three treatment groups. immunohistochemistry labeling for prrsv in lung tissue was negative in all sham inoculated control pigs. sera collected at à 1, 7, and 13 dpe were tested for prrsv antibody. most swine infected with rjxwn06 (group 2) had seroconverted by 7 dpe (mean s/p ¼0.51, sd¼0.30), and all had seroconverted by 13 dpe (mean s/p ¼1.14, sd ¼0.26); however, the rjxwn06 contact animals (group 3) had not yet seroconverted by 5 dpe (mean s/p ¼0.00, sd ¼0.00) but did by 11 dpe (mean s/p¼0.99, sd¼0.12). vr-2332 infected pigs (group 4) were seronegative at 7 dpe (mean s/p ¼0.09, sd ¼0.12); but positive by 13 dpe (mean s/p¼ 1.05, sd ¼0.37). the sham inoculated control animals (group 1) remained negative for prrsv nucleocapsid antibody throughout the study. all serum and balf samples were screened on marc-145 cells for infectious virus, and vi positive samples were then titered. a significant increase in virus load (100-1000 fold) for the rjxwn06 challenge and rjxwn06 contact pigs was found in the serum when compared to the vr-2332 challenge group (fig. 6a ). no significant differences were found in serum virus titer between the rjxwn06 challenge and rjxwn06 contact groups at any time points even though the contact pigs were exposed to rjxwn06 infected pigs at 2 dpe. at necropsy, no significant differences in balf titers were found among the rjxwn06 challenge, rjxwn06 contact, or vr-2332 challenge groups (fig. 6b ). control serum and balf samples were vi negative throughout the experiment ( fig. 6a and b) . to examine the levels of prrsv rna and to strengthen the case for the apparent difference in viral loads between the hp-prrsv and vr-2332 inoculated animals seen above, qrt-pcr was completed on all serum samples as well as balf and lymph node tissue (fig. 7) . the level of viral rna detected in serum samples mirrors the results obtained by virus isolation on marc-145 cells (fig. 7a, pr0 .005 at all dpe). the qrt-pcr results from balf showed little or no significant difference between the infected lymph node weight (lnw) to body weight (bw) ratios showed pronounced lymphadenopathy in all infected animals (groups 2-4) as compared to control animals (group 1)(p¼ 0.02 or higher significance), but no statistical significance between prrsv infected swine. (b) thymus weight (tw) to bw ratios showed thymic atrophy in all infected animals (groups 2-4) as compared to control animals (p o 0.0001), and statistically significant differences between hp-prrsv inoculated (po 0.0001) and naturally infected (p ¼ 0.0003) animals compared to vr-2332 inoculated swine. groups (fig. 7b ). tbln samples suggested that swine inoculated with vr-2332 had significantly less viral rna present than those inoculated (p¼0.0205) or infected (p¼ 0.0017) with hp-prrsv (fig. 7c ). sham inoculated control serum, balf, and tbln tissue samples were viral rna negative throughout the experiment ( fig. 7a -c ). bacteria were isolated from the balf of 8 of the 12 rjxwn06 inoculated pigs (group 2) and 2 of the 4 rjxwn06 contact pigs (group 3) but from only 1 of 8 pigs in each of the sham inoculated control (group 1) and vr-2332 inoculated (group 4) groups. in the rjxwn06 challenge group pasteurella multocida was isolated from four pigs (ranging from 50 to 4 300 cfu/100 ml), p. multocida ( 4300 cfu/100 ml) and actinobacillus suis ( 4300 cfu/ 100 ml) were isolated from two pigs, p. multocida, a. suis and streptococcus suis (approximately 50, 100, and 25 cfu/100 ml, respectively) from one pig, and staphylococcus aureus and klebsiella pneumoniae (approximately 150 and 20 cfu/100 ml, respectively) were isolated from one pig. in the contact group, p. multocida and a. suis were isolated from one pig (4300 cfu/100 ml each) and p. multocida, a. suis, and arcanobacterium pyogenes (approximately 50, 4300, and 4300 cfu/100 ml, respectively) were isolated from a second pig. s. aureus and escherichia coli (4300 cfu/100 ml each) were isolated from 1 vr-2332 inoculated pig, and bordetella bronchiseptica (65 cfu/100 ml) was isolated from 1 sham inoculated control pig. results on 10 cytokines measured in serum, balf and tbln homogenates are presented in figs. 8 and 9 and the changes in cytokine levels relative to sham-inoculated controls are listed in table 3 . pigs directly inoculated with rjxwn06 (group 2) had significantly elevated average serum levels of ifna, il-1 b, il-2, il-10 and ifng, ranging from 6 to 11 times the levels detected in sera of sham inoculated controls (group 1). in pigs inoculated with rjxwn06, serum ifna levels were significantly elevated at 4 dpe in comparison to both controls and vr-2332 exposed pigs (po0.05) and remained significantly elevated at 7 dpe versus both groups. serum levels of il-10 were elevated by 7 dpe, with il-2, il-10, il-12 and ifng all signficantly elevated at 11 dpe compared to both controls and vr-2332 exposed pigs (po 0.05). compared with sham inoculated controls, contact pigs exposed to rjxwn06 (group3) developed significant serum level elevations (3 to 89 times greater) in 3 out of 5 innate immunity cytokines measured (ifna, tnfa and il-1 b) and elevated levels (3 to 21 times greater) for all 5 cytokines measured associated with adaptive immunity (il-2, il-4, il-10, il-12 and ifng). in pigs exposed to rjxwn06 by contact transmission, serum ifna levels were signifcantly elevated at 2, 5 and 9 dpe in comparison to controls (po0.05). in addition, contact pigs had significant elevations in il-1 b at 9 and 11 dpe (po0.05). contact pigs also had significantly elevated levels of all adaptive immunity cytokines at 9 and 11 dpe (p o0.05) with il-10 also significantly elevated at 5 dpe. in contrast, none of the 10 cytokines measured had significant elevations in serum levels detected in pigs inoculated with the north american prototype strain vr-2332 (group 4) when compared with sham inoculated controls. similarly, when compared to sham inoculated controls, balf of swine inoculated with rjxwn06 (group 2) had significantly elevated (7 to 22 times greater) levels of 3 out of 5 innate immunity cytokines measured (tnfa, il-1 b and il-8) and elevated levels (2 to 54 times greater) for all 5 cytokines measured associated with adaptive immunity (il-2, il-4, il-10, il-12 and ifng). compared with sham inoculated controls, contact swine exposed to rjxwn06 (group 3) had significantly elevated balf cytokine levels (4 to 21 times greater) in 4 out of 5 innate immunity cytokines measured (ifna, tnfa and il-8) and elevated levels (3 to 30 times greater) for all 5 cytokines measured associated with adaptive immunity (il-2, il-4, il-10, il-12 and ifng). in contrast, swine inoculated with vr-2332 (group 4) had significantly elevated balf cytokine levels (4 times greater) for only 2 out of 5 innate immunity cytokines measured (il-1b and il-8) and elevated levels (10 to 12 times greater) for 2 of 5 cytokines measured associated with adaptive immunity (il-10 and ifng). in this study, we compared the pathogenicity of chinese hp-prrsv strain rjxwn06 to north american prototype vr-2332 in u.s. high health swine under controlled conditions. the clinical disease induced in this study by the hp-prrsv virus was similar to what has been reported in asia for phfd and for experimental infections with the wild-type or rescued virus ( zhou et al., 2009; zhou et al., 2008) . likewise, the clinical disease induced by the vr-2332 virus was similar to that observed in previous reports (faaberg et al., 2010; rossow et al., 1994) . clinical disease and pathology were much more severe in the rjxwn06 group and their contacts than in the vr-2332 group. overall, gross pathology lesions in the rjxwn06 challenged and contact groups were much more extensive and not restricted solely to the respiratory tract and lymph nodes as was the case with the vr-2332 challenged pigs. the high occurrence of bacterial co-infections in the rjxwn06 challenge and contact swine likely played a prominent role in the difference in pathology and clinical disease between groups. bacterial co-infections in pigs naturally infected with prrsv have been documented, with susceptibility attributed to factors including prrsv strain differences, host genetics, management practices and environmental factors (rossow, 1998) . in this study, we used swine of high health status in a controlled research environment that were from 2-and 3-way crosses of commercial genetic lines. we believe the incidence and magnitude of bacteria isolated from the rjxwn06 challenge and contact groups when compared to the vr-2332 and control groups suggest the differences in secondary bacterial infection susceptibility are specific to viral strain. in this study, we observed a 30% mortality rate in rjxwn06 infected pigs by 13 dpe, which is less mortality than the original report (zhou et al., 2009) , but similar to other hp-prrsv strains used (li et al., 2007; lv et al., 2008; zhou et al., 2008) . possible explanations for differences may be the route and dose of inoculation, the age of pigs, the hp-prrsv strain utilized, and the time course of study. in addition, since the hp-prrsv lineage may exacerbate subclinical bacterial infections, it is possible different endemic bacterial infections played a role in apparent different mortality rates. in natural infections with chinese hp-prrsv, pulmonary interstitial hyperplasia with hemorrhage and edema is described, which suggests an acute septicemic process due to a secondary bacterial pathogen (tian et al., 2007; zhou and yang, 2010) . upon histopathologic examination of tissues from this study, north american pigs directly inoculated with rjxwn06, as well as contact pigs, had an interstitial pneumonia that was significantly more severe than the vr-2332 inoculated group, which is consistent with the severity of disease reported in china. although pulmonary lesions were more severe in the rjxwn06 challenge and contact pigs, the amount of antigen labeling was not significantly different from the vr-2332 inoculated swine. since levels of proinflammatory cytokines, including tnfa, il-1 b and il-8, were significantly increased in the balf of rjxwn06-inoculated and contact pigs, the host response to hp-prrsv may play a role in the augmented lung pathology seen. in addition, the increased incidence of secondary bacterial infections in rjxwn06 challenge and contact pigs may have contributed to increased cytokine production and resultant immunopathology. there are numerous reports about the interplay of prrsv with the swine immune system that describe variable responses to infection at the cellular and cytokine level (miguel et al., 2010; thanawongnuwech et al., 2004; thanawongnuwech and thacker, 2003; wang et al., 2011) . although a large part of this variability may result from differing methods, challenge viruses, outbred pigs, and experimental designs, there are consistent findings emerging among the studies of increases in levels of selected cytokines associated with both innate and adaptive immunity. here we report a comprehensive assessment of the effects of prrsv infection on levels of cytokines critical to innate (ifna/b, tnfa, il-1 b, il6 and il-8) and adaptive (il-2, il-4, il-10, il-12 and ifng) immune systems in serum, balf and tbln homogenates. it was demonstrated that infection with a highly pathogenic strain of prrsv elicited a significant elevation of all adaptive immunity cytokines measured in balf, as well as a majority of these cytokines in serum and tbln homogenates of the same groups of pigs. this observation is consistent with previous reports of table 3 changes in cytokine levels in sampled tissues relative to non-challenged control pigs (p-values in comparison to non-challenged controls). serum values are an area under the curve overall average level measured in samples collected after exposure to virus. geometric means back-calculated from log10 means were used to determine relative changes in tbln and balf cytokine levels compared to sham-inoculated controls. (sun et al., 2012) ]. in contrast, in pigs infected in this study with rjxwn06, we observed significant elevations of serum ifna as the first cytokine to peak following exposure to rjxwn06 in either the directly challenged or contact pigs, with the zenith occurring 4 and 2 dpe, respectively. however, consistent with a previous study (gomez-laguna et al., 2009 ), elevated serum ifna had little to no apparent effect on virus clearance as the viremia peaked in the rjxwn06 challenge and contact pigs on dpe 7 and 4, respectively, and remained above the serum virus levels of vr-2332 pigs until the end of the study (11 and 13 dpe). it is well documented that prrsv infection will increase the susceptibility of swine to co-infection with various bacteria (brockmeier et al., 2001; thanawongnuwech et al., 2004; thanawongnuwech and thacker, 2003; thanawongnuwech et al., 1997; thanawongnuwech et al., 2001; xu et al., 2010) , and based on previous findings with other hp-prrsv strains (xu et al., 2010) and our current rjxwn06 findings, it is clear that exposure to hp-prrsv greatly increases the likelihood of secondary bacterial infection due to commensal or pathogenic organisms typically found in the swine upper respiratory tract. whether the significant elevations of multiple cytokines that were measured in serum, balf and tbln following exposure to rjxwn06 were a direct result of the virus, an indirect effect mediated by the secondary bacterial infections, from extensive host tissue damage, or a combination of all of these events cannot be determined from our experiments. however, the pattern of multiple cytokines being elevated nearly simultaneously in serum (8 of 10 cytokines in rjxwn06 contact pigs and 5 of 10 cytokines in direct inoculated pigs) has not previously been reported and was not detected in vr-2332 infected swine. cytokines are a diverse collection of peptides that elicit a wide range of biological responses and are characteristically understood in the context of an immune response wherein inflammation and immunity are carefully orchestrated by sequential secretion of cytokines that coordinate innate and adaptive immune responses. macrophages and stressed or damaged cells typically initiate a cytokine cascade through secretion of chemokines and proinflammatory cytokines in order to initiate the innate immune response at sites of acute infection or damage. generally, they act locally at nano-to picogram levels with short half-lives and transient activity. however, inflammatory cytokine cascades classically comprise a sequential appearance and disappearance of proinflammatory cytokines (e.g., tnfa, il-1 and il-6) intended to activate immune cells and their recruitment to generate additional cytokines and chemokines. this initial wave of cytokine production is usually followed by anti-inflammatory cytokine production (mainly the il-10 family) to moderate or down regulate the pro-inflammatory cytokines. cytokine cascades are therefore usually sequential with transiently detectable levels in peripheral blood; any dysregulation of these cascades can lead to adverse immunopathological responses. while significant elevations of several cytokine levels in tissues such as balf and tbln were expected, near simultaneous elevation of several cytokines in serum was not entirely expected as part of a normal host immune response. moreover, the levels of cytokines detected were in several instances significantly elevated (usually several times more) over levels detected with the low virulence north american prototype prrsv strain, vr-2332 . prolonged elevations of serum ifng levels have been reported in swine infected with prrsv, a finding in contrast to serum ifng in pigs infected with influenza or respiratory coronavirus, where minimal transient detectable levels are observed (wesley et al., 2006) . in swine infected with rjxwn06, it appears the normal sequence of cytokine production (e.g., ifng, tnfa, and il-2 often being the first cytokines produced in response to a viral infection, followed by il-6 and il-10) leading to effective virus clearance and a normal immune response was dysregulated given that significant elevations of several serum cytokines levels were still evident at 7 to 11 dpe. two recent studies have reported hp-prrsv infection in swine results in down regulation of a key toll-like receptor adapter gene, sarm1 (sterile aand armadillomotif-containing protein) (zhou et al., 2011; zhou et al., 2012) . sarm1 normally dampens the proinflammatory immune response by attenuating nf-kb activation and decreasing expression of il1, il-8 and tnfa (carty et al., 2006) . previous studies in pigs infected with strains of hp-prrsv were reported to have swollen livers and petechial hemorrhages on the kidneys as well as immunohistochemical staining evidence of viral antigen in the liver and kidneys (among other tissues) (li et al., 2007; tian et al., 2007) . impaired hepatic and renal function as a consequence of viral or secondary bacterial disease could have a significant effect on clearance of the cytokines detected in serum, and contribute to an apparent severe cytokine release syndrome. whether our findings represent a parallel condition in swine to severe cytokine release syndromes or cytokine storms reported in humans that have been attributed to various causes cannot be proven with our data (descotes and gouraud, 2008; tarrant, 2010) . however, given prrsv causes polyclonal b cell activation, autoimmunity, lymphoid hyperplasia and hypergammaglobulinemia in pigs (lemke et al., 2004) , and it modulates multiple intracellular signaling pathways [reviewed in (sun et al., 2012) ], it is not surprising to find an exaggerated immune stimulation in swine infected with a particularly virulent strain of prrsv. early studies with cytokine administration to livestock species identified potential toxicities associated with systemic cytokine administration. interferon-g was found to have beneficial activity on immune function but was too toxic for practical usage due to febrile responses observed within 24 h of a single dose of 1.25 mg/ kg of body weight (roth and frank, 1989) . similarly, administration of 166 ng of bovine il-1 b/kg of body weight every 8 h for 4 days caused transient fever, inappetence, increased pulse and respiratory rate, and diuresis (goff et al., 1992) . blood cytokines have been proposed as biomarkers of in vivo toxicity associated with new drug development (tarrant, 2010) . under most disease scenarios where there is rapid resolution of the infection by the host immune response, much of the biological activity of cytokines will occur in the locally infected tissues and elevated levels of the cytokines may not be detected in serum. we cannot definitively state whether the elevated cytokine syndrome reported here contributes to the pathophysiology of the disease caused by this highly pathogenic prrsv isolate. however, the association between the elevated levels of multiple cytokines and the severe morbidity and high mortality reported is consistent with a multiple cytokine toxicity syndrome in humans associated with various therapeutics (tarrant, 2010) . adverse reactions ranging from mild-to-moderate flu-like reactions to severe cytokine release syndromes have been observed with many therapeutic proteins in current use in human medicine, and some result in severe and even potentially life-threatening syndromes (reviewed in (tarrant, 2010) ). macrophage activation syndrome and cytokine storm are different names for two syndromes that share many features including a massive inflammatory response, elevated serum cytokine levels, multiorgan system disease and often death (behrens et al., 2011) . although these syndromes may be clinically indistinguishable, the cytokines that predominate in each may differ with tnfa being dominant in bacterial sepsis and ifng predominate in the macrophage activation syndrome (behrens et al., 2011) . the exact pathophysiology of the systemic toxicity in these syndromes is not fully defined. in pigs infected by natural contact with rjxwn06 inoculated pigs, we detected significant serum elevations in ifna, tnfa, il-1 b, il-2, il-4, il-10, il-12 and ifng. however, given that prrsv infects the macrophage cell line in pigs, the elevated serum cytokine levels in pigs infected with hp-prrsv may represent both conditions (macrophage activation syndrome and cytokine storm) and contribute to the multiorgan damage and high mortality reported here. marc-145 cells were cultured in minimum essential medium (mem, safc 56416c) with 10% fetal bovine serum at 37 1c, 5% co 2 . wild-type (wt) type 2 prrsv strain vr-2332 (genbank u87392), passage 6 on marc-145 cells, was titrated, and used for the swine study. virus (rescued rjxwn06; rjxwn06) was rescued from a cloned cdna of chinese highly pathogenic type 2 prrsv strain jxwn06 in marc-145 cells [pwsk-jxwn; genbank ef641008, (zhou et al., 2009) ] and passaged 3 times on marc-145 cells for use in the swine study. the in vivo swine study described here was performed at the national animal disease center under approval from its animal care and use committee. thirty-two 10-week-old cross-bred pigs were obtained from a u.s. high-health status herd and were found to be free of prrsv antibodies by herdchek elisa, influenza virus antibodies by np elisa, and negative for porcine circovirus type 2 by quantitative real-time pcr (data not shown). one day prior to inoculation, pigs were bled, weighed and randomly assigned to one of four groups. group 1 (n ¼8) consisted of sham inoculated control pigs, which received an intranasal 2 ml sham inoculum of mem on day 0. group 2 pigs (n ¼12) were challenged intranasally with 2 ml of 1 â 10 6 50% tissue culture infective dose (tcid 50 )/ml of chinese prrsv strain rjxwn06 in animal biosafety level 3-agriculture (bsl3-ag) housing, where they remained for the duration of the experiment. group 3 consisted of naïve pigs (n ¼4) that were placed in contact with group 2 two days after group 2 was inoculated (dpe). group 4 pigs (n ¼8) were challenged intranasally on day 0 dpe with 2 ml of 1 â 10 6 tcid 50 /ml of type 2 prototype strain vr-2332 . groups 1 and 4 were housed in separate isolation rooms in an absl2 facility. clinical monitoring of pigs was performed daily throughout the study. specifically, observations were made regarding the pig's mental alertness, body condition, appetite, activity level, and clinical signs of respiratory or systemic disease. serum was collected on à 1, 4, 7, 11, and 13 dpe (sera from group 3 pigs was collected on à 3, 2, 5, 9 and 11 dpe), and pigs were weighed on à 1, 7 and 13 dpe (group 3 were weighed on à 3, 5 and 11 dpe). necropsy was scheduled on dpe 13 (dpe 11 for group 3), or sooner if pigs died or were euthanized due to severe disease. at necropsy, a thorough post-mortem examination was performed, and a complete set of samples was collected for evaluating disease severity. tracheobronchial lymph nodes (tbln) and thymic tissue were weighed (lnw and tw, respectively) and compared to respective body weights (bw) to derive a tissue mass index (lnw/bw, tw/bw) measuring the effect of prrsv infection on organ weight (mengeling et al., 2003) . upon removal, lungs were examined and extent of macroscopic lung lesions was estimated, as previously described, and reported as a percentage of lungs affected (halbur et al., 1996) . sections of tissues (lung, tracheobronchial lymph node, trachea, thymus, heart, tonsil, spleen, iliac lymph node, mesenteric lymph nodes, ileum, bone marrow, kidneys, liver, inguinal lymph node, cerebrum, brainstem, cerebellum and ventral midbrain) were collected into 10% neutral-buffered formalin for histopathology and immunohistochemistry. tbln were collected for rna extraction and cytokine protein assays. bronchoalveolar lavage fluid (balf) was collected after removing whole lungs from pigs and aseptically lavaging with 50 ml antibiotic-free mem. tissues were processed by routine histopathologic procedures and slides were stained with hematoxylin and eosin. a boardcertified veterinary pathologist blinded to treatment groups evaluated microscopic lesions. lung sections were scored on a 5-point scale that accounted for distribution and severity of interstitial pneumonia: 0-no lesions, 1-mild, focal to multifocal interstitial pneumonia (o50% of lung section affected), 2-moderate, multifocal to coalescing (50-75% of lung section affected), 3-severe, patchy to coalescing and extensive (75-90% of lung section affected), and 4-severe and diffuse ( 490% of lung section affected). immunohistochemistry prrsv-specific antigen was detected in lung tissues using a previously described immunohistochemical (ihc) method with minor modifications (halbur et al., 1995) . briefly, tissue sections were deparaffinized and hydrated in distilled water. slides were quenched in 3% hydrogen peroxide for 10 min, rinsed three times in distilled water and treated in 0.05% protease for 2 min. slides were then rinsed three times in distilled water. a primary monoclonal antibody (mab) cocktail of one part 1:800 prrsv sdow17 (rti, brookings, sd) and one part 1:500 prrsv sr30 (rti, brookings, sd) was applied to the slides and were incubated at room temperature for 1 h. bound mabs were stained with peroxidase-labeled anti-mouse igg followed by chromogen using the dako lsab2-hrp detection system (dako, carpinteria, ca) according to the manufacturer's instructions. 3,3 0 -diaminobenzidine (dab; vector laboratories, burlingame, ca) was applied to the slides for 5 min. the slides were rinsed in deionized water and counterstained with gill's hematoxylin. prrsv labeling was graded on a 4 point scale of 0: none, 1: mild scattered signals (1 to 20 cells in entire section), 2: moderate scattered signals (less than or equal to 50% of high power fields (hpf) containing immunolabeling) and 3: abundant scattered signals (greater than 50% of hpf contain labeling and/or there are at least 2-3 groups of 30 cells or more with staining). virus isolation was attempted on all serum and balf samples as described previously (faaberg et al., 2010) . those samples that were positive by virus isolation were then titered by serial dilution on marc-145 cells to determine the quantity of virus present to produce a cytopathic effect in 50% of inoculated tissue culture cells (tcid 50 ). quantitative rt-pcr (qrt-pcr), as previously described (faaberg et al., 2010) , was used to determine the amount of viral rna per ml of serum and balf, and per gram of tbln homogenized tissue prepared as described below. serum samples were tested on study days à1, 7 and 13 for evidence of seroconversion with the prrs 2xr enzyme-linked immunosorbent assay (herdchek elisa; idexx laboratories). a sample was considered positive for antibodies to prrsv nucleocapsid protein if the sample-to-positive (s/p) ratio was equal to or greater than 0.4. levels of tnfa, il-1 b, il-2, il-4, il-6, il-8, il-10, il-12p70 and ifng cytokine levels (pg/ml) were measured in serum, balf and tbln samples diluted 1:2 with dilution buffer supplied by the manufacturer using a searchlight (aushon biosystems, woburn, ma, usa) customized multiplex immunoassay (m-elisa) following the manufacturer's protocol. m-elisa samples were assayed in duplicate. the elisa limits of detection for tnfa, il-1 b, il-2, il-4, il-6, il-8, il-10, il-12p70 and ifng were 0.2, 2.4, 0.78, 0.39, 1.6, 0.098, 0.001, 0.098, and 0.78 pg/ml respectively. approximately 1 g of tbln was homogenized in 750 ml of lysis buffer containing 0.5% triton x-100, 150 mm nacl, 15 mm tris, 1 mm cacl 2 , and 1 mm mgcl 2 , ph 7.4, using a tissue homogenizer (biospec products, bartlesville, ok) (greenberger et al., 1995) . homogenates were incubated on ice for 30 min, then centrifuged at 1258 x g for 10 min. supernatants were collected, passed through a 0.45micron filter (gelman sciences, ann arbor, mi), then stored at à20 1c prior to assessment of cytokine levels. ifna protein was measured with a porcine ifna specific elisa by using f17 monoclonal antibody (mab) and k9 mab (r&d systems inc.) as previously described (miller et al., 2009) . mab k9 was conjugated with horseradish peroxidase (hrp) using a peroxidase labeling kit (roche molecular biochemical, indianapolis, in). immulux hb flat-bottomed 96-well plates (dynex technology, chantilly, va) were coated overnight at 4 1c with f17 at a concentration of 3 mg/plate in coating buffer (100 mm carbonate buffer, ph 9.6, sigma inc., st. louis, mo). after blocking with 1% non-fat dried milk, 0.05% tween 20 in phosphate buffered saline (pbs) for 1 h at 37 1c, the plates were washed three times with 0.05% tween 20 in pbs. samples (50 ml) were added into each well containing 50 ml of 1% non-fat dried milk, 0.05% tween 20 in pbs and incubated for 2 h at 37 1c. following three washes, 100 ml of peroxidase conjugated k9 was added to each well. after 1 h incubation, at 37 1c, and three washes, 100 ml of substrate solution, tetramethylbenzidine (kpl inc., gaithersburg, md), was added to each well. after 30 min, the reaction was stopped with tetramethylbenzidine stop solution (kpl inc., gaithersburg, md) and the optical density was measured at 450 nm by an elisa plate reader. quantified recombinant porcine ifna (rifna, r&d systems inc., minneapolis, mn) was used as a standard, and ifna concentrations were calculated based upon a standard curve. one unit/ml of rifna is equivalent to 26 pg/ml. bacterial culture was performed by plating 100 ml of balf both on a casman's agar plate supplemented with 0.01% nicotinamide adenine dinucleotide (nad) and 5% horse serum, and on a 5% sheep's blood agar plate. both agar plates were incubated for 48 h at 37 1c. bacterial identification was performed by 16s rrnaspecific pcr and dna sequencing. 16s rrna-specific pcr and dna sequencing whole-cell bacterial lysates, used as templates, were prepared by suspending a colony, $ 2 mm in diameter or the equivalent, in 50 ml of sterile water. the mixture was boiled for 10 min, placed on ice until chilled, and centrifuged at 16,000 â g for 1 min to pellet cell debris and stored at à 20 1c. supernatant (5 ml) was used as the template in each pcr. the forward primer (5 0 -agagtttgatcctggctcag-3 0 ), designated univ16s-3, is homologous to a highly conserved sequence from the 5 0 end of the 16s rrna gene and the reverse primer (5 0 -gcggctgctggcacg-3 0 ), designated univ16s-4, is homologous to a highly conserved sequence between the third and fourth variable regions of the 16s rrna gene. this previously described primer set generates an amplicon of approximately 520 bp (register and yersin, 2005) . reactions were carried out in a volume of 50 ml and contained 2 u amplitaq polymerase (applied biosystems, foster city, ca), 5 ml 10x buffer ii (100 mm tris-hcl , ph 8.3, 500 mm kcl), 5 ml dimethyl sulfoxide, 1.5 mm mgcl 2 , 0.5 mm primers, and 200 mm deoxynucleoside triphosphates. an initial denaturation step of 5 min at 95 1c was followed by 35 cycles of 20 s at 94 1c, 30 s at 55 1c, and 3 min at 72 1c, with a final extension step of 10 min at 72 1c. 10 ml of each pcr was analyzed by agarose gel electrophoresis and pcr products were purified with spin columns (promega, madison, wi) and sequenced directly by fluorescence-based cycle sequencing with amplitaq and bigdye terminators on an abi 377 sequencer at the national animal disease center genomics unit. sequences were analyzed using geneious 5.0 software (biomatters ltd, auckland, new zealand). quantitative virus copy numbers and serum cytokine levels were analyzed using a mixed linear model for repeated measures (proc mixed, sas 9.2 for windows, sas institute, cary, nc, usa). linear combinations of the least squares means estimates for each variable were used in a priori contrasts after testing for either a significant (po0.05) effect of prrsv challenge strain (vr-2332, rjxwn06, rjxwn06 contacts or sham inoculated controls). comparisons were made between groups at each time-point using a 5% level of significance (po0.05) to assess statistical differences. log10 transformed virus copy numbers and cytokine levels in balf and tbln homogenates were analyzed by analysis of variance using a general linear model for unbalanced data (proc glm, sas 9.2 for windows, sas institute, cary, nc, usa). a 5% level of significance (po0.05) was used to assess statistical differences. geometric mean back transformations were made for final data presentation in figures and tables. mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the u.s. department of agriculture. usda is an equal opportunity provider and employer. highly pathogenic porcine reproductive and respiratory syndrome virus repeated tlr9 stimulation results in macrophage activation syndrome-like disease in mice effects of intranasal inoculation with bordetella bronchiseptica, porcine reproductive and respiratory syndrome virus, or a combination of both organisms on subsequent infection with pasteurella multocida in pigs the human adaptor sarm negatively regulates adaptor protein trif-dependent toll-like receptor signaling clinical immunotoxicity of therapeutic proteins immune responses of pigs after experimental infection with a european strain of porcine reproductive and respiratory syndrome virus vaccine efficacy of porcine reproductive and respiratory syndrome virus chimeras in vivo growth of porcine reproductive and respiratory syndrome virus engineered nsp2 deletion mutants porcine respiratory and reproductive syndrome virus variants, vietnam and china physiological effects of exogenous administration of interleukin-1b in cows cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus changes in lymphocyte subsets and cytokines during european porcine reproductive and respiratory syndrome: increased expression of il-12 and il-10 and proliferation of cd4( à )cd8(high) acute phase response in porcine reproductive and respiratory syndrome virus infection genomic characterization of virulent, attenuated, and revertant passages of a north american porcine reproductive and respiratory syndrome virus strain neutralization of il-10 increases survival in a murine model of klebsiella pneumonia immunohistochemical identification of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model identification of virulence determinants of porcine reproductive and respiratory syndrome virus through construction of chimeric clones lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the mid-eastern region of china an infectious cdna clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus expression of inflammatory cytokines and toll-like receptors in the brain and respiratory tract of pigs infected with porcine reproductive and respiratory syndrome virus role of toll-like receptors in activation of porcine alveolar macrophages by porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations analytical verification of a pcr assay for identification of bordetella avium porcine reproductive and respiratory syndrome experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and 10-week-old pigs recombinant bovine interferon-g as an immunomodulator in dexamethasone-treated and nontreated cattle molecular epidemiology of prrsv: a phylogenetic perspective examination of the selective pressures on a live prrs vaccine virus interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus blood cytokines as biomarkers of in vivo toxicity in preclinical safety assessment: considerations for their use increased production of proinflammatory cytokines following infection with porcine reproductive and respiratory syndrome virus and mycoplasma hyopneumoniae interleukin-10, interleukin-12, and interferon-gamma levels in the respiratory tract following mycoplasma hyopneumoniae and prrsv infection in pigs effect of porcine reproductive and respiratory syndrome virus (prrsv) (isolate atcc vr-2385) infection on bactericidal activity of porcine pulmonary intravascular macrophages (pims): in vitro comparisons with pulmonary alveolar macrophages (pams) differential production of proinflammatory cytokines: in vitro prrsv and mycoplasma hyopneumoniae co-infection model emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark highly pathogenic porcine reproductive and respiratory syndrome the arterivirus replicase immune responses in piglets infected with highly pathogenic porcine reproductive and respiratory syndrome virus attenuation of porcine reproductive and respiratory syndrome virus strain mn184 using chimeric construction with vaccine sequence infection with porcine reproductive and respiratory syndrome virus stimulates an early gamma interferon response in the serum of pigs genetic variation and pathogenicity of highly virulent porcine reproductive and respiratory syndrome virus emerging in china secondary infection with streptococcus suis serotype 7 increases the virulence of highly pathogenic porcine reproductive and respiratory syndrome virus in pigs porcine reproductive and respiratory syndrome in china the 30-amino-acid deletion in the nsp2 of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china is not related to its virulence molecular characterization of transcriptome-wide interactions between highly pathogenic porcine reproductive and respiratory syndrome virus and porcine alveolar macrophages in vivo molecular characterization of porcine sarm1 and its role in regulating tlrs signaling during highly pathogenic porcine reproductive and respiratory syndrome virus infection in vivo highly virulent porcine reproductive and respiratory syndrome virus emerged in china the authors would like to recognize ann vorwald, sarah anderson, deb adolphson and amanda burow for their excellent technical assistance, and jason huegel, brian pottebaum, and jason crabtree for their exceptional animal care. we also appreciate the suggestions on manuscript layout and data organization made by crystal loving. key: cord-013174-whg64w0w authors: bhatta, tarka raj; ryt-hansen, pia; nielsen, jens peter; larsen, lars erik; larsen, inge; chamings, anthony; goecke, nicole b.; alexandersen, soren title: infection dynamics of swine influenza virus in a danish pig herd reveals recurrent infections with different variants of the h1n2 swine influenza a virus subtype date: 2020-09-10 journal: viruses doi: 10.3390/v12091013 sha: doc_id: 13174 cord_uid: whg64w0w influenza a virus (iav) in swine, so-called swine influenza a virus (swiav), causes respiratory illness in pigs around the globe. in danish pig herds, a h1n2 subtype named h1n2dk is one of the main circulating swiav. in this cohort study, the infection dynamic of swiav was evaluated in a danish pig herd by sampling and pcr testing of pigs from two weeks of age until slaughter at 22 weeks of age. in addition, next generation sequencing (ngs) was used to identify and characterize the complete genome of swiav circulating in the herd, and to examine the antigenic variability in the antigenic sites of the virus hemagglutinin (ha) and neuraminidase (na) proteins. overall, 76.6% of the pigs became pcr positive for swiav during the study, with the highest prevalence at four weeks of age. detailed analysis of the virus sequences obtained showed that the majority of mutations occurred at antigenic sites in the ha and na proteins of the virus. at least two different h1n2 variants were found to be circulating in the herd; one h1n2 variant was circulating at the sow and nursery sites, while another h1n2 variant was circulating at the finisher site. furthermore, it was demonstrated that individual pigs had recurrent swiav infections with the two different h1n2 variants, but re-infection with the same h1n2 variant was also observed. better understandings of the epidemiology, genetic and antigenic diversity of swiav may help to design better health interventions for the prevention and control of swiav infections in the herds. the influenza a virus (iav) is a negative-sense, single-stranded, eight-segmented rna virus belonging to the family orthomyxoviridae [1] . the main antigenic proteins are encoded by the surface gene segments hemagglutinin (ha) and neuraminidase (na). the six internal gene segments encode for polymerase b2 (pb2), polymerase b1 (pb1), polymerase a (pa), nucleoprotein (np), matrix (m1 and m2), and non-structural protein (nep-ns1) respectively [2, 3] . there are 18 different ha (h1 to h18) and 11 different na (n1 to n11) subtypes. most of these subtypes can be found in aquatic birds (h1-h16 and n1-n9), whereas only a few subtypes are found in mammals [1, 4] . in pigs, circulation of iav, so-called swine influenza a virus (swiav), is currently mainly limited to three different subtypes including h1n1, h1n2 and h3n2 [5] [6] [7] . avian-like swine h1n1 swiav, was first detected in european pig herds in the late 1970s [8, 9] and caused epizootic disease outbreaks that resolved shortly within a few weeks [10, 11] . however, several recent studies have shown that the dynamics of swiav have changed to a more enzootic form where the virus persists in the herds for months or even years [12] [13] [14] [15] [16] [17] . the altered dynamics of swiav from a short-term epizootic disease to continuous circulation in the herds, is probably a consequence of increased herd sizes and the continuous supply of naïve individuals that maintain the infection [18, 19] . in european pig herds, an average prevalence of 31% has been estimated for swiav infection [6] . the most prevalent subtypes identified in europe in recent years were the eurasian avian-like swine h1n1 (53.6%), the pandemic a/h1n1 2009 (h1n1pdm09) (10.3%), the human-like reassortant swine h1n2 (13%), and the human-like reassortant swine h3n2 (9.1%) [6] . in addition, a number of studies have also shown evidence of reassortants with internal gene segments of h1n1pdm09 and the surface gene segments of predominant enzootic swiav [20] [21] [22] [23] . in denmark, the human-like reassortant h1n2 has never been detected. however, another h1n2 reassortant is widespread among danish pig herds. this subtype is termed "h1n2dk" and has the ha gene of the eurasian avian-like h1n1 subtype and the na gene of the h3n2 human-like swiav [24] . in addition to the subtypes mentioned above, introductions of human seasonal iav occurs regularly, increasing the risk of novel swiav reassortants, and making the disease more difficult to control in the herds [23] . in addition, similar to other rna viruses, swiav has a high mutation rate, which drives the viral evolution and helps the virus evade the immune system by creating novel variants with modified antigenicity [25] [26] [27] . mutations in the antigenic sites (cb, sa, sb, ca1 and ca2) of the ha protein can lead to the virus being able to escape the binding of neutralizing antibodies [28, 29] . moreover, mutations in b-cell epitopes and t-cell epitopes of other iav proteins might also impact the host immunity [30] [31] [32] [33] . finally, mutations favouring altered n-linked glycosylation (nlg) sites near/within ha and na antigenic sites can also affect the binding of antibodies [34] . according to the danish agriculture and food council, denmark produces approximately 30 million pigs annually from around three thousand herds of which approximately 10 million are exported as weaners to other countries such as poland and germany [35, 36] . in contrast, denmark imports a very limited number of live pigs. in denmark and other countries worldwide swiav is one of the causes of respiratory infection in pigs [37] . swiav infections lead to destruction of the epithelial cells and impairs the immune system, thereby making the host more susceptible to infection by other viruses and bacteria. co-infection of pigs with distinct variants of swiav and other respiratory pathogens (like pasteurella multocida, mycoplasma hyopneumoniae, haemophilus parasuis, actinobacillus pleuropneumoniae, porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus) are known to cause enhanced disease compared to single pathogen infections, and are all part of the porcine respiratory disease complex (prdc), which can lead to massive economic losses [11, [38] [39] [40] . the aim of the present study was to monitor the molecular epidemiology of swiav circulating in pigs between two weeks of age to 22 weeks of age in a danish pig herd. a newly developed high-throughput real-time pcr (rtpcr) system (fluidigm, south san francisco, usa), which consists of 21 rtpcr assays targeting 18 selected respiratory and enteric viral and bacterial pathogens [41] was applied for initial detection of swiav in nasal swab samples. moreover, next generation sequencing (ngs) was used to characterize the complete genome of swiav during natural infections, and to examine the genetic variability in the antigenic sites of the virus ha and na proteins. in this study, only nasal swabs were collected and thereby the study did not include the introduction of a needle, which according to the danish law on animal experimentation (lbk number 474 of 15 may 2014) is the minimum intervention that requires a specific license. a trained veterinarian was involved in the sampling and data collection, and farmer consent was obtained before the sample collection. the study was designed as an observational cohort study to monitor/screen pig health (particularly swiav infection) in a danish farrow-to-finish continuous-flow pig herd. the herd had 985 sows and a farrowing area divided into a number of units. nursery pigs were housed at a separate site with 2000 pen places, and finisher pigs were raised at a third site with 1050 pen places. according to the danish spf (specific pathogen free) herd health declaration (seges svineproduktion 2020), the herd was declared positive for mycoplasma hyopneumoniae (m. hyo), actinobacillus pleuropneumoniae (app) type 6 and 12 and porcine reproductive and respiratory syndrome (prrs) type 2 virus, and negative for app type 2, prrs type 1 virus, brachyspira hyodysenteriae, atrophic rhinitis, demodex mites and lice. the herd had contract with a large abattoir and processing company in denmark, which in 2015 initiated the "raised without antibiotics" (rwa) concept [42] . at present (march 2020), 50 danish herds including this herd are included in the rwa program, where the producers are payed a premium price to reflect the cost of the increased workload, intensified hygiene measures and other interventions to prevent disease in the effort to reduce the use of antimicrobial agents. the herd produced and recruited gilts internally. after weaning, pigs selected for gilt recruitment were housed in a separate room at the sow site, and vaccinated with a live prrs type 2 virus vaccine (ingelvac ® prrs vet.) at 7 and 10 weeks of age, and additionally with m. hyo, porcine circovirus-2 (pcv2) (porcilis ® pcv m hyo) and swiav (respiporc flu3) vaccines at 7, 10 and 24 weeks of age. all adult sows were vaccinated against swiav simultaneously four times a year (respiporc flu3). due to clinical signs like nasal discharge and swiav positive laboratory testing, piglets were vaccinated at four days of age with respiporc flu3 (0.4 ml, off label use). during the first week after weaning, pigs were vaccinated against m. hyo and pcv2 (porcilis ® pcv m hyo). the prevalence of different iav strains in danish pig herds has been reported to be around 45% [43] . based on this data, the iav prevalence in this herd was assumed to be between 10% and 45%. by using a minimum sample size of 40, this should, at a very minimum, give at least one iav-positive sample with a high probability of getting iav-positive samples from 4-18 pigs. it was assumed from the outset of the study that some piglets may be lost to follow-up or that some may die and hence it was decided to use an initial sample size of 47. all live-born piglets born within three consecutive farrowing days (n = 518) were ear-tagged at birth with consecutive unique id numbers. from every 10th piglet (id number 10, 20, etc.) nasal swabs were obtained (at five to nine day intervals) at week 2 (n = 46), week 3 (n = 38), week 4 (n = 39), week 5 (n = 45), week 6 (n = 44), week 8 (n = 42), week 12 (n = 41) and week 22 (n = 13). overall, 47 individual piglets/pigs were sampled throughout the study (table s1 ). due to the variable weaning age (week 4 or week 5) of the piglets, the week 4 sampling was done either at the farrowing unit (week 4 f, n = 24) or in the nursery unit (week 4 n, n = 15) situated at separate buffer stables at the sow site. at week 5, all weaned piglets were moved into separate nursery units and housed with other pigs from the same herd of the same age until approximately 12 weeks of age. thereafter, the nursery pigs were transferred to a finisher site (~5 kilometre away) where they were housed until slaughter at approximately 22 weeks of age. this finisher site received piglets from the nursery units of the same herd. the samples were collected from the 25 april 2018 until the 20 september 2018. small-or medium-sized sterile rayon swabs (medical wire, wiltshire, uk) were used to collect the nasal swab samples. the swab was inserted into one nostril of the individual piglet and was then turned a full 360 • . samples were stored in 2 ml phosphate buffered saline (pbs) at 4-8 • c until delivered to the laboratory within two days after sampling. at weeks 2 and 4, piglets were clinically examined before obtaining the nasal swabs. clinical signs of nasal discharge and conjunctivitis were recorded for each individual pig. all samples were processed at the centre for diagnostics (cfd), technical university of denmark (dtu). the pcr analysis was carried out at cfd-dtu, while the ngs was carried out at statens serum institute (ssi), denmark. all the collected samples were vortexed for 1 min, the rayon swab was then removed from the tube and 400 µl of the fluid transferred to a 2 ml eppendorf (ep) tube. the 2 ml ep tubes were centrifuged at 9000× g for 5 min at room temperature. finally, 200 µl supernatant was used for nucleic acid extraction. nucleic acid (both dna and rna) was extracted using the qiacube ht extraction robot (qiagen, hilden, germany) and the cador pathogen 96 qiacube ht kit (qiagen) according to the manufacturer's instructions. the extracted nucleic acids were stored at −80 • c for further use. the extracted nucleic acids were subjected to reverse transcription using a high capacity cdna rt kit (applied biosystems, foster city, ca usa). a final volume of 10 µl reaction mix was prepared by mixing 1 µl of 10x rt buffer, 0.4 µl of 100 mm dntp mix, 1 µl of 10x random hexamer, 0.5 µl of multiscribe rt enzyme, 2.1 µl of nuclease free water and 5 µl of extracted nucleic acids. finally, cdna synthesis was carried out in a t3 thermocycler (biometra, fredensborg, denmark) with the given cycling conditions: 25 • c for 10 min, 37 • c for 120 min followed by 85 • c for 5 min and finally paused at 4 • c. the cdna sample was pre-amplified using 2x taqman preamp master mix (applied biosystems). a total volume of 10 µl was prepared by mixing 2.5 µl of cdna with 5 µl of 2x taqman preamp master mix (applied biosystems) and 2.5 µl of a 200 nm primer mix (containing the different sets of primers used for the detection of different pathogens) as previously described [41] . in brief, pre-amplification was carried out in a t3 thermocycler (biometra) using the program 95 • c for 10 min followed by 14 cycles of 95 • c for 15 s and 60 • c for 4 min. finally, the amplification was paused at 4 • c and the pre-amplified product was stored at −20 • c for further use. initially, the collected samples were screened for iav by using the high-throughput rtpcr platform biomark (fluidigm, south san francisco, usa) and the 192.24 dynamic array (da) integrated fluidic circuit (ifc) chip (fluidigm). a 4 µl sample mix was prepared by mixing 2.2 µl of pre-sample mix (prepared by mixing 2 µl of 2x taqman gene expression mastermix (applied biosystem) and 0.2 µl of 20x sample loading reagent (fluidigm) for each sample) and 1.8 µl of pre-amplified sample. similarly, 2 µl primer/probe stock was mixed with 2 µl of 2x assay loading reagent (fluidigm). three µl of the assay mix and 3 µl of sample mix was loaded into the respective inlets of the 192.24da ifc chip. the 192.24 da ifc chip was placed in the ifc controller rx for loading and mixing for approximately 30 min. finally, the chip was inserted into the high-throughput rtpcr platform biomark (fluidigm) for thermal cycling with the following cycling condition: 50 • c for 2 min, 95 • c for 10 min followed by 40 cycles of 95 • c for 15 s and 60 • c for 60 s. all samples were tested in duplicates. positive and non-template (nuclease-free water) controls were included. amplification curves and cycle threshold (ct) values were obtained on the biomark system and finally analysed using fluidigm real time pcr analysis software 4.1.3 (fluidigm) as previously described [41] . samples found positive for iav using the high-throughput rtpcr system, were selected for rna extraction. the extraction was carried out for the selected nasal swabs using the qiacube extraction robot (qiagen) and the rneasy mini kit (qiagen) according to the manufacturer's instructions as described previously [16] . rna was eluted in 60 µl rnase-free water and stored at −80 • c. detection of iav in the extracted rna was performed in the rotor-gene q (qiagen) pcr platform using a previously published rtpcr assay targeting the matrix gene of iav [15, 44] . confirmed iav positive samples with ct values < 30 (using rotor-gene pcr) were used for full genome amplification of iav. a one-tube reaction amplifying each gene segment of iav was performed using a modified version [45] of a previously published assay [46] . five µl of the amplified one-tube full genome iav pcr products along with 10 µl of 1kb dna ladder (invitrogen, carlsbad, ca usa) were run on 0.8% agarose gels (invitrogen) to check if the bands representing all 8 gene segments of iav were visible on a bio-rad gel documentation system (hercules, ca, usa). only fully amplified iav pcr products were selected and were purified using a high pure pcr product purification kit (roche, mannheim, germany) following the manufacturer's protocol. the purified pcr products were sent for ngs on the illumina miseq sequencing platform at the state serum institute (copenhagen, denmark). sequence analysis was performed in clc genomic workbench 11.0.1. software (qiagen). all reads obtained from each of the samples were initially trimmed to remove short and low quality reads and primers/adaptors and then consensus sequences of each gene segment was constructed using the function "map reads to reference", using a panel of 22 sequences representing each different lineage of each gene segment known to be present in denmark. the consensus sequences of each gene segment were then aligned using the muscle algorithm [47] , and then examined for similarities using the function "create pairwise comparison". moreover, the function, blastn, was used to compare the generated consensus sequences with the online ncbi genbank database [48, 49] . further analysis was done by selecting some of the closest swiav sequences from the ncbi genbank. phylogenetic analysis was done after aligning all sequences of each gene using clustal-w [50] and using the maximum likelihood (ml) method with the best fitting substitution model in mega [51] . the ha subtype numbering was done using the influenza research database (ird) tool at http://www.fludb.org [52, 53] . the different amino acids present in the ha antigenic sites (cb, sa, sb, ca1 and ca2) were calculated by comparing our sequences with reference sequences [28, [54] [55] [56] . b-and t-cell epitopes were analysed by aligning our sequences with reference sequences [57] [58] [59] [60] using clustal-w [50] . differences present at seven antigenic sites (1, 2a, 2b, 2c, 2d, 3 and 4) of the na gene segment were calculated by comparing our sequences with reference sequences [61] [62] [63] [64] . netnglyc 1.0 (http://www.cbs.dtu.dk/services/netnglyc/) from dtu bioinformatics (department of bio and health informatics) [65] was used for the prediction of n-linked glycosylation sites in the ha gene segments only on the n-x-s/t sequons (excluding p at x) with a score threshold >0.5. fisher's exact test was used to determine any association of data using the analysis tool 2by2.xls at http://itve.dk/ [66] . the results of the high-throughput rtpcr analysis of nasal swab samples, including an assay for detection of the matrix gene of iav, allowed for estimation of the prevalence of swiav at all sampling times. swiav was detected in pigs throughout the sampling period starting from week 2 until week 22, indicating continuous circulation of iav in the herd ( table 1 ). the prevalence of swiav increased from the first sampling (week 2) (4.3%) until week 4 (33.3%). among week-4-old piglets, 41.7% (22.1-63.4% at 95% confidence interval) were iav-positive in the farrowing unit while 20% (4.3-48.1% at 95% confidence interval) were iav-positive in the nursery unit (weaned at week 4 or week 5). after weaning (week 5), the prevalence stabilized and then decreased, reaching the lowest prevalence at week 12 (2.4%). after the pigs had been transferred to the finisher site, the prevalence increased again, reaching 30.8% at 22 weeks of age. in total, 76.6% of the pigs were iav-positive at least once during the study period (table 1 ). occurrence of iav is defined as the detection of iav in nasal swabs in one or more consecutive weeks in the same pig, whereas recurrence is defined as the detection of iav from the same pig at two or more non-consecutive weeks [67] . of 47 pigs, 36 (76.6%) were found to be positive for iav at least once, whereas 11 (23.4%) pigs were found to be negative throughout the study (table s1 ). all the iav positive and negative pigs were housed together in mixed pens. among the positives, 7 of the 36 pigs (19.4%) were found to have recurrent iav (table s1 ), whereas 29 of 36 pigs (80.6%) were found to have only a single occurrence of iav. of these 29 pigs, 23 (63.9%) pigs were iav-positive only at a single sampling time, whereas nine pigs (25%) tested positive at two consecutive sampling time points (table s1 ). among the 46 piglets sampled in week two, 33 (71.7%) had nasal secretion and 20 piglets (43.5%) had conjunctivitis. however, only two (5.1%) piglets had nasal secretion, while four piglets (10.3%) had conjunctivitis in week 4. nasal secretion was recorded in both of the iav-positive piglets at week 2, while it was only present in two of the 13 positive piglets at week 4. similarly, conjunctivitis was observed in one out of two positive piglets at week 2 and was only present in one out of 13 positive piglets at week 4. no association between iav infection in the individual pigs and clinical signs of nasal secretion and conjunctivitis at week 2 and 4 was observed (supplementary tables s2, s3 , s4 and s5). fifteen samples that had a ct value < 30 in the iav rtpcr assay were selected for one-tube full genome amplification. of these, 11 samples (table s6 ) displayed clear bands representing all the iav gene segments on the agarose gel and were selected for iav whole genome sequencing (wgs) ( table s6 ). all the generated sequences for eight full gene segments from all the 11 pig samples have been deposited in ncbi genbank with accession numbers mt946974 -mt947061. eleven full length (1701 nt) ha gene segments from the study were used for phylogenetic analysis. twenty three additional ha reference sequences representing avian, human, classical swine and h1n1pdm09 subtypes were downloaded from ncbi genbank and included in the analysis. phylogenetic analysis revealed that all the 11 ha gene segments were of eurasian avian-like h1nx origin (1c.2 lineage using the global swine h1 clade classification system) [68] . all nine h1 sequences obtained from pigs sampled between week 4 and 8 were grouped together in one cluster and had pairwise sequence differences of 0 to 0.6% at the nucleotide level and 0 to 0.8% at amino acid level. this cluster had the highest sequence identity to a/swine/germany/holdorf-idt12357/2010(h1n2) (accession no.: kr699687) in a blastn search with~97% identity at the nucleotide level and~96.5% at the amino acid level [21] . the two h1 sequences obtained from pigs sampled at week 22 were 100% identical but clustered separately from the h1 sequences obtained from the younger pigs ( figure 1 ). these h1 genes had the highest sequence identity to a/swine/denmark/10-1725-1/2011(h1n2) (accession no.: kr700049) in the blastn search with identities of 95.4% at the nucleotide level and 96.3% at the amino acid level [21] . as mentioned earlier, sequences from two pigs shedding swiav at two non-consecutive sampling times were obtained (pig id 250 sampled at week 4 and week 8, pig id 380 sampled at week 5 and week 22; table s6 ). in the phylogenetic analysis, the ha sequences of pig id 250, obtained at weeks 4 and 8 were located in the same cluster and were~0.5% (9/1701) divergent at the nucleotide level and < 1% (5/566) divergent at the amino acid level. in contrast, the ha sequences of pig id 380 at weeks 5 and 22 were~12% (198/1701) divergent at the nucleotide level and 10% (56/566) divergent at the amino acid level. from the phylogenetic analysis of pig id 380 it was clearly seen that the ha sequence obtained at the first sampling were located in the cluster defined by the viruses from the younger pigs, whereas the ha sequence obtained at the last sampling (after the pigs had been transferred to the finisher site) were located outside this cluster, thereby representing another h1n2 variant (figure 1 ). the majority of the differences between the two different samplings (week 4 and week 8) for pig id 250 and week 5 and week 22 for pig id 380 were located in the antigenic sites of the ha protein between amino acid 78-240. three different amino acid differences were found in the antigenic sites (v144a and k145n at ca2 and s160h at the sa region) of pig id 250 at week 4 and week 8. whereas, 16 different amino acids differences were found in the antigenic sites of pig id 380 at week 5 and week 22 (table 2) . sequences from different individual piglets sampled at week 4 have one different amino acid (s160h) at the sa region and two different amino acids (v144a and k145n) at the ca2 region. similarly, sequences from pigs of 8 weeks of age had only one different amino acid (s160h) at the sa region. the two ha sequences from week 22 were found to have identical amino acids in the antigenic sites. the cleavage site with one arginine (psiqsr: 324-329) and fusion peptide sequence (glfgaiagfieggwtgmidgwyg: 330-352) were found to be conserved in all 11 full-length h1 ha sequences [28, 69] . the ha2 region of all the ha sequences were found to be more conserved compared to the ha1 region, as they were only approximately 0-9% divergent at the nucleotide level and 0-2% divergent at the amino acid level. however, the ha1 region of all the ha sequences were 0-13.6% divergent at the nucleotide level and 0-14.8% divergent at the amino acid level. eleven full-length (1410 nt) na gene segments were used for phylogenetic analysis (figure 2 ). twenty seven additional na reference sequences representing danish h1n2, european h1n2, h3n2 and asian and american h1n2 na were also used. the phylogenetic tree shown in figure 2 revealed that the n2 na gene segments from the present study were most identical to the na segment of the danish h1n2 and european h3n2 subtypes. the n2 sequences obtained from pigs sampled between week 4 and week 8 grouped together in one cluster (danish hxn2 type) and had pairwise sequence differences of 0 to 0.4% at the nucleotide level and 0 to 0.4% at the amino acid level. this cluster had the highest sequence identity to a/swine/germany/holdorf-idt12357/2010 (h1n2) (accession no.: kr699688) in the blastn search with an identity of 97.7% at the nucleotide level and >98% identity at amino acid level, when performing a pairwise comparison [21] . in contrast, the two n2 sequences obtained from pigs at week 22 were identical and were positioned apart from the cluster formed by the sequences obtained from the younger pigs. similar to the ha segment, this cluster had the highest sequence identity to a/swine/denmark/10-1725-1/2011 (h1n2) (accession no.: kr700051) in the blastn search, with an identity of 96.2% at the nucleotide level and >95% identity at the amino acid level [21] (figure 2 ). the n2 sequences obtained from pig id 250 at weeks 4 and 8 were found to be <0.1% (1/1410) divergent at the nucleotide level and 0.2% (1/469) divergent at the amino acid level. however, the two n2 na sequences obtained from pig id 380 at weeks 5 and 22 were found to be~14.5% (205/1410) divergent at the nucleotide level and~13.6% (64/469) divergent at the amino acid level. amino acids present in the antigenic sites (1, 2a, 2b, 2c, 2d, 3 and 4 ) of the n2 na sequences of pig id 250 at week 4 and week 8 were found to be identical. while the n2 sequence obtained from pig id 380 at week 5 was similar to the ones from pig id 250, the sequence from pig id 380 at week 22 were found to be~35.3% (12/34) divergent in the antigenic sites of n2. similarly, n2 sequences of pig id 380 at antigenic site 2d were found to be most divergent, at 75% (3/4), whereas no changes were found in the antigenic site 1 ( table 3 ). all 11 n2 na sequences have eight highly conserved amino acids (r118, d151, r152, r224, e276, r292, r371 and y406) at the inner shell of the na active site, which interact directly with sialic acids. similarly, ten highly conserved amino acids (e119, r156, w178, s179, d198, i222, e227, e277, n294 and e425) [61, 71, 72] were also present in an outer shell of the na active site. hence, all the amino acids present at na active sites were found to be conserved. table 3 . comparison of amino acid sequences of neuraminidase (na) antigenic sites of pig id 380 sampled at week 5 and week 22 from the pig herd. amino acid positions were numbered using an n2 numbering system. amino acid change all the six internal gene segments (pb2, pb1, pa, np, m1-m2 and nep-ns1) were compared with available online ncbi genbank reference sequences. all the internal gene segment sequences obtained from younger pigs (≤ 8 weeks) were found to be most identical (>98%) with the internal gene segments of h1n1pdm09 origin. in contrast, only four of the internal gene segments (pb2, pb1, pa and np) of virus sequences from older pigs (week 22) were of h1n1pdm09 origin. the remaining two internal gene segments (m1-m2 and nep-ns1) of sequences from older pigs (week 22) clustered with the h1n2dk and european h3n2 swiav subtypes, indicating that these viruses are the result of reassortment events between h1n2-like viruses and the h1n1pdm09 subtypes. in addition, respective internal gene segments obtained from pig id 250 at week 4 and week 8 were compared and were found to be 0-0.3% different at the nucleotide level and 0-0.4% at the amino acid level. a similar comparison of respective internal gene segments was also done for pig id 380 at weeks 5 and 22 and were found to be 3.7-22.1% different at the nucleotide level, while at the amino acid level they were 1.2-23.6% different ( table 4 ). the m1-m2 gene segments of pig id 380 at week 5 and 22 were found to be 6.5% divergent at the nucleotide level as shown in table 4 . similarly, the nep-ns1 gene segments of pig id 380 at weeks 5 and 22 were found to be most divergent (20.5%) ( table 4 ) and also clearly supported by two distinct clusters in the phylogenetic tree ( figure 3 ). nep-ns1 gene sequences obtained from pigs sampled between week 4 and week 8 made one cluster close to the h1n1pdm09 subtypes. in contrast, the nep-ns1 sequences obtained from pigs sampled at week 22 made a separate cluster and resembled the h1n2dk and european h3n2 subtypes (figure 3 ). table 4 . pairwise comparison of six internal gene segments of pig id 380 sampled at week 5 and week 22 both at the nucleotide and the amino acid level. week clearly supported by two distinct clusters in the phylogenetic tree ( figure 3 ). nep-ns1 gene 378 sequences obtained from pigs sampled between week 4 and week 8 made one cluster close to the 379 h1n1pdm09 subtypes. in contrast, the nep-ns1 sequences obtained from pigs sampled at week 22 380 made a separate cluster and resembled the h1n2dk and european h3n2 subtypes (figure 3 ). b-and t-cell epitopes present in all the swiav sequences between week 4 and week 8 were identical. one amino acid change (r487k) was observed in the b-cell epitope present in the ha2 part of the ha gene segment after comparing ha sequences from pig id 380 at week 5 and week 22. similarly, one (e101d) and four different amino acids (l86f, s91n, e219v and s220t) were found in the t-cell epitope sequence of np and ha gene segments, respectively. t-cell epitopes of other gene segments were found to be identical among the compared isolates. six to seven nlg sites were present in all the ha sequences obtained in this study of which five (at positions 21, 33, 276, 483 and 541) were well conserved between all analysed sequences. all the ha sequences obtained from pigs sampled between week 4 and week 8 had one separate nlg site at position 94, whereas the ha sequences obtained from pigs sampled at week 22 have two other nlg sites at position 125-126 and at position 165. the nlg site at position 94 was located near the ca2 antigenic site in the globular head domain of the ha protein sequence, whereas the nlg site at position 125-126 was located near the sa antigenic site (table 5) . similarly, the nlg site at position 165 was located within the sa antigenic site of the ha protein sequence. table 5 . n-linked glycosylation (nlg) sites of h1 hemagglutinin (ha) gene segments obtained from all the pigs from week 4 to week 8 and week 22. "+, ++, +++" indicates the nlg potential with score threshold > 0.5. "*" indicates that the nlg site is located in between amino acid 125-126 for h3 numbering system. the study was designed to describe the infection dynamics of swiav in one danish pig herd by following 47 pigs from 2 weeks of age until slaughter (approximately 22 weeks of age). using a high-throughput rtpcr system, we were able to determine the prevalence of iav, and by the use of ngs, we characterized the genetic and antigenic diversity of circulating h1n2 swiav. based on the analysis, it was found that the prevalence of iav in pigs reached a maximum around weaning (4-5 weeks) and then decreased until 12 weeks and then increased again at 22 weeks of age. at least two different h1n2 variants were circulating in the herd, with one of the h1n2 variants circulating at the sow and nursery sites, and the other circulating in the finisher site. finally, we also demonstrated that individual pigs could have recurrent iav infections either with a very similar h1n2 variant (pig id 250) or with two divergent h1n2 variants (pig id 380). this confirms previous findings that some pigs can have prolonged swiav infections and be subjected to re-infection, even with closely related swiav [15, 45] . the understanding of the epidemiology and the genetic and antigenic diversity of swiav in pigs may help to unravel the layers of swiav infection dynamics and viral evolution from birth to slaughter, thereby helping to design better health interventions for the prevention and control of swiav in the herds. the relative low prevalence of iav before week 4 might be due to the presence of maternally-derived antibodies (mdas), which in this herd were stimulated by the sows being vaccinated with respiporc flu3 four times annually [16, 74] . however, the mdas wane over time, and several studies have shown results indicating that piglets are no longer completely protected from around three to four weeks of age [15, 16, 74] . moreover, the loss of mda occurs at the same time as the piglets are weaned into the nursery, thereby mixing different litters of pigs, creating the optimal environment for iav circulation. this has also been observed in previous studies [15, 16, 45] . similarly, a higher prevalence of iav at week 4 also indicated that the piglet immune system did not respond to the vaccination at day 4, which is in accordance with a previous study showing no effect of early piglet vaccination against swiav [45] . one of the explanations for this is that the presence of mda may hinder an active immune response in the piglets, but it could also be due to the reduced vaccine dose used (0.4 ml) [75] in the piglets under study. after week 5, most of the weaned pigs likely developed immunity to the circulating iav subtype and at the same time no new pigs were introduced into the nursery, resulting in a lower prevalence of iav in accordance with other findings [76] . the increase in prevalence observed at week 22 at a separate finisher site, was most likely due to infections with a second h1n2dk variant that might have been circulating continuously in the finisher site. this site was managed as a multi-aged, continuous-flow pig herd. this h1n2dk variant from the finisher site differed significantly in antigenic regions from the h1n2dk variant circulating at the sow and nursery sites. mutations in the antigenic sites of eurasian avian-like swine h1 have previously been linked to a lack of cross protection and emphasize that the diversity within the subtypes, especially in the h1 avian-like viruses, have now reached a level where it makes no sense to consider viruses of the same subtypes as belonging to the same serotype [15] . however, the lack of a significant cross-reaction should be confirmed by hi-testing, which was not performed in this study. moreover, the na gene and the internal gene cassette were also different between the two h1n2dk variants, which could also impact the cross-protective immunity between different swiav variants of the same subtype [77, 78] . specifically, the internal gene cassette of the h1n2dk variant that infected the pigs in the sow and nursery sites had a complete internal gene cassette of h1n1pdm09 origin, whereas the h1n2dk variant circulating in pigs in the finisher site had an m1-m2 and nep-ns1 gene of eurasian avian-like h1nx origin. however, the protective role of immunity against the internal gene segments are still controversial [58, 79] . pig id 250 was infected twice with the same h1n2dk variant, which only showed minor genetic differences between samplings. however most of the differences were located in antigenic sites. hence, it can be speculated that even a small number of mutations could facilitate re-infection with the same subtype, thereby confirming the results of a previous study [45] . in summary, it can be concluded that re-infections can occur with both similar and different variants within the same subtype. the presence of prolonged (2-3 weeks consecutive) and recurrent (non-consecutive) shedding of iav within week 4 to week 8 pigs also indicated reinfection with the same subtype and this was documented by sequencing. a number of previous studies have shown reinfection with the same strain, leading to prolonged iav shedding [12, 45, 74] . the presence of mda may play role in the prolonged iav shedding, as mda may hinder an active immune response [45, 80] . pig id 380 was infected with two different h1n2dk variants, and most of the mutations were located in antigenic sites as mentioned above. similarly, the acquisition of nlg sites near/within sa and ca2 antigenic sites of the ha sequence may lead to a shielding effect on the antigenic sites and probably the emergence of new antigenic variants. a range of studies have shown that the shielding effect on ha antigenic sites may lead to an evolution of the ha sequence and be responsible for escaping the pre-existing immunity in the hosts [81] [82] [83] [84] . the presence of these major differences between variants within the same subtype emphasizes the presence of a massive genetic drift of eurasian avian-like h1 in danish herds [27] , which in turn could have consequences for vaccine efficacy as the current swiav vaccine available against the h1n1 subtype has not been updated since 2002-2003. in our study, the presence of clinical signs of nasal discharge and conjunctivitis in pigs harbouring iav at week 4 was not very evident. reduced levels of clinical signs could be due to the presence of mda. similarly, previous exposure or a low level of exposure to the virus might preclude clinical signs in pigs [85] [86] [87] . in contrast with other studies, our study did not find any association between iav infection and nasal secretion [15, 16] . however, clinical signs were only recorded at two stages (at week 2 and week 4) of the total samplings in this study. similarly, we did not find any association between iav infection and conjunctivitis, which is also supported by other studies [15, 16] . in conclusion, the complexity of swiav infection dynamics in pigs from the farrowing unit to the finisher unit has been demonstrated. a high infection pressure of swiav was identified during the end of the stay in the farrowing unit and the start of the nursery unit. in addition, it has been shown that the prolonged persistence of iav in pigs could be due to re-infection with iavs that are closely related to each other. similarly, re-infections with different strains within the same lineage can also be expected, as the genetic changes affect important antigenic epitopes. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/12/9/1013/s1, table s1 : occurrence and recurrence of iav in pigs from week 2 to week 22 in the pig herd using high-throughput rtpcr. "+" indicates a positive case of iav whereas '-' indicates a negative case of iav. green boxes indicate the non-consecutive detection of iav, yellow boxes indicate the consecutive detection of iav, whereas blue boxes indicate the single detection of iav. "na" indicates the unavailability of the sample for iav detection test. table s2 : association between iav infection and nasal secretion at week 2. table s3 : association between iav infection and nasal secretion at week 4. table s4 : association between iav infection and conjunctivitis at week 2. table s5 : association between iav infection and conjunctivitis at week 4. table s6 : nasal swabs selected for whole genome sequencing (wgs). new world bats harbor diverse influenza a viruses the biology of influenza viruses the evolution of epidemic influenza ecology and evolution of avian influenza viruses genetic and antigenic evolution of swine influenza viruses in europe and evaluation of their zoonotic potential european surveillance network for influenza in pigs: surveillance programs, diagnostic tools and swine influenza virus subtypes identified in 14 european countries from molecular subtyping of european swine influenza viruses and scaling to high-throughput analysis evidence for the natural transmission of influenza a virus from wild ducks to swine and its potential importance for man review of influenza a virus in swine worldwide: a call for increased surveillance and research van reeth, k. swine influenza. in diseases of swine the epidemiology and evolution of influenza viruses in pigs swine influenza virus infection dynamics in two pig farms; results of a longitudinal assessment study of the persistence of activity of the h1n1 influenza virus in swine intensive units out of epidemical phases influenza a virus infection dynamics in swine farms acute influenza a virus outbreak in an enzootic infected sow herd: impact on viral dynamics, genetic and antigenic variability and effect of maternally derived antibodies and vaccination longitudinal field studies reveal early infection and persistence of influenza a virus in piglets despite the presence of maternally derived antibodies global migration of influenza a viruses in swine high turnover drives prolonged persistence of influenza in managed pig herds population dynamics of swine influenza virus in farrow-to-finish and specialised finishing herds in the netherlands reassorted pandemic (h1n1) 2009 influenza a virus discovered from pigs in germany molecular epidemiology and evolution of influenza viruses circulating within european swine between reassortant pandemic (h1n1) 2009 virus in pigs triple-reassortant influenza a virus with h3 of human seasonal origin, na of swine origin, and internal a (h1n1) pandemic 2009 genes is established in danish pigs genetic and biological characterisation of an avian-like h1n2 swine influenza virus generated by reassortment of circulating avian-like h1n1 and h3n2 subtypes in denmark why are rna virus mutation rates so damn high? evasion of influenza a viruses from innate and adaptive immune responses substantial antigenic drift in the hemagglutinin protein of swine influenza a viruses molecular basis of the structure and function of h1 hemagglutinin of influenza virus structural basis of influenza virus neutralization vaccination potential of b and t epitope-enriched np and m2 against influenza a viruses from different clades and hosts cross-reactive human b cell and t cell epitopes between influenza a and b viruses conserved epitopes of influenza a virus inducing protective immunity and their prospects for universal vaccine development pre-existing immunity against swine-origin h1n1 influenza viruses in the general human population glycosylation of hemagglutinin and neuraminidase of influenza a virus as signature for ecological spillover and adaptation among influenza reservoirs structural development in danish pig production statpearls dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study interaction between mycoplasma hyopneumoniae and swine influenza virus swine influenza viruses: a north american perspective development of a high-throughput real-time pcr system for detection of enzootic pathogens in pigs raised without antibiotics monitoring of influenza a virus in pig final development and evaluation of a one-step real-time rt-pcr assay for universal detection of influenza a viruses from avian and mammal species limited impact of influenza a virus vaccination of piglets in an enzootic infected sow herd single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza a viruses muscle: a multiple sequence alignment method with reduced time and space complexity basic local alignment search tool using the basic local alignment search tool (blast). csh protoc clustal w and clustal x version 2.0 mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets influenza research database: an integrated bioinformatics resource for influenza research and surveillance influenza research database: an integrated bioinformatics resource for influenza virus research the predicted antigenicity of the haemagglutinin of the 1918 spanish influenza pandemic suggests an avian origin predicting the antigenic structure of the pandemic (h1n1) 2009 influenza virus hemagglutinin the antigenic structure of the influenza virus a/pr/8/34 hemagglutinin (h1 subtype) detailed mapping of the linear b cell epitopes of the hemagglutinin (ha) protein of swine influenza virus t and b cell immune responses to influenza viruses in pigs identification of cross-reacting t-cell epitopes in structural and non-structural proteins of swine and pandemic h1n1 influenza a virus strains in pigs identification of swine influenza virus epitopes and analysis of multiple specificities expressed by cytotoxic t cell subsets structure of the catalytic and antigenic sites in influenza virus neuraminidase antigenic and biological characterization of influenza virus neuraminidase (n2) with monoclonal antibodies amino acid sequence changes in antigenic variants of type a influenza virus n2 neuraminidase location of antigenic sites on the three-dimensional structure of the influenza n2 virus neuraminidase prediction of glycosylation across the human proteome and the correlation to protein function measure of association and effect multiple genome constellations of similar and distinct influenza a viruses co-circulate in pigs during epidemic events a phylogeny-based global nomenclature system and automated annotation tool for h1 hemagglutinin genes from swine influenza a viruses peptides and membrane fusion: towards an understanding of the molecular mechanism of protein-induced fusion molecular evolution and phylogenetics the 2.2 a resolution crystal structure of influenza b neuraminidase and its complex with sialic acid sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase estimation of the number of nucleotide substitutions when there are strong transition-transversion and g+c-content biases effect of strain-specific maternally-derived antibodies on influenza a virus infection dynamics in nursery pigs effect of maternally derived antibodies on the clinical signs and immune response in pigs after primary and secondary infection with an influenza h1n1 virus protection against a european h1n2 swine influenza virus in pigs previously infected with h1n1 and/or h3n2 subtypes evaluation of the antigenic relatedness and cross-protective immunity of the neuraminidase between human influenza a (h1n1) virus and highly pathogenic avian influenza a (h5n1) virus prior infection of pigs with a recent human h 3 n 2 influenza virus confers minimal cross-protection against a e uropean swine h 3 n 2 virus protective antibodies against influenza proteins dynamics of influenza a virus infections in permanently infected pig farms: evidence of recurrent infections, circulation of several swine influenza viruses and reassortment events prediction of biological functions on glycosylation site migrations in human influenza h1n1 viruses a perspective on the structural and functional constraints for immune evasion: insights from influenza virus a carbohydrate side chain on hemagglutinins of hong kong influenza viruses inhibits recognition by a monoclonal antibody effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza a/h3n2 virus hemagglutinin differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity cytokines in the pathogenesis of influenza avian and swine influenza viruses: our current understanding of the zoonotic risk this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we want to acknowledge the herd owner for providing access to his property for sampling, and the herd-veterinarian providing assistance during sampling. similarly, we want to acknowledge the laboratory technicians hue thi thanh tran and jonathan rahlff rogersen for their help with laboratory tasks. the authors declare no conflict of interest. the funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. key: cord-266199-smlq11y9 authors: dhakal, santosh; renukaradhya, gourapura j. title: nanoparticle-based vaccine development and evaluation against viral infections in pigs date: 2019-11-06 journal: vet res doi: 10.1186/s13567-019-0712-5 sha: doc_id: 266199 cord_uid: smlq11y9 virus infections possess persistent health challenges in swine industry leading to severe economic losses worldwide. the economic burden caused by virus infections such as porcine reproductive and respiratory syndrome virus, swine influenza virus, porcine epidemic diarrhea virus, porcine circovirus 2, foot and mouth disease virus and many others are associated with severe morbidity, mortality, loss of production, trade restrictions and investments in control and prevention practices. pigs can also have a role in zoonotic transmission of some viral infections to humans. inactivated and modified-live virus vaccines are available against porcine viral infections with variable efficacy under field conditions. thus, improvements over existing vaccines are necessary to: (1) increase the breadth of protection against evolving viral strains and subtypes; (2) control of emerging and re-emerging viruses; (3) eradicate viruses localized in different geographic areas; and (4) differentiate infected from vaccinated animals to improve disease control programs. nanoparticles (nps) generated from virus-like particles, biodegradable and biocompatible polymers and liposomes offer many advantages as vaccine delivery platform due to their unique physicochemical properties. nps help in efficient antigen internalization and processing by antigen presenting cells and activate them to elicit innate and adaptive immunity. some of the nps-based vaccines could be delivered through both parenteral and mucosal routes to trigger efficient mucosal and systemic immune responses and could be used to target specific immune cells such as mucosal microfold (m) cells and dendritic cells (dcs). in conclusion, nps-based vaccines can serve as novel candidate vaccines against several porcine viral infections with the potential to enhance the broader protective efficacy under field conditions. this review highlights the recent developments in nps-based vaccines against porcine viral pathogens and how the nps-based vaccine delivery system induces innate and adaptive immune responses resulting in varied level of protective efficacy. viruses are the obligate intracellular nano-sized particles, which depend on host cell machinery for propagation and survival. they carry deoxyribonucleic acid (dna) or ribonucleic acid (rna) as their genomic material. there are several viruses from both dna and rna virus families that infect and produce disease in pigs [1] . there are many economically important swine viral infections which cause considerable morbidity and mortality, and responsible for significant economic losses to the pork industry (table 1 ). depending on their cellular and tissue tropisms, viruses cause pathological changes and clinical signs associated with respiratory system, reproductive and gastrointestinal tracts, nervous system, skin and extremities, alone or in combinations [1, 2] . porcine reproductive and respiratory syndrome virus (prrsv), an enveloped and positive-stranded rna virus of arteriviridae family, causes porcine reproductive and respiratory syndrome (prrs) [3] . prrs is responsible for over one billion dollar loss per year through direct and indirect costs in the us swine industry [4] . two entirely distinct genotypes of prrsv circulate in european (genotype 1/prrsv 1) and north american countries (genotype 2/prrsv 2) and cause tremendous economic loss. prrsv is transmitted through oral-nasal secretions and semen. the clinical signs include fever, anorexia, mild to severe respiratory problems, abortion and reproductive failures. it is the most common pathogen associated with porcine respiratory disease complex (prdc) [3] . swine influenza (flu) constitutes another persistent health challenge to the global pig industry. flu infection is caused by influenza a virus of orthomyxoviridae family which has negative-sense, single-stranded, segmented rna genome. influenza virus is transmitted through direct contact with infected animals or contaminated fomites, aerosols and large droplets [5] . the clinical signs of influenza infection include fever, anorexia, loss of weight gain and respiratory problems. influenza associated economic losses are due to morbidity, loss of body weight gain, increased time to market, secondary infections, medication and veterinary expenses [6] . influenza of swine origin occasionally infect humans and can even lead to pandemics as of 2009 [7] . porcine epidemic diarrhea virus (pedv), transmissible gastroenteritis virus (tgev) and porcine deltacoronavirus (pdcov) are enteric pathogens of young pigs [8] . these viruses belong to coronaviridae family and have positive-sense, single-stranded rna genome. tgev did serious economic damage to the swine industry in 1990s but with the advent of vaccines it has been largely controlled [8] . pedv still results in high morbidity and mortality in neonatal piglets with clinical signs like severe diarrhea, vomiting, dehydration and death. in 2013/14, pedv outbreak in the us led to over a billion-dollar loss [9] . rotaviruses are double-stranded rna viruses of reoviridae family, cause enteric infections in pigs. rotavirus of groups a, b, c, e and h are involved in porcine enteric infections. some of these porcine rotaviruses also have zoonotic potential [10] . foot and mouth disease (fmd) is another highly contagious, acute viral disease in pigs. the etiologic agent, fmd virus (fmdv), is a positive-sense, single-stranded rna virus of picornaviridae family [11] . fmdv is transmitted through direct contact with infected animals or contaminated sources. clinical signs include high fever, appearance of vesicular lesions on the extremities, salivation, lameness and death. fmdv causes frequent epizootics in many parts of the world resulting in severe economic loss, food insecurity and trade restrictions [11] . classical swine fever (csf) or hog cholera can result in high morbidity and mortality in pigs. it is caused by csf virus (csfv), an enveloped, positive-sense, singlestranded virus of flaviviridae family. transmission of csfv occurs through oral-nasal routes after contact with infected pigs or contaminated resources and even vertically from infected sows to piglets [12] . clinical signs include fever, anorexia, respiratory problems, neurological disorders, reproductive failures and death. csf is a notifiable disease to world organization for animal health (oie). the economic losses are associated with production loss, trade limitations and tremendous expenditures in eradication programs [13] . for example, the 1997/98 outbreak of csfv in the netherland resulted in death of 9 million pigs and economic losses of 2.3 billion dollars [14] . united states is free of csfv; however, this virus is endemic in many parts of the world including central and south america, africa and asia. porcine circovirus 2 (pcv2), a single-stranded dna virus of circoviridae family, causes multi-systemic disease referred as porcine circovirus-associated disease (pcvad). pcv2 is transmitted horizontally as well as vertically. direct contact is the most efficient way of horizontal transmission of this virus. the clinical signs of pcv2 infection include poor weight gain, respiratory problems, dermatitis, enteritis, nephropathy and reproductive failures [15] . five genotypes of pcv2 (pcv2a to pcv2e) are identified and circulate with high prevalence in swine herds causing significant economic losses worldwide [16] . porcine parvovirus (ppv) is the common cause of reproductive failure in swine herds. this single-stranded dna virus of parvoviridae family is transmitted through oral-nasal routes. stillbirths, mummification, embryonic death, and infertility (smedi syndrome) are linked to ppv infection. conventionally, ppv was considered genetically conserved but recent evidences suggest that several virulent strains have emerged due to its high mutation rate [17] . aujeszky's disease or pseudorabies in pigs is caused by suid herpesvirus 1, a double stranded dna virus belonging to herpesviridae family. the causative agent is spread primarily through direct animal-to-animal (nose-to-nose or sexual) contact. pseudorabies is characterized by nervous disorders, respiratory problems, weight loss, deaths in younger piglets and reproductive failures; and is one of the most devastating infectious diseases in pig industry [18, 19] . african swine fever (asf) causes hemorrhagic infection with high morbidity and mortality. the etiologic agent, asf virus (asfv), is a double stranded dna virus of asfarviridae family [20] . virus transmission occurs through direct contact with infected animals, indirect contacts with fomites or through soft tick species of the genus ornithodoros. clinical disease may range from asymptomatic infection to death with no signs. acute infections are characterized by high fever, anorexia, erythema, respiratory distress, reproductive failure in pregnant females and death [20] . asf is oie notifiable disease. united states is free of asfv, however, this virus is endemic in domestic and wild pig population in many parts of the world with possibility of transmission to the us and other nonendemic regions through animal trades [13] . the economic losses are associated with production loss, trade limitations and tremendous expenditures in eradication programs [13] . besides the rna and dna viruses described above, many other emerging and re-emerging viruses such as porcine hepatitis e virus, porcine endogenous retrovirus, porcine sapovirus, japanese encephalitis virus, encephalomyocarditis virus and others cause variable degree of impact in swine health and economic losses in pig industry globally [2, 21, 22 ]. different types of vaccines that are available against economically important swine viruses are listed in table 2 . vaccines against prrsv are being used in the us since 1980s [23] . both inactivated and modified-live virus vaccines are available and used globally. these vaccines are effective in reducing clinical disease and viremia mainly against homologous but not against heterologous infections [24] . therefore, different strategies are ongoing to develop live, inactivated, subunit and mucosal prrsv vaccines to induce better immunity and broader protection [23, [25] [26] [27] . swine influenza vaccines are also most effective when the vaccine strains closely match with the circulating strains [5, 28] . to increase the immunity and protection, vaccines containing multiple strains of influenza a virus (iav) and autogenous vaccines are widely used [5, 28] . cocirculation of multiple lineages of iav and frequent antigenic drift are responsible for reduced field efficacy of current swine influenza vaccines. moreover, the most commonly used whole inactivated iav vaccines administered via intramuscular route do not induce adequate mucosal antibody and cellular immune responses, suffer maternal antibody interference in young piglets and even can cause enhanced respiratory disease [5, 28] . the emergence of highly virulent strains of pedv in recent years has highlighted the need of safe and effective vaccines against porcine enteric coronaviruses that prevents clinical disease, mortality and virus shedding in neonates [8] . modified live vaccines against rotavirus are available for use in pigs against rotavirus a but their efficacy under field conditions is questionable indicating the need of alternatives for porcine rotavirus management [10] . the available inactivated vaccines provided great help in prevention and control of fmd outbreaks in many countries. however, the development of these vaccines needs high level biocontainment facilities. further, the fmdv serotypes undergo continuous antigenic drift and escape the vaccine-induced immunity [29] . thus, fmd vaccines with less stringent regulatory procedures and multi-serotype protective efficacy are needed in the future. safe and highly efficacious live-attenuated vaccines are available against csfv but differentiation of infected from vaccinated animals (diva) is not possible with these vaccines, which limit their use during outbreak control or disease eradication programs [30] . inactivated whole virus or subunit vaccines based on pcv2a are highly adopted in pig farms and are efficacious in reducing clinical signs and improving the production parameters. however, infections are still widespread in vaccinated farms [16, 31] . further, the replacement of pcv2a to pcv2b and recently to pcv2d is in part contributed by the selection pressure exhibited by pcv2a-based vaccines [32] which highlights the need of vaccines that protect against multiple genotypes. the currently used inactivated vaccines of porcine parvo virus protect against old ppv strains but not against the newly emerging strains demanding for more efficacious vaccines [17, 33] . fortunately, pseudorabies has been eradicated in many countries including the us by using inactivated and attenuated vaccines together with stringent biosecurity measures. however, it is still a problematic disease in many countries including china and is also maintained in feral swine populations in other countries [18, 34] . the frequent emergence of virulent strains even in the vaccinated herds demands updated vaccine technology to achieve efficient control and ultimate global eradication of pseudorabies [19, 34] . vaccine is not available so far against asfv, and the control measures depend entirely on early identification and culling of infected herds and adoption of strict sanitary measures [35] . vaccine development is hindered by the antigenic diversity and multitude of immune-evasion strategies used by the virus. an effective vaccine will definitely help in control and eradication of asfv from endemic countries and prevent its geographical expansion [20] . [5, 28] porcine epidemic diarrhea (ped) rna particle, inactivated and live-attenuated virus (in asia) protective immune response in sows better mucosal immunity [8, 9] foot and mouth disease (fmd) inactivated virus less stringent requirements in vaccine production protection against multiple serotypes [29] classical swine fever (csf) live-attenuated virus diva potential [30] porcine circovirus associated disease (pcvad) inactivated, recombinant subunit multi-genotype protection [16, 31] porcine parvovirus infection inactivated virus protection against novel strains [17, 33] pseudorabies inactivated, live-attenuated virus protection against novel emerging strains [18, 19] african swine fever (asf) none novel cross-protective vaccine [20] 3 importance of nanoparticle-based vaccine delivery platforms development of vaccines has made significant impact on reducing the viral infectious disease burden in both humans and animals. however, there are still many diseases for which either we do not have vaccines or need substantial improvements over existing ones [36, 37] . in the past few decades, nanoparticles (nps)-based technologies have elicited significant interests in the development of novel vaccine candidates as they offer multiple benefits over inactivated virus or subunit soluble antigens. nps-based vaccines (nanovaccines) are prepared either by encapsulating vaccine components within the nps or by decorating the particle surface with viral antigens. nps protect antigens from proteolytic degradation, prolong their bioavailability and maintain slow and sustained antigen release. all of these properties help in induction of better immune responses compared to soluble antigen vaccines [38] . the different mechanisms used by various nps to facilitate immune modulation of antigen presenting cells (apcs) are depicted graphically in figure 1 . briefly, nps can enhance antigen adsorption and uptake by apcs; they can also facilitate antigen processing mechanisms; nps can induce maturation of dcs and promote antigen cross-presentation through major histocompatibility complex (mhc) class i to cd8 + t cells; and induce production of different innate cytokines that regulate humoral and cellular immune responses. nps-loaded antigens are readily phagocytosed by apcs; soluble antigens are not [39] . moreover, dendritic cells (dcs), the key player involved in bridging innate and adaptive immunity, preferentially internalize nps compared to microparticles (> 1000 nm). for example, when poly(lactic-co-glycolic acid) (plga) particles of size 300 nm to 17 âµm encapsulating ovalbumin were tested on mouse bone-marrow derived dendritic cells, 300 nm sized particles were taken up efficiently compared to larger ones [40] . the 300 nm sized plga nps resulted in greater activation of dcs and stronger antigen-specific t cells responses in immunized mice compared to soluble antigens and larger particles [40] . besides controlled delivery of antigens, nps also provide adjuvant-like functions. vaccine adjuvants either work as antigen delivery systems facilitating antigen uptake and presentation by apcs or they activate innate immune receptors for cytokine production and maturation/migration of dcs [41] . adjuvant-induced innate immune responses determine the type of adaptive immune responses generated such as t helper 1 (th1) versus t helper 2 (th2)-biased immunity [42] . alum, the most widely used adjuvant in humans, is safe and inexpensive. its compatibility has been proved favorable with different vaccine antigens. however, despite inducing potent antibody responses, alum is a weak-inducer of cell-mediated immunity. adverse reactions are observed at injection site with alum-based adjuvants [43, 44] . in veterinary vaccines, oil-in-water emulsions or saponins are the most common adjuvants. these can also cause adverse reactions at the injection sites [45, 46] . while number of adjuvants are available for parenteral vaccinations, very limited options are available for intranasal (in) or other alternative routes of immunization [41, 47, 48] . nps can serve as an alternative adjuvant for human and animal use as they act both as antigen delivery system and activate the innate immune responses [49] [50] [51] . further, the modern vaccination approach has shifted from traditional whole pathogen-based antigens to small fraction (subunit) of the pathogen. however, purified whole inactivated pathogen and subunit or recombinant antigens by themselves are poorly immunogenic and require a potent immunostimulatory platform to augment the immune response. this can be achieved through nps-based technologies [47, 52] . nps-based platforms can be used to deliver multiple antigens or antigen/adjuvant combinations, which improves antigen uptake and concurrent activation of apcs leading to innate immune programming [53, 54] . co-delivery of cpg oligodeoxynucleotide and tetanus toxoid in nanospheres induced significantly greater t cell proliferative response and 5 to 16 times greater igg antibody isotypes in mice after subcutaneous immunization compared with the group that received tetanus toxoid and cpg oligodeoxynucleotide in soluble form [54] . likewise, co-delivery of melanoma antigen and toll-like receptor (tlr) 4 agonist in plga nps induced therapeutic anti-tumor effects that are mediated through potent cd8 + t cell activation [55] . nps can be surface modified to target microfold (m) cells, macrophages or dcs, and could be used for mucosal vaccination through oral, nasal or other mucosal routes of immunization. in mice, surface coating of plga nps encapsulating hepatitis b virus vaccine antigens with lectin resulted in efficient targeting of oral delivered nps to mucosal m cells and induced secretary iga antibody response in mucosal surfaces [56] . likewise, dcs targeted chitosan nps loading plasmid dna encoding nucleocapsid protein of severe acute respiratory syndrome coronavirus (sars-cov) induced better nucleocapsid protein-specific mucosal iga antibody response compared to soluble unentrapped antigens after nasal immunization in mice [57] . a targeted t-cell mediated immune response is critical in protection against intracellular pathogens such as viruses. beneficially, nps-delivered antigens are useful in antigen cross-presentation to cytotoxic t lymphocytes (ctls) and development of robust cell-mediated immune response [58, 59] . plga-based particulate vaccines are shown to induce efficient t-cell immunity in mice and pigs [60] [61] [62] [63] . similarly, rodent and pig studies have shown that polyanhydride nps-based vaccines also enhance cellular immunity [50, 64] . thus, immunogenic properties of different polymer-based nps could be exploited to improve the efficacy of vaccines for use against porcine viral infections. in this review, only studies conducted in pigs related to the development and evaluation of nps-based vaccine candidates by using virus-like particles (vlps), biodegradable polymers, polysaccharides and liposomes against porcine viral infections are included (table 3) . vlps are constructed using viral structural proteins, which can self-assemble but are non-infectious as they lack the viral genomic material. vlps mimic the virion and can effectively induce innate and adaptive immune responses [65] . vlps are produced using different bacterial, insect, yeast or mammalian expression systems [66] . due to their smaller size and particulate nature, vlps-based vaccines are processed and presented not only through mhc class ii but also through mhc class i pathway leading to the generation of antibodies as well as ctl responses [67, 68] . the potential use of vlps in porcine viral vaccine development is evident through the success in commercialization of human papilloma virus (hpv), hepatitis b virus and malaria vaccines by adapting this technology [69] . in one study, prrsv vlps containing five (gp5, gp4, gp3, gp2a and m) and two (gp5 and m) viral surface proteins were generated using the baculovirus expression system. prrsv vlps vaccine was mixed at 1:1 ratio with mycobacterium tuberculosis whole cell lysate (m. tuberculosis wcl) adjuvant and administered into pigs. vlps-vaccinated pigs were partially protected with 2-log reduction of virus titers in lungs. vlps-vaccinated pigs also had enhanced ifn-î³ response compared to mock challenge pigs [70] . however, in another study, when pigs were vaccinated in with prrsv vlps expressing n, m, gp5 and e proteins, enhanced viremia accompanied with higher level of ifn-î± cytokine response was observed [71] . the contrasting results in prrsv vlps study suggest the need for further research to fully evaluate the potential of vlps-based prrsv vaccines for swine. influenza-associated vlps expressing ha, na and m1 proteins of pandemic 2009 (h1n1) virus were inoculated twice intramuscularly with or without emulsigen (mvp lab, usa) adjuvant to pigs. this vaccine induced robust serum igg, mucosal iga and virus neutralizing antibody responses in pigs. after homologous virus challenge, vlps-vaccinated pigs had significantly reduced pneumonic lesions and virus titers were substantially lowered in upper and lower respiratory tracts compared to mock vaccinated animals [72] . many studies have been conducted with the goal to develop vlps-based fmdv vaccine using various expression systems encoding different viral antigens. rabbit hemorrhagic disease virus (rhdv) vlps expressing t-cell epitope of 3a protein of fmdv (rhdv-3a-vlps) was generated. this vlps vaccine induced maturation of bone marrow derived dendritic cells in vitro [73] . pigs immunized im with rhdv-3a-vlps together with montanide isa 206 adjuvant (seppic, france) induced higher serum igg and iga antibody responses. this vaccine also increased number of ifn-î³ secreting cells and lymphoproliferative responses in pbmcs compared to vaccine delivered without adjuvant and in rhdv-3a-vlps inoculated pigs; however, challenge experiments were not performed [73] . guo et al. constructed fmdv vlps expressing capsid proteins vp0, vp1 and vp3 and immunized pigs by im route [74] . vlps-vaccinated pigs produced virus-specific neutralizing antibodies and ifn-î³ response in peripheral blood mononuclear cells (pbmcs) as good as the inactivated fmdv vaccine control. after challenge with homologous virus, vlps-vaccinated pigs did not show specific clinical signs [74] . in another study, vp1 epitope peptides (ep141-160) of fmdv were inserted into the coat protein genes of male-specific coliphage (ms2) (cp-ep141-160 vlps) and injected im to pigs. this formulation resulted in induction of virus neutralizing antibodies and protected 60% of the immunized pigs compared to only 20% protection in peptide alone vaccinated animals. however, the protection was lower than inactivated vaccine (80%) indicating the need 50:90 of further improvement in this vlps either by using longer sequence of epitope or addition of other adjuvants [75] . vlps generated by insertion of vp1 epitopes of fmdv into porcine parvovirus vp2 were administered im to pigs. this vlps-vaccine induced higher virus neutralizing antibodies compared to synthetic peptide vaccine and resulted in better protection to challenge fmdv infection [76] . vlps have also been developed and tested against porcine neurotropic viruses [21, 22] . porcine encephalomyocarditis virus (emcv) vlps containing structural protein p1, nonstructural protein 2a and protease 3c were generated. after im administration together with montanide ims 1313 n vg adjuvant (seppic), vlps-vaccine induced sustained production of virus neutralizing antibodies comparable to commercial vaccine control. there was absence of any severe injection site reactions in vlps-vaccinated pigs [21] . this suggests the potential of developing vlps-based vaccine against emcv disease in pigs. likewise, in a recent study, japanese encephalitis virus genotype i (gi) vlps encoding premembrane (prm) and envelope (e) proteins were constructed. after subcutaneous immunization, this vaccine formulation induced robust neutralizing antibody response and protection against both homologous gi and heterologous giii jevs viruses. this finding indicates the cross-protection potential of vlps-based jev vaccine in pigs [22] . early study on pcv2 vlps used full length cap protein in escherichia coli expression system [77] . pigs vaccinated against pcv2 using cap vlps and isa 201 adjuvant (seppic) by im route induced cap-specific igg antibodies. vaccinated animals were apparently healthy with normal body weight gain and absence of any clinical signs of disease [77] . li et al. [78] showed induction of cap-specific igg antibodies in pigs vaccinated by subcutaneous (sc) injection of cap vlps. vaccinated pigs demonstrated reduced fever, viremia and mild pathological changes in lungs and lymph nodes compared to unvaccinated challenge animals. in another study, vlps co-expressed with cap protein and porcine gm-csf were administered im to pigs. this vaccine formulation induced significantly higher virus neutralizing antibodies in pigs. after virus challenge, vlps-vaccinated pigs had normal body weight gain compared to cap protein alone and commercial pcv2 vaccine groups. virus clearance, however, was observed in equally in vlps as well as other control vaccine groups [79] . only a single vlps-based vaccine study for porcine parvovirus was found [80] . ppv-vlps expressing major structural protein vp2 were administered im with double oil emulsion (doe) mineral oil adjuvant to weaned pigs. there was an induction of significantly higher neutralizing antibodies in vlps-vaccinated animals compared to inactivated vaccine group. further, when gilts immunized with this formulation were challenged with virulent ppv, virus was not detected in any of the fetuses. thus, ppv-vlps can be a potential vaccine candidate to prevent ppv-induced reproductive failure [80] . in summary, vlps of various origin can be used to develop more efficient vaccines against porcine viral infections. further studies are needed to evaluate their immunogenicity and protective efficacy under field conditions. plga is a co-polymer of lactic acid and glycolic acid. it is the most widely explored synthetic polymer in vaccine studies. it is a safe and non-toxic compound, and its hydrolysis products are readily assimilated into existing metabolic pathways [81] . plga nanoparticles are prepared either by oil in water emulsification or nanoprecipitation methods [82, 83] . plga nps bear a net negative charge. they enter apcs through pinocytosis and endocytosis, undergo reversal of charge and endolysosomal escape of entrapped vaccine cargo leading to antigen processing in cytoplasm, resulting in cross-presentation of antigen to cd8 + t cells through mhc class i pathway [59, 82] . plga nps are involved in maturation of dcs of mice and human origin, and controlled release of entrapped antigens leading to efficient expansion and differentiation of memory t-cells [84, 85] . in rodent studies, induction of robust t-cell immunity is observed with plga nps-based vaccines containing various vaccine antigens [55, 82] . further, plga is approved for drug deliveries in humans by the us food and drug administration (fda) and european medicine agency (ema) [82] . plga nps enhance antigen uptake and induce maturation of porcine apcs [62, 86, 87] . single dose of in immunization with plga nps-encapsulated inactivated/ killed prrsv antigen (nps-kag) induced activation of innate natural killer (nk) cells, î³î´ t-cells and secretion of innate cytokine ifnî± [86] . nps-kag vaccine also induced greater frequency of cd8 + t cells; increased secretion of ifn-î³; lowered frequency of t-regulatory cells; and reduced secretion of inflammatory cytokines compared to control kag-vaccinated animals [86, 88] . in a subsequent study, when nps-kag was co-administered in with m. tuberculosis wcl adjuvant, a balanced th1/ th2 immune response and augmentation of mucosal iga antibody response was observed. after heterologous prrsv challenge, pigs that received nps-based vaccine showed no clinical signs and also had significant reduction in lung virus load [87, 89] . plga nps were also used to encapsulate highly conserved influenza peptides and evaluated for efficacy in pigs after in administration. plga nps-based subunit vaccine resulted in induction of epitope-specific t-cell response but not the antibody response [61] . the t-cell biased immune response was also observed in pigs after in immunization with plga nps-encapsulated inactivated/killed influenza virus (plga-kag) vaccine in pigs [62] . in plga-kag vaccine administered animals observed reduced fever; lowered pneumonic lesions; and increased virus clearance from lungs after heterologous virus challenge compared to kag vaccine controls [62] . in another study, pedv kag was encapsulated in plga nps and used to immunize pregnant sows by in route. this nanovaccine induced higher virus-specific igg and neutralizing antibodies in serum and greater igg, iga and neutralizing antibody responses in colostrum. it also induced greater cell proliferation and ifn-î³ responses in restimulated pbmcs compared to kag vaccine controls. importantly, piglets born to nps-vaccinated sows had higher virus neutralizing antibodies and were better protected against homologous virus challenge than kag controls [90] . these studies suggest that plga nps can be used as an efficient means of enhancing virus-specific cell-mediated immune responses in pigs. polyanhydrides are another type of synthetic polymer widely studied for vaccine deliveries [91] . polyanhydride nps are synthesized by polycondensation or emulsification processes and are biodegradable, biocompatible and safe for vaccine delivery [91, 92] . they activate innate immune responses in a manner similar to lipopolysaccharides (lps) [93] . the surface-eroding nature of polyanhydride nps provides safe microenvironment for the encapsulated antigens and facilitates slow and sustained antigen release [92, 94] . induction of better antibody and cell-mediated immune responses by polyanhydride npsbased vaccines has been reported against viral, bacterial and parasitic infections [48, 91] . inoculation of polyanhydride nps-based siv kag vaccine (kag-nanovaccine) by in route enhanced cell-mediated but not the antibody responses in pigs [64] . after heterologous virus challenge, kag-nanovaccine group had six to eightfold reduction of nasal virus shedding compared to kag vaccine controls [64] . in a subsequent study, when kag-nanovaccine formulation was supplemented with cpg-odn adjuvant, both cell-mediated as well as mucosal iga antibody responses were improved [95] . after heterologous virus challenge, cpg-odn-adjuvanted kag-nanovaccine provided better protection through a significant reduction in influenza-induced fever, 16-fold reduction of nasal virus shedding and 80-fold reduction in lung virus titers compared to pigs immunized with five-times greater quantity of soluble killed antigen (kag) vaccine [95] . this study also indicates the dose-sparing ability of polyanhydride nps. thus, polyanhydride nps can also be used to induce better cellular as well as humoral immune responses in pigs. chitosan, alginate and other polysaccharides have also attracted attention as materials for nps formulation and drug delivery studies. chitosan is a natural polymer derived from deacetylation of chitin and is composed of glucosamine and n-acetylglucosamine residues [96] . due to the availability of amino and carboxyl groups in an acidic microenvironment, chitosan nps have net positive surface charge which makes them highly mucoadhesive and increases their half-time of antigen retention on mucosal surfaces [97, 98] . further, chitosan nps can reversibly open the epithelial cell tight junctions thereby improving paracellular and intracellular antigen transport across mucosal epithelial surfaces [99, 100] . chitosan nps also enhance antigen uptake by apcs, induce apc maturation and active secretion of innate cytokines [101, 102] . thus, chitosan nps form an attractive mucosal vaccine delivery vehicle. chitosan-based nps are used in pigs to deliver adjuvants such as bee venom and plasmid encoding porcine il-2 and il-4/il-6 genes, which improved induction of better virus-specific immune responses of respective vaccines against prrsv and pcv2 [103, 104] . chitosan nps enhance antigen uptake by porcine apcs and activate them to produce innate cytokines including ifn-alpha, tnf-alpha and il-1î² [105] . chitosan nps encapsulated siv kag (cnps-kag) vaccine administered twice through in route without any additional adjuvant in pigs induced the cross-reactive mucosal iga antibodies. chitosan nps-based vaccine also induced ifn-î³ response in pbmcs and tracheobronchial lymph nodes (tbln) better than kag vaccine controls. this vaccine formulation substantially reduced the challenge heterologous virus titers by up to 100-fold in both the upper and lower respiratory tracts compared to soluble kag vaccine. this finding emphasizes the potential benefits of using chitosan nps in future development of mucosal swine influenza vaccine for pigs [105] . recently, dendrimer-like-alpha-d-glucan (nano-11) nps derived from sweet corn variety sugary-1 was examined as an alternative, safe, cost-effective and potent adjuvant [106, 107] . nano-11 are positively charged nps which efficiently adsorb negatively charged antigens through electrostatic interactions. rodent studies have shown that nano-11 nps enhance antigen uptake by dcs, induce their maturation, activate them to produce pro-inflammatory cytokines and help in induction of antigen-specific antibodies [106, 107] . in a recent study, we observed that nano-11 nps with or without addition of siv killed antigen (kag) can stimulate porcine apcs and produce cytokines such as ifn-î±, tnf-î± and il-1î² [108] . pigs immunized via in route with nano-11 nps adsorbed siv kag at two-to-one ratio (nano-11 + kag) resulted in cross-reactive mucosal iga responses better than kag controls. moreover, pigs immunized im with nano-11 adsorbed ovalbumin (nano-11 + ova) had significantly greater igg1 and igg2 antibodies in serum compared with pigs vaccinated with ova alone [108] . these findings highlight the possibility of using cornderived nano-11 nps as a potential adjuvant in porcine viral vaccine development. liposomes can encapsulate both hydrophilic and hydrophobic molecules in aqueous and non-aqueous phases of their vesicles [109] . liposome vesicles protect antigens from enzymatic degradation, enhance antigen internalization by apcs and maintain controlled release of antigens [110] . liposome-encapsulated antigens can enhance both cellular and humoral immune responses [110, 111] . in a pig study, liposome nps were used as an im adjuvant for a pcv2 dna vaccine [112] . liposome nps-adjuvant induced higher neutralizing antibodies and ifnî³ response in pigs and reduced viremia of a challenge virus compared to alum-adjuvanted vaccine, providing the evidence that liposome nps can be a potent adjuvant in pigs [112] . in our recent study, we used liposome nps to encapsulate ten highly conserved peptides of different influenza viruses of human and pig origin and immunized pigs through in route co-administered with monosodium urate (msu) crystal adjuvant [113] . the liposome-adjuvant based vaccine enhanced the mucosal iga antibody response and induced peptide and virusspecific t-helper/memory cells and ifnî³ responses resulting in reduced fever and modest reduction in virus titers in the respiratory tract of pigs [113] . these studies highlight the fact that liposome-based nps can be used as an attractive vaccine delivery platform against porcine viral infections. virus infections have significant impact on pig industry worldwide. use of available vaccines have definitely helped in achieving strong control over some of the porcine viral infections such as food and mouth disease, transmissible gastroenteritis, classical swine fever and pseudorabies. vaccination also helped in reducing the clinical signs and increasing the production parameters in pcv2-associated disease. however, for many other porcine viruses, further improvements in existing vaccine platforms and development of novel vaccine delivery systems are necessary to: (1) induce better mucosal and cell-mediated immunity; (2) protect against emerging and re-emerging strains; (3) enhance the breadth (heterologous, cross-genotype and heterosubtypic) of immunity; and (4) differentiate between infected and vaccinated animals. nps-based vaccine delivery platforms such as vlps, biodegradable polymers and liposomes have great potential as they-(1) protect vaccine antigens from degradation; (2) facilitate antigen uptake and processing by apcs; (3) impart adjuvant potential; (4) can be used in mucosal and other alternate routes of immunizations; and (5) induce effective mucosal and cellular cross-protective (broader) immunity. research efforts are ongoing to develop porcine viral vaccines using nps-based technologies. however, more collaboration(s) and in-depth studies are warranted to make this innovative vaccine antigen delivery technology successful and practical for application in food animal industry. to date, almost all of the immunomodulatory mechanisms of nps-based vaccine delivery platforms have been studied in rodent disease models, which may or may not reflect the situation in pigs or other domestic animal species [114] . likewise, proper understanding of effect of size, charge and other physicochemical properties of nps after delivery through different routes of immunization in pigs is necessary to make efficient translation of this robust nps-based vaccine technology. similarly, studies should also focus on nps stability at different storage conditions and immunogenicity over a long period of time as they will directly associate with commercial aspect of the vaccine product. recent advances in nps-based adjuvant and vaccine delivery platforms in pigs demonstrate great promise to yield better candidate vaccines against many porcine viral infections with enhanced efficacy in the field. these nanovaccine technologies can also be adopted to develop effective vaccines against viral infections in other animal species, and knowledge gained could be exploited for improving the efficacy of existing human viral vaccines. the important viral infections of pigs emerging and re-emerging swine viruses porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system assessment of the economic impact porcine reproductive and respiratory syndrome virus on united states pork producers optimal use of vaccines for control of influenza a virus in swine assessing production parameters and economic impact of swine influenza, prrs and mycoplasma hyopneimoniae on finishing pigs in a large production system the role of swine as "mixing vessel" for interspecies transmission of the influenza a subtype h1n1: a simultaneous bayesian inference of phylogeny and ancestral hosts vaccines for porcine epidemic diarrhea virus and other swine coronaviruses lactogenic immunity and vaccines for porcine epidemic diarrhea virus (pedv): historical and current concepts porcine rotaviruses: epidemiology, immune responses and control strategies the pathogenesis of foot-and-mouth disease in pigs a review of classical swine fever virus and routes of introduction into the united states and the potential for virus establishment african and classical swine fever: similarities, differences and epidemiological consequences the classical swine fever epidemic 1997-1998 in the netherlands: descriptive epidemiology epidemiology and transmission of porcine circovirus type 2 (pcv2) porcine circovirus type 2 (pcv2) vaccines in the context of current molecular epidemiology molecular epidemiology and evolution of porcine parvoviruses pseudorabies virus in wild swine: a global perspective vaccines against pseudorabies virus (prv) a review of african swine fever and the potential for introduction into the united states and the possibility of subsequent establishment in feral swine and native ticks immunogenicity and safety of virus-like particle of the porcine encephalomyocarditis virus in pig genotype i of japanese encephalitis virus virus-like particles elicit sterilizing immunity against genotype i and iii viral challenge in swine improved vaccine against prrsv: current progress and future perspective challenges for porcine reproductive and respiratory syndrome virus (prrsv) vaccinology live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction mucosal vaccines to prevent porcine reproductive and respiratory syndrome: a new perspective influenza a virus vaccines for swine foot-and-mouth disease vaccines classical swine fever vaccines-state-of-the-art ten years of pcv2 vaccines and vaccination: is eradication a possibility? pcv2d-2 is the predominant type of pcv2 dna in pig samples collected in the u.s. during biology of porcine parvovirus (ungulate parvovirus 1) control of swine pseudorabies in china: opportunities and limitations african swine fever: a re-emerging viral disease threatening the global pig industry vaccination greatly reduces disease, disability, death and inequity worldwide veterinary vaccines and their importance to animal health and public health role of sustained antigen release from nanoparticle vaccines in shaping the t cell memory phenotype biodegradable nanoparticles as vaccine adjuvants and delivery systems: regulation of immune responses by nanoparticle-based vaccine biodegradable particles as vaccine delivery systems: size matters modes of action for mucosal vaccine adjuvants vaccine adjuvants: putting innate immunity to work alum adjuvant: some of the tricks of the oldest adjuvant mechanism of immunopotentiation and safety of aluminum adjuvants adjuvants in veterinary vaccines: modes of action and adverse effects adjuvants for veterinary vaccines-types and modes of action recent progress in mucosal vaccine development: potential and limitations respiratory nanoparticle-based vaccines and challenges associated with animal models and translation chitosan nanoparticles act as an adjuvant to promote both th1 and th2 immune responses induced by ovalbumin in mice poly(anhydride) nanoparticles act as active th1 adjuvants through toll-like receptor exploitation liposome-based adjuvants for subunit vaccines: formulation strategies for subunit antigens and immunostimulators vaccine delivery using nanoparticles potent antigen-specific immune responses stimulated by codelivery of cpg odn and antigens in degradable microparticles enhancement of immune responses by co-delivery of a cpg oligodeoxynucleotide and tetanus toxoid in biodegradable nanospheres co-delivery of cancer-associated antigen and toll-like receptor 4 ligand in plga nanoparticles induces potent cd8+ t cell-mediated anti-tumor immunity m-cell targeted biodegradable plga nanoparticles for oral immunization against hepatitis b dendritic cell targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein rapid endo-lysosomal escape of poly(dl-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery enhanced and prolonged crosspresentation following endosomal escape of exogenous antigens encapsulated in biodegradable nanoparticles cytotoxic t cell vaccination with plga microspheres interferes with influenza a virus replication in the lung and suppresses the infectious disease entrapment of h1n1 influenza virus derived conserved peptides in plga nanoparticles enhances t cell response and vaccine efficacy in pigs biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs induction of potent antigen-specific cytotoxic t cell response by plga-nanoparticles containing antigen and tlr agonist polyanhydride nanovaccine against swine influenza virus in pigs virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development virus-like particle engineering: from rational design to versatile applications phagocytic processing of exogenous particulate antigens by macrophages for presentation by class i mhc molecules efficient major histocompatibility complex class i presentation of exogenous antigen upon phagocytosis by macrophages major findings and recent advances in virus-like particle (vlp)-based vaccines development of a porcine reproductive and respiratory syndrome virus-like-particlebased vaccine and evaluation of its immunogenicity in pigs intranasal immunization of pigs with porcine reproductive and respiratory syndrome virus-like particles plus 2â�², 3â�²-cgamp vaccigrade adjuvant exacerbates viremia after virus challenge pandemic h1n1 influenza virus-like particles are immunogenic and provide protective immunity to pigs chimeric calicivirus-like particles elicit specific immune responses in pigs foot-and-mouth disease virus-like particles produced by a sumo fusion protein system in escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle promising ms2 mediated virus-like particle vaccine against foot-and-mouth disease immunogenicity of adenovirus-derived porcine parvovirus-like particles displaying b and t cell epitopes of foot-and-mouth disease characterization of porcine circovirus type 2 (pcv2) capsid particle assembly and its application to virus-like particle vaccine development construction and immunogenicity of recombinant porcine circovirus-like particles displaying somatostatin a novel subunit vaccine co-expressing gm-csf and pcv2b cap protein enhances protective immunity against porcine circovirus type 2 in piglets a novel recombinant virus-like particle vaccine for prevention of porcine parvovirus-induced reproductive failure poly lactic-co-glycolic acid (plga) as biodegradable controlled drug delivery carrier plga-based nanoparticles: an overview of biomedical applications the preparation of sub-200 nm poly(lactide-co-glycolide) microspheres for site-specific drug delivery delivery of a peptide via poly(d, l-lactic-co-glycolic) acid nanoparticles enhances its dendritic cell-stimulatory capacity duration of antigen availability influences the expansion and memory differentiation of t cells biodegradable nanoparticle-entrapped vaccine induces cross-protective immune response against a virulent heterologous respiratory viral infection in pigs adjuvanted poly(lactic-co-glycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs plga nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus vaccine helps in viral clearance in pigs an innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination poly (d, l-lactide-co-glycolide) nanoparticle-entrapped vaccine induces a protective immune response against porcine epidemic diarrhea virus infection in piglets recent advances in polyanhydride based biomaterials amphiphilic polyanhydrides for protein stabilization and release activation of innate immune responses in a pathogen-mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants structural and antigenic stability of h5n1 hemagglutinin trimer upon release from polyanhydride nanoparticles evaluation of cpg-odn-adjuvanted polyanhydride-based intranasal influenza nanovaccine in pigs chitosan-based gastrointestinal delivery systems strong adhesion and cohesion of chitosan in aqueous solutions contact timeand ph-dependent adhesion and cohesion of low molecular weight chitosan coated surfaces effect of chitosan on the permeability of monolayers of intestinal epithelial cells (caco-2) effect of chitosan on epithelial permeability and structure the effect of antigen encapsulation in chitosan particles on uptake, activation and presentation by antigen presenting cells immunogenic properties of a bcg adjuvanted chitosan nanoparticle-based dengue vaccine in human dendritic cells nasal delivery of chitosan/alginate nanoparticle encapsulated bee (apis mellifera) venom promotes antibody production and viral clearance during porcine reproductive and respiratory syndrome virus infection by modulating t cell related responses enhancement of immune response of piglets to pcv-2 vaccine by porcine il-2 and fusion il-4/6 gene entrapped in chitosan nanoparticles mucosal immunity and protective efficacy of intranasal inactivated influenza vaccine is improved by chitosan nanoparticle delivery in pigs alpha-d-glucan nanoparticulate adjuvant induces a transient inflammatory response at the injection site and targets antigen to migratory dendritic cells dendrimer-like alpha-d-glucan nanoparticles activate dendritic cells and are effective vaccine adjuvants corn-derived alphad-glucan nanoparticles as adjuvant for intramuscular and intranasal immunization in pigs liposomes as vaccine delivery systems: a review of the recent advances mucosal vaccine development based on liposome technology liposomeencapsulated antigens are processed in lysosomes, recycled, and presented to t cells development of porcine circovirus 2 (pcv2) open reading frame 2 dna vaccine with different adjuvants and comparison with commercial pcv2 subunit vaccine in an experimental challenge liposomal nanoparticle-based conserved peptide influenza vaccine and monosodium urate crystal adjuvant elicit protective immune response in pigs intranasal delivery of influenza antigen by nanoparticles, but not nkt-cell adjuvant differentially induces the expression of b-cell activation factors in mice and swine porcine epidemic diarrhea virus: an emerging and reemerging epizootic swine virus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the research reviewed in this article was supported by agriculture and food research initiative competitive grant no. 2013-67015-20476 from the usda-nifa and nanovaccine institute (2015-2018), iowa state university to rgj. salaries and research supports were provided by the state and federal funds appropriated to oardc. we thank dr. steven krakowka for scientific editing of the manuscript. authors' contributions sd wrote the article; gjr edited and revised the article. both authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-325433-a2fynm75 authors: riggs, shannon m. title: chapter 17 guinea pigs date: 2009-12-31 journal: manual of exotic pet practice doi: 10.1016/b978-141600119-5.50020-2 sha: doc_id: 325433 cord_uid: a2fynm75 publisher summary this chapter deals with the health and medical care issues of guinea pigs. guinea pigs have wide bodies with short limbs. they have a short, flat nose, laterally placed eyes, and hairless external pinnae. the dentition of the guinea pig is described as aradicular hypsodont. guinea pigs are best housed in well-ventilated, wire-sided cages with solid bottoms. if housed indoors, guinea pig enclosures do not require a cover, as these animals do not typically jump or climb. heavy food containers are recommended to make dumping of the receptacle more difficult. all food containers should be easy to disinfect and should be cleaned regularly, because guinea pigs have a habit of soiling their food bowls. these animals, native to the andes mountains, are very susceptible to hyperthermia and should never be housed in temperatures greater than 80°f. high humidity can also exacerbate a guinea pig's sensitivity to elevated temperatures by increasing the heat index. guinea pigs often do not exhibit clinical signs early in a disease process. therefore, a thorough physical examination can be extremely useful in determining the overall health status of the animal. c h a p t e r 1 7 guinea pigs shannon m. riggs guinea pigs, cavia porcellus, are members of the caviidae family of the order rodentia. guinea pigs are native to mountainous regions of south america where they were domesticated as long as 3000 years ago. wild species of guinea pigs, or cavies, still inhabit columbia, peru, venezuela, argentina, brazil, and paraguay. domesticated cavies in these countries are used for food. in the wild, guinea pigs live in small groups and, therefore, are often more comfortable in the presence of other guinea pigs when maintained as companion animals. extensive breeding has resulted in numerous varieties of coat color and characteristics. the most common breeds are the american (or english) which has a short, smooth coat and the multicolored teddy (figure 17-1); abyssinian ( figure 17 -2), which has a medium length coat in a whorled pattern; and the peruvian, which has a very long, smooth coat. guinea pigs have wide bodies with short limbs. a distinctive anatomic characteristic of species in the family caviidae is the number of digits on the front and rear feet (4 digits front feet and 3 digits rear). tails are usually very short or absent. the guinea pig has a short, fl at nose, laterally placed eyes, and hairless external pinnae. adult guinea pigs usually weigh between 700 and 1200 g, with the males being slightly larger than females. the average life span of the companion guinea pig is approximately 5 to 7 years. the dentition of the guinea pig is described as aradicular hypsodont (e.g., all teeth have a relatively long crown and are "open rooted"). 1 the maxilla is slightly wider than the mandi-ble, and the occlusal angle of the premolars and molars is marked compared to other rodent species. the dental formula of the guinea pig is 2(i 1/1, c 0/0, pm 1/1, m 3/3) = 20. the maxillary incisors are much shorter than those set in the mandible. the molars and premolars are not easily visualized without special instrumentation because of the small size of the oral cavity and tendency for the involution of the buccal surface. females are sexually mature at 6 weeks of age, whereas males on average reach puberty approximately 4 weeks later. gestation is long, when compared to other rodents, at 68 days. 2 as a result of this long gestation period, young are precocial when born. juvenile pigs usually eat solid foods by 4 or 5 days of age. 3 litter sizes range from 1 to 6, with an average of 3 to 4 young. 2 a female guinea pig should deliver her fi rst young before she is 6 months of age. if birth has not occurred before 6 months of age, the pubic symphysis becomes mineralized, with future pregnancies resulting in an inability of the sow to naturally deliver the babies. female guinea pigs that become pregnant after 6 months of age invariably require cesarean section deliveries. guinea pigs are best housed in well-ventilated, wire-sided cages with solid bottoms. wire-bottom cages may also be used; however, care must be taken to ensure that the mesh is small enough that a limb cannot become entrapped. an area of solid fl ooring should be provided, as uninterrupted time on wire mesh may predispose the guinea pig to pododermatitis. adequate space is needed in the enclosure for the guinea pig to move about unencumbered with enough space for a hide box. hide boxes or a secluded space is required for prey species (e.g., rodents) to reduce stress that may lead to disease problems. substrate products that contain aromatic oils (e.g., cedar and pine shavings) should not be used, as they can act as contact and respiratory irritants. appropriate bedding materials include recycled newspaper products, shredded paper, and aspen shavings. the enclosure should be cleaned thoroughly on a regular basis (e.g., 2 times per week) because unsanitary conditions predispose the guinea pig to pododermatitis, respiratory, and other health problems. if housed indoors, guinea pig enclosures do not require a cover, as these animals do not typically jump or climb. however, the sides of the enclosure should be high enough to prevent escape (approximately 25 cm). 2 heavy food containers are recommended to make dumping of the receptacle more diffi cult. all food containers should be easy to disinfect and cleaned regularly, as guinea pigs have a habit of soiling their food bowls. most guinea pigs readily accept drinking water from a sipper bottle, which will decrease spillage and will keep feces, urine, and bedding from contaminating the water. these animals, native to the andes mountains, are very susceptible to hyperthermia and should never be housed in temperatures greater than 80° f. high humidity can also exacerbate a guinea pig's sensitivity to elevated temperatures by increasing the heat index. all animals are very sensitive to environmental and/or nutritional changes. therefore, if changes have to be made, gradual exposure of the animal to the changes is recommended. an appropriate guinea pig diet includes a formulated, pelleted diet for that species, high-quality hay (e.g., timothy, orchard grass, oat) ad libitum, and ample fresh vegetables. as the animal's food intake is more dependent on volume consumed rather than calories consumed, a pet fed a predominantly pelleted diet (higher nutritional concentration) has a tendency to become obese. fruits and grains, if they are offered at all, should comprise a very small portion (<10%) of the total diet and offered only as treats. because guinea pigs lack the enzyme l-gulonolactone oxidase, they are unable to synthesize ascorbic acid from glucose. therefore, guinea pigs require supplemental vitamin c in their diets. although commercial guinea pig pellets are manufactured with vitamin c, the supplement often degrades rapidly, especially if the pellets are subjected to high heat and humidity. vitamin c placed in drinking water also degrades rapidly and should be changed daily. to ensure that a guinea pig is receiving a proper amount of vitamin c, it is necessary to supplement a diet of pellets and hay with plenty of fresh foods or often a specifi cally manufactured vitamin c supplement tablet (oxbow, inc., murdock, ne). many green, leafy vegetables, such as kale, mustard greens, dandelion greens, parsley, and many others, are excellent sources of ascorbic acid (box 17-1). the vitamin c requirement of an adult, nonbreeding guinea pig is 10 mg/kg/day. 2,3 in the united states, guinea pigs are not routinely vaccinated for infectious diseases. however, owners should be encouraged to have annual examinations that include an oral examination and a complete blood count. being prey species in the wild, guinea pigs are adept at hiding illness, and routine evaluations by a qualifi ed veterinarian may help in detecting abnormalities early. many health problems of guinea pigs are related to improper husbandry. during a routine veterinary visit, the owners should be asked to provide a detailed description of the animal's housing environment, including substrate, frequency of cleaning, ambient temperature, and exercise time. the diet history is also important. owners should be asked not only what the guinea pig is offered, but also in what proportions and of what foods the animal actually eats. most guinea pigs are quite docile and do not require aggressive restraint. often a hand on the animal's dorsum is adequate to restrain a guinea pig patient on the examination table ( figure 17 -3). when transporting a guinea pig, support the body with one hand under the thorax and abdomen while placing the other hand on the back to prevent the patient from falling or jumping ( figure 17-4) . if chemical restraint is required, gas anesthesia with isofl urane or sevofl urane is generally well tolerated. anesthetic gases can be delivered via mask or induction chamber. mild sedation can be achieved with an intramuscular injection of a combination of midazolam (0.2-0.5 mg/kg) and butorphanol (0.2-0.5 mg/kg) intramuscularly. guinea pigs often do not exhibit clinical signs early in a disease process. therefore, a thorough physical examination can be extremely useful in determining the overall health status of the animal. before beginning a physical examination, it is important to observe the animal before it has been stressed by restraint. a healthy guinea pig should be alert and aware of its surroundings. as guinea pigs are often shy animals, they may attempt to hide or escape. the examiner should use a thorough, systematic approach to focus on the respiratory character and rate, posture, and attitude of the animal. it is also important to have any instruments (e.g., transilluminator, stethoscope, thermometer, blood-collecting supplies) that may be necessary to decrease the amount of handling time for the patient. obtaining an accurate body temperature, heart rate, and respiratory rate is best accomplished at the beginning of the examination, as these parameters will invariably change with handling. an accurate weight should be obtained using an electronic gram scale. a "hands-on" physical examination should begin with the head; the veterinarian should assess the eyes for symmetry or discharge and check that the external pinnae of the guinea pig are hairless, as normal. the external ear canals often contain a small to moderate amount of dark, ceruminous debris. the nasal planum should be dry and fl at, whereas palpation of the ventral mandible may reveal deformities secondary to overgrowth of molar and premolar apices. the guinea pig coat varies somewhat with breed but, in general, should be smooth and shiny. guinea pigs often have a mild to moderate amount of dark sebaceous debris on the skin of the dorsum. older male guinea pigs may develop a focal accumulation of this debris at the base of the vertebral column, which may be referred to as the "grease gland." 4 thoracic auscultation and abdominal palpation can be performed as in other patients. heart and respiratory rates will vary depending on the degree of stress a patient experiences. as pulse and respiratory rates can be very rapid, careful auscultation is necessary to detect subtle abnormalities (e.g., murmurs, crackles, wheezes). auscultation of gut sounds is also an important part of the guinea pig physical exam. a healthy guinea pig should have 1 to 2 borborygmi per minute. the practitioner should keep in mind that stress will decrease gastrointestinal (gi) motility; therefore, a stressed animal will often have a decrease in borborygmi. structures normally found on abdominal palpation include kidneys, urinary bladder, cecum, and intestines. fecal pellets are often palpable in the colon. careful examination may reveal the presence of abnormalities such as gi distention, masses, or, in females, ovarian cysts. limbs and joints should be carefully evaluated because thickened or painful joints may be indicative of a vitamin c defi ciency. a complete oral examination is an important part of the guinea pig exam. because of the potential stress associated with the oral exam, this evaluation should be reserved for the end. the oral cavity of the guinea pig is very narrow with a small opening, making visualization diffi cult ( figure 17 -5) . instruments such as an otoscope with cone or a human nasal speculum will increase visualization of the caudal oral cavity ( figure 17 -6). however, many dental lesions may be overlooked using these methods, and anesthesia is often required to adequately determine oral health. obtaining blood samples from guinea pigs for routine diagnostics is a challenging procedure. peripheral venipuncture sites available for most mammals (e.g., lateral saphenous vein and cephalic vein) are diffi cult to visualize in guinea pigs and only allow for the collection of small sample volumes. the jugular vein may be used; however, the short neck of the guinea pig and poor tolerance of the aggressive restraint by the patient restrict the availability of this site for blood collection. often to obtain an adequate blood sample, it is necessary to anesthetize the patient. once anesthetized, the large central vessels may be accessed with less stress on the patient and phlebotomist. acceptable venipuncture sites while the patient is anesthetized include the cranial vena cava ( figure 17 baseline blood work is an important tool in the routine monitoring of health as well as in the diagnosis of disease. a complete blood count is essential for assessing red blood cell and white blood cell parameters (box 17-2). guinea pigs have heterophils rather than neutrophils as the predominant circulating granulocyte. heterophils lack myeloperoxidase, the enzyme that causes purulent, liquid exudates. therefore, the debris contained in guinea pig abscesses is often found to be very thick and caseous, a fact that must be understood when treating these conditions. a normal guinea pig white blood cell differential will consist of primarily heterophils and lymphocytes, usually with a greater proportion of lymphocytes. the remaining leukocyte types (e.g., monocytes, eosinophils, basophils) are normally present in very low numbers. early stages of a guinea pig's infl ammatory response are often characterized by a shift in the differential white cell ratio (e.g., increased heterophils, decreased lymphocytes) rather than an increase in total leukocyte count. this ratio shift makes evaluating the entire leukogram essential for health evaluation (box 17-3). the platelet count is also an important marker of infl ammation in guinea pigs and other small mammal species. large increases in the platelet count (>1,000,000/μl) can be seen without an increase in the total white blood cell count. a cell type that is unique to guinea pigs is the kurloff cell. these large lymphocytes contain a cytoplasmic inclusion (e.g., a kurloff body). kurloff cells are noted most often in the peripheral blood of reproductive age females, although they are also identifi ed in male guinea pigs. the number of circulating kurloff cells will increase in response to exogenous estradiol and testosterone administration, although the effects were more dramatic with estradiol administration. 7, 8 other studies have shown the disappearance of kurloff cells in spayed female and castrated male guinea pigs. 8 the function of the kurloff cell is not completely understood, and these cells appear to lack lysosomes. at this time, there is no evidence that the kurloff cell has phagocytic activity. 8 the activity of kurloff cells appears to most closely correlate with that of natural killer cells found in other species. 7 plasma biochemical analysis is necessary for evaluation of organ function as well as plasma glucose and proteins. it is important to interpret the results of a chemistry panel in conjunction with history, physical exam fi ndings, and the results of other diagnostic tests. reference intervals for biochemical parameters are shown in box 17-4. urinalysis is a useful diagnostic test in guinea pigs with signs of upper or lower urinary tract disease. urine samples may be obtained by free catch, fl oor catch, or cystocentesis. if urine is to be cultured, it is ideal to use the cystocentesis method of collection. ultrasound guidance and/or mild sedation of the patient will aid the veterinarian and/or veterinary technician in obtaining the urine sample. the urine of guinea pigs is typically yellow to amber in color, but it may be darker and more orange depending on the patient's diet. pigments in the urine can sometimes be mistaken for hematuria, so differentiation is important. because guinea pigs are herbivores, the ph of guinea pig urine is alkaline, usually 8.0 to 9.0. crystalluria may be seen but is not a normal fi nding. if crystals are found in a urine sample, the guinea pig patient should be examined for urinary calculi. to minimize rotation, care should be taken to extend the limbs symmetrically when positioning the patient. because guinea pigs have stocky builds with short limbs, and because they resent aggressive restraint, sedation or anesthesia is helpful in obtaining diagnostic radiographs as well as in reducing the patient's stress ( figure 17 -11). table 17 -1 is a guideline for radiographic techniques used in common small mammal radiographic studies. dental malocclusion is a common disease problem in guinea pigs. radiographs of the skull are helpful in assessing the degree of malocclusion as well as potential bone involvement. in addition to lateral and dorsoventral views of the skull, right and lateral oblique views can help to localize lesions. magnifi ed views of the skull can be obtained by placing the patient on an elevated platform under the x-ray beam without changing the distance between the x-ray cassette and beam. ultrasound is another imaging modality that is very useful in the diagnosis of common guinea pig disease processes, such as ovarian cysts (figure 17 -12) and urinary tract calculi. as with radiographs, sedation or anesthesia can assist in reducing patient stress and improve the quality of images. guinea pigs often have a large amount of gas accumulation in the gi tract, which obscures the ultrasound image, sometimes making this imaging technique of limited value. advanced imaging techniques, such as computed tomography (ct) and magnetic resonance imaging (mri), are useful tools in the diagnosis of disease in guinea pig patients. limitations of these modalities include limited availability, expense, and decreased image quality compared with that of larger patients. mri has the added limitation of requiring a significant amount of time (often >45 min) under anesthesia with limited monitoring ability. regardless of the present limitations, veterinarians should be aware that these techniques are available and that reference material exists that depicts normal anatomy. 9 as guinea pig owners continue to demand high-quality care for their pets, these imaging techniques will likely become more commonplace in small mammal practice for these patients. microbiologic samples can be obtained for diagnosis of various guinea pig infections. exudates from nasal or ocular secretions can be examined for abnormal fl ora. some bacterial organisms (e.g., bordetella bronchiseptica) are diffi cult to culture. laboratories need to be advised as to the organisms in question so they can perform more specifi c diagnostic tests to obtain organism identifi cation. abscesses are another disease condition that may require culture for proper treatment. because guinea pigs form caseous abscesses, the purulent debris itself is typically not useful for bacterial isolation. for the best chance of identifying organisms within an abscess, a portion of the abscess capsule should be submitted to the laboratory. both aerobic and anaerobic bacterial cultures should be requested, especially if it is suspected that the abscess may have originated from a dental problem. roundworms and coccidia are seen in guinea pigs and can be identifi ed by fecal fl otation or fecal direct smear, as in other species. cryptosporidiosis has also been reported. 10 identifi cation of cryptosporidium organisms usually requires acid-fast staining or immunofl uorescent antibody testing in addition to fecal fl otation. ectoparasites (e.g., mites, lice, fl eas) commonly infest guinea pigs. visualization of the parasites and/or their waste, as well as skin scrapings, microscopic examination of hair follicles, and tape preparations can be useful in the identifi cation of these parasites. the term enterotoxemia refers to the overgrowth of toxinproducing bacteria in the gi tract, particularly clostridium diffi cile. this can occur with stress, an abrupt change in diet, gi stasis, or inappropriate antibiotic administration. the gi fl ora of guinea pigs is predominantly gram positive, and administration of antibiotics with a primarily gram-positive spectrum (e.g., beta-lactam antibiotics, macrolides, lincosamides) can lead to the depletion of normal gut fl ora, allowing colonization by opportunistic bacteria (e.g., gram-negative organisms, clostridium spp.). the dentition of the guinea pig is described as aradicular hypsodont, meaning all teeth grow throughout the animal's life and are open-rooted. 1 the dental formula of the guinea pig is i 1/1, c 0/0, p 1/1, m 3/3. the mandible of these animals is wider than the maxilla. the occlusal angle of the molars and premolars in the guinea pig is quite severe when compared with that of rabbits and other rodents (figure 17-13 ). guinea pigs lack the layer of yellow enamel present on the rostral surface of the incisors in most other rodents. dental malocclusion is a common disease process in guinea pigs. the development of malocclusion can be the result of multiple etiologies or a combination of factors (e.g., improper diet, genetics, trauma). incisor malocclusion alone is rare in guinea pigs, so a thorough oral examination is necessary to determine the presence of cheek teeth malocclusion. when the molars and premolars do not occlude properly, overgrowth of the crowns and reserve crowns takes place. with improper wear, sharp points can form on the buccal aspects of the maxillary cheek teeth and the lingual aspects of the mandibular cheek teeth. the mandibular cheek teeth can overgrow to such an extent that they entrap the tongue, making eating and drinking diffi cult (figure 17-14) . clinical signs can include inappetence or dysphagia, decreased fecal output, ptyalism, poor coat quality, and leth-argy. abscesses commonly occur at the apical aspects of overgrown molars and premolars. because the premolars and molars are elodont (open-rooted), it is possible that these teeth can overgrow and impinge on the nasolacrimal duct, causing ocular and nasal discharge. the reserve crowns can also overgrow into the nasal cavity, where oral bacteria may be seeded, causing rhinitis and sinusitis. diagnosis of dental disease is based on physical and oral examination fi ndings. careful palpation of the ventral mandible and maxilla may reveal bony protuberances corresponding to overgrowth of the apical surfaces of the cheek teeth. proper instrumentation is very important for adequate visualization of the oral cavity. dental speculums and pouch dilators are invaluable aids in obtaining a good view of the molars and premolars (figure 17-15) . however, many abnormalities can be overlooked in a conscious animal, so anesthesia is often required to obtain a thorough oral examination. visualization of the oral cavity and occlusal surfaces can be facilitated by the use of an endoscope (figure 17-16) . most endoscopes available in veterinary practice have a degree of angulation of the fi eld of view, allowing greater focal area. abnormal oral examination fi ndings may include an uneven occlusal surface or angle of the cheek teeth, formation of sharp points with or without associated ulceration of the oral mucosa, food impaction, and abnormal spaces (diastema) between teeth. imaging, including routine radiographs, magnifi ed skull radiographs, and computed tomography can be incorporated to better evaluate the extent and seriousness of the process (figure 17-17) . radiographic studies should include dorsoventral, lateral, and right and left oblique views. treatment is centered on restoring a normal occlusal plane to the teeth, a procedure that should be performed while the animal is under general anesthesia. unstable animals should be provided with supportive care before the anesthetic event. a high-speed surgical dental handpiece can be used to restore a normal occlusal plane and to reduce any sharp points. handheld trimmers are not acceptable, as they tend to crush teeth and can cause fractures and pulp exposure. 1 dremel tools are also not considered acceptable because the small size of the guinea pig oral cavity will not allow for appropriate trimming. with any dental instruments, care should be observed to minimize oral soft tissue trauma. when a guinea pig is diagnosed with dental malocclusion, it is important to convey to the owners that this will be a lifelong problem for their pets. with many cases, routine occlusal adjustments will be necessary for the remainder of the animal's life. tyzzer's disease is the common term for bacterial enteritis caused by clostridium piliforme. infection with this organism is more commonly described in small rodents such as mice and hamsters, although it has also been described in guinea pigs as well as other mammalian groups. 11 tyzzer's disease often presents acutely, with signs such as diarrhea, depression, and poor coat quality, often progressing rapidly to death. transmission is via the fecal-oral route. guinea pigs can be infected with c. piliforme without showing clinical signs. animals that are carrying the organism but showing no signs of illness have still been found to shed organisms. 12 animals that carry the organism and then become immunocompromised (e.g., stress, immunosuppressive drug therapy) often develop clinical disease. clostridium piliforme is an intracellular bacterium that will not grow on routine culture media, so antemortem diagnosis is diffi cult. tyzzer's disease begins as an intestinal infection, but later spreads to the liver hematogenously, causing areas of necrosis. 11, 13 positive identifi cation of the organism on necropsy requires examination of hematoxylin and eosin or silver staining of affected tissues. treatment should consist of fl uid and nutritional support as well as appropriate antibiotic therapy. unfortunately, the progression of the disease is rapid, making treatment unrewarding. prevention is key, focusing on owners' reducing housing stress, providing a proper diet, and maintaining a clean environment. cryptosporidiosis has been described as causing disease in guinea pigs in a laboratory setting. 10 diagnosis was made histologically from affected animals that lost weight, had diarrhea, and suffered an acute death. cryptosporidium organisms were found in the brush border of the intestinal tract from the duodenum to cecum, with associated infl ammation. 10 suspected coronavirus infection has been described in young guinea pigs. 14 clinical signs in affected animals included anorexia, weight loss, and diarrhea. 14 approximately half of the affected animals in the reported outbreak recovered. 14 necropsies performed on animals that died or were euthanized showed lesions consistent with coronavirus infection resulting from an acute to subacute necrotizing enteritis involving primarily the distal ileum. 14 coronavirus-like virions were identifi ed on transmission electron microscopy of feces collected from the lower gi tract at necropsy. 14 outbreaks of salmonellosis have occurred in guinea pig colonies. some of the organisms that have been isolated include s. enteritidis, s. dublin, s. fl orida, s. poona, and s. bredeney. 15, 16 salmonellosis has been described to affect guinea pigs in acute and chronic disease processes. with acute salmonellosis, guinea pigs often die after a brief illness characterized by nonspecifi c signs of illness and diarrhea. 15 usually only a few pathogenic abnormalities are found at necropsy, and confi rmation of a diagnosis is dependent on culture of affected tissues. chronic salmonellosis is a wasting disease, lasting several weeks. splenomegaly, hepatomegaly, and mesenteric lymphadenopathy are common postmortem fi ndings in affected animals. 15 guinea pigs that recover can remain chronic, intermittent shedders of salmonella organisms. yersinia pseudotuberculosis causes several disease syndromes in guinea pigs. 15, 17 the most common presentation affects the mesenteric and colonic lymph nodes, infi ltrating these lymph nodes with caseous nodules. clinically, affected guinea pigs will lose weight, have diarrhea, and develop a generalized lymphadenopathy. transmission of the disease can be either vertical or horizontal. as stated previously, guinea pigs require dietary supplementation of ascorbic acid because they lack the enzyme l-gulonolactone oxidase necessary for synthesis of this compound. although diets formulated for guinea pigs are supplemented with ascorbic acid, it is important to remember that it breaks down very rapidly, usually within the fi rst 90 days after production. if a guinea pig is not receiving vitamin c supplementation in its diet, the veterinarian can assume that the diet is defi cient in this important nutrient. ascorbic acid is a necessary component of collagen, and defi ciencies are often noted as manifestations of abnormal collagen synthesis. 13 clinical signs of hypovitaminosis c can include lameness, hemorrhage, lethargy, anorexia, poor coat quality, and bruxism. diagnosis of hypovitaminosis c is based largely on clinical signs and history. radiographs will reveal changes to the costochondral junctions and widening of the epiphyses of long bones. recommended treatment includes fl uids and nutritional support, pain control, and parenteral vitamin c supplementation. attention should also be paid to improvement of the guinea pig's diet at home. in addition to causing skeletal and cartilage abnormalities, vitamin c defi ciency has been known to reduce immune function. in vitro, vitamin c defi ciency was demonstrated to cause a reduction in migration of macrophages in guinea pigs. 18 urogenital dystocia dystocia most frequently occurs in primiparous sows that are bred after approximately 6 months of age. at this time, the symphysis between the pubic bones becomes fused and will not expand to allow the passage of fetuses. cesarean section must be performed in these cases to save the sow and the young. factors other than age that can predispose a sow to dystocia include large fetuses in relation to sow size, uterine inertia, and obesity. 13 urolithiasis occurs commonly in pet guinea pigs, and the common clinical signs associated with the disease include stranguria and pollakiuria, vocalizing when urinating, and hematuria. the underlying cause(s) of this condition is not completely understood but is likely associated with a genetic predisposition and/or the presence of a high-calcium diet. other less common underlying etiologies associated with urinary calculi formation include ureteral neoplasms (e.g., papilloma). 19 calculi are primarily composed of calcium carbonate, although magnesium ammonium phosphate hexahydrate and calcium phosphate calculi will also occur. 20 radiographic or ultrasonic imaging can be used to confi rm the location of the urinary calculi, which may be present in the renal pelvis, ureters, urinary bladder, or urethra (figure 17-18) . urinary tract calculi often require surgical removal. ideally, once removed, the calculus and/or a portion of the bladder wall should be cultured. it may also be helpful to analyze the composition of the stone to help determine ways of preventing recurrence. ovarian cysts are a common fi nding in middle-aged to older female guinea pigs. this disorder has a reported prevalence of 76% in female guinea pigs aged 1.5 to 5 years. 13, 21 multiple cysts can be present on one or both ovaries. clinical signs related to cystic ovaries can include abdominal distention, lethargy, and anorexia related to the space-occupying nature of the cysts. ovarian cysts that are actively producing hormones can produce bilaterally symmetrical alopecia of the fl anks. ovarian cysts can be quite large (up to several centimeters) and can often be identifi ed on physical examination. abdominal ultrasound is the most useful diagnostic tool for a defi nitive diagnosis. the most defi nitive treatment for cystic ovaries is ovariectomy or ovariohysterectomy, curing the current problem and preventing recurrence. in animals where surgery is not a viable option, ultrasound-guided percutaneous aspiration of ovarian cysts can be performed as a palliative treatment. however, without removal of the ovaries, the cysts will recur, requiring repeated procedures. pregnancy toxemia typically occurs in pregnant sows during the last 2 weeks of gestation. as with other species, pregnancy toxemia is the result of a negative energy balance and the metabolism of fat. sows that experience pregnancy toxemia are typically overweight and become anorexic. clinical signs include lethargy, dyspnea, and anorexia, usually progressing to death within a few days. 13 therapy of affected sows should center on providing nutritional support, correcting electrolyte imbalances, and preventing opportunistic infections. prognosis is generally considered poor, as many sows fail to respond to treatment. neoplasia of the reproductive tract is not commonly reported in guinea pigs, but several tumor types have been described. of the described reproductive neoplasms, the vast majority occur in female guinea pigs. uterine leiomyoma (often associated with ovarian cysts) 22 and leiomyosarcoma, ovarian teratoma, and granulosa cell tumor are reported neoplasms of the reproductive tract. 21 diagnosis, as in any other patient, should be based on the results of cytologic or histopathologic sampling. benign neoplasms may be resolved with ovariohysterectomy, but further diagnostics to determine the extent of local invasion should be performed before surgery takes place. a thorough diagnostic work-up, including imaging techniques (e.g., abdominal ultrasound, thoracic radiographs), should be performed to look for metastases of malignant tumor types. mycoplasma caviae has been isolated from the reproductive tracts of guinea pigs. 15 these guinea pigs are often unaffected by the organism, but metritis has been suspected to be associated with infection. as with other species, mycoplasma spp. is suspected to cause reproductive problems (e.g., abortion and decreased fertility). respiratory disease is a common presenting complaint in guinea pig patients. clinical signs can vary from sneezing and upper respiratory signs to severe dyspnea and death. bordetella bronchiseptica is one of the most common respiratory bacterial agents associated with pneumonia in guinea pigs. 15 many guinea pigs are carriers of the organism, which will cause clinical disease if the animal is stressed. a thorough history, obtained from the patient's owner, often reveals interactions with other species that are also subclinical carriers of b. bronchiseptica (e.g., rabbits, dogs). 13 clinical signs noted in guinea pigs infected by the bordetella organism include nasal discharge, dehydration, tachypnea, and lethargy. 23 diagnosis should be based on clinical and radiographic signs and history. confi rmation of the diagnosis can be determined with enzyme-linked immunosorbent assay (elisa) and indirect immunofl uorescence diagnostic tests, or culture of exudates. 24 treatment/preventive options for b. bronchiseptica infections in guinea pigs include an autogenous bacterin vaccine as well as three commercially available vaccines for bordetella spp. (porcine b. bronchiseptica and human b. pertussis), which were found to offer some protection against the development of bronchopneumonia in experimentally infected guinea pigs. 25 streptococcus pneumoniae can cause pleuropneumonia, pleuritis, and peritonitis in guinea pigs. 26 serologic detection of s. pneumoniae antibodies using elisa have been described in previously published papers. 26 on postmortem examination, lesions found to be associated with s. pneumoniae infection included pleuritis, pleural effusion, lung abscessation, otitis media, pericarditis, and others. 20 streptococcus pneumoniae was also identifi ed from septic arthritis lesions in a group of guinea pigs. 26 five guinea pigs in a laboratory colony demonstrated multiple enlarged joints from which pure cultures of s. pneumoniae were isolated. several other individuals in this colony had previously died with typical s. pneumoniae lesions (e.g., pleuritis, pericarditis) and lesions consistent with hypovitaminosis c. the group with septic arthritis also had scorbutic changes on necropsy. 26 streptobacillus moniliformis, the causative agent of rat-bite fever in humans, was isolated from a laboratory guinea pig with pneumonia. this organism is of particular importance because of its zoonotic potential. 27 adenovirus has been shown to cause necrotizing bronchopneumonia in guinea pig populations, although it can also produce a transient, subclinical infection. 28, 29 clinical signs include depression and dyspnea, but guinea pigs often die acutely without clinical signs. 30 identifi cation of adenovirus dna from diseased lung tissue was achieved using a polymerase chain reaction technique in one study. 28 development of this pcr assay for identifi cation of the virus has determined that the guinea pig adenovirus is distinct. 28 histopathologic examination of affected tissues of animals infected with adenovirus will reveal the presence of characteristic intranuclear inclusion bodies. 29 the most commonly reported neoplasm of the respiratory tract in guinea pigs is bronchogenic papillary adenoma. the prevalence of the tumor is as high as 30% in guinea pigs over the age of 3 years. 13 given the relatively more common occurrence of pneumonia in guinea pigs, bronchogenic papillary adenoma can often be misdiagnosed. for this reason, thoracic radiographs of guinea pigs with respiratory disease are highly recommended. yersinia pseudotuberculosis can cause septicemic pneumonia in guinea pigs. 15 death usually occurs rapidly, after the development of coughing and dyspnea. at necropsy, severe congestion of the lungs is found grossly and through histologic evaluation of the tissues. trixacarus caviae are sarcoptoid mites that commonly affect guinea pigs (figure 17-19) . affected guinea pigs are intensely pruritic, sometimes to the extent of seizure development. as with other mite infestations, diagnosis is based on the identifi cation of the parasites on skin scrapings. once defi nitive diagnosis has been made, treatment with ivermectin and selamectin (6 mg/kg q2-4wk) is usually effective. chirodiscoides caviae are fur mites diagnosed in guinea pigs. because it rarely causes clinical disease, treatment is usually unnecessary. 20, 31 gyropus ovalis and gliricola porcelli are species of lice that are commonly identifi ed in guinea pigs (figure 17-20) . infested guinea pigs may be pruritic, but are usually unaffected. 20 guinea pigs severely infested with these mites may demonstrate poor coat quality and alopecia. patchy hair loss without associated pruritus may be attributed to dermatophytosis, most commonly trichophyton mentagrophytes 2 (figure 17-21) . lesions are circular and scaled and usually occur on the face and head. 20 the diagnosis of dermatophytosis is made by a positive fungal culture. because of the zoonotic potential of these fungal organisms, care should be streptococcus zooepidemicus lancefi eld's group c is the causative agent of cervical lymphadenitis. this disease will cause severe swellings of the lymph nodes in the cervical region in guinea pigs. affected guinea pigs will frequently exhibit no other clinical signs but may become septicemic, with lesions affecting the heart, lungs, kidney, and skin. 20 the most effective treatment for cervical lymphadenitis is complete surgical excision of the affected lymph nodes, followed by appropriate antibiotic therapy based on culture and sensitivity testing. lancing and draining the abscesses is often not curative, as the abscesses form thick capsules that harbor organisms, leading to recurrence. yersinia pseudotuberculosis has also been shown to cause cervical lymphadenitis in guinea pigs. 17 although the affected guinea pigs are usually not ill, the concern is that rupture of the abscesses will release large amounts of this potentially zoonotic organism into the environment. another potential causal agent of cervical lymphadenitis is streptobacillus moniliformis. 32 one report exists of a cervical mass, initially believed to be cervical lymphadenitis, which was histologically determined to be a thyroid papillary adenoma. 33 the mass was apparently nonfunctional and the guinea pig appeared otherwise healthy, but surgical resection was curative. guinea pigs housed in cages with wire fl ooring are predisposed to developing ulcerated lesions on the plantar surfaces of their feet. mild lesions may appear as hyperemic, swollen areas of the weight-bearing surfaces. these lesions can progress to ulcerations with secondary infections (figure 17-22 ). vitamin c defi ciency has also been considered a predisposing factor for the development of pododermatitis, as affected animals may be in pain and reluctant to move, resulting in the development of pressure sores. 20 ulcerated, infected lesions should be managed with appropriate antibiotics and antiinfl ammatory drugs, but the focus of treatment should be improving husbandry (e.g., providing appropriate fl ooring/bedding, vitamin c supplementation). trichofolliculomas are the most common cutaneous tumor seen in guinea pigs. 20 these benign tumors often occur on the dorsum and are typically round and hairless (figure 17-23 ). another cutaneous abnormality that has been described in guinea pigs is cutaneous vascular malformation. 34 the lesion described was a raised, ulcerated plaque on the animal's fl ank, which bled intermittently. ultimately, the cutaneous vascular malformation resulted in fatal hemorrhage. histologically, the lesion was described as an expansile mass extending into the skeletal muscle and consisting of multiple vascular spaces of varying sizes. these vascular spaces were lined with endothelial cells. compared to the incidence of neoplasia in other mammalian species, the incidence of neoplasia in guinea pigs appears low or is underreported. however, there have been several reported cases of neoplasia in guinea pigs. as more guinea pig owners seek quality veterinary care for their pets, reports of neoplasia will increase. lymphoma is the most commonly reported neoplasia in guinea pigs. 20 clinical signs associated with guinea pig neoplasia include lymphadenopathy, splenomegaly, and hepatomegaly. leukemic and aleukemic forms of guinea pig lymphoma have been identifi ed. thyroid carcinoma has been reported in an adult guinea pig that demonstrated multiple masses in the ventral cervical region. 35 as there was no evidence of neoplastic disease elsewhere on postmortem examination, this was considered a primary tumor. mesothelioma has been reported in the abdomen of an adult guinea pig. 36 the guinea pig had died of complications associated with pneumonia, and the mesothelioma was diagnosed through a necropsy examination. the mass consisted of diffuse nodules on the serosal surfaces of numerous organs in the abdominal cavity. guinea pigs are native to cooler regions of south america and are therefore relatively intolerant of temperatures above 80° f, lower if the environment is also humid. guinea pigs should be housed in well-ventilated enclosures at temperatures between 65° and 75° f to prevent heat stress. clinical signs of heat stress include rapid, shallow respirations, lethargy, poor peripheral perfusion, and ptyalism. 13 treatment includes reducing the animal's core body temperature with cool water baths or applying alcohol to the feet and ears; in addition, fl uid therapy (either intravenous or subcutaneous) is recommended to improve perfusion. the prognosis for this condition is guarded. chlamydophila psittaci has been identifi ed as a disease-causing agent in guinea pigs; it usually causes a mild, self-limiting conjunctivitis. this intracellular bacterial disease usually occurs in young guinea pigs 4 to 8 weeks of age but has been reported in adults as well. 37 in one outbreak, the typical conjunctival abnormalities were present with other, more signifi cant, clinical signs, such as rhinitis, abortion, and pneumonia. 38 a defi nitive diagnosis can be achieved through giemsa staining, immunofl uorescent antibody testing of conjunctival scrapings, and serologic testing. 37 guinea pigs have been reported to develop pathologic changes related to ingestion of forssk fern. 39, 40 in these studies, guinea pigs fed fresh fern developed hematuria and hemorrhage of the bladder wall. 40 one guinea pig fed dried fern developed proliferative urocystica and adenoma of the bladder, a fi nding sometimes considered precancerous in human patients. this guinea pig did not show clinical signs associated with the bladder abnormalities. 39 rabies virus infection is uncommon in rodent species, but it has been described and should be considered a differential diagnosis for an ill guinea pig with suspect contact to wildlife, especially raccoons. a recent report of a rabies in a privately owned guinea pig described abnormal behavior (biting the owner) 26 days after the guinea pig had possible interactions with a raccoon. 41 in this guinea pig, rabies virus antigen was detected by immunofl uorescent antibody testing in the sublingual salivary gland, tongue, and buccal tissues, implying that the guinea pig could have transmitted the virus via a bite wound. sick guinea pigs are often anorexic and therefore dehydrated. restoration of normal hydration status is crucial for the successful treatment of many disease processes. replacement of fl uid defi cit and maintenance of normal hydration can be achieved by administering crystalloids substances through subcutaneous, intraperitoneal, intravenous, or intraosseous routes. because of the diffi culty in placing and maintaining catheters in peripheral veins and bones, the most common route for fl uid administration is subcutaneous. subcutaneous fl uid administration is generally well tolerated. fluids are administered under the skin of the cranial, dorsal thorax (figure 17-24) . butterfl y catheter needles are useful because they allow the patient to move around without pulling out the injection needle. maintenance fl uid rates for guinea pigs are 80-100 ml/kg/day. another important aspect of management for the sick guinea pig is nutritional support. anorexic guinea pigs can experience a change in their normal gi fl ora in as little as 8 to 12 hours. this change of gi fl ora can lead to ileus, colic, overgrowth of pathogenic bacteria, and enterotoxemia. commercial products are available that are palatable and high in fi ber. these products help to maintain gut motility (oxbow critical care for herbivores, oxbow hay company, murdock, ne). patients will often eat directly from a dish or 60-ml catheter tip syringe. for patients that are more resistant to eating, a technique that is useful, in my experience, is to remove the plungers from 1-ml or 3-ml syringes and fi ll them individually using a catheter tip syringe. although this method may seem tedious, it allows the delivery of small boluses of food to be incrementally dispensed. the gi tract of guinea pigs can be very sensitive to the effects of certain classes of antibiotics. the gi fl ora of guinea pigs is primarily gram positive, and administration of antibiotics with a primarily gram-positive spectrum can result in overgrowth of gram-negative and anaerobic organisms. enteral administration of penicillins (e.g., amoxicillin, ampicillin), macrolides (e.g., erythromycin, lincomycin), and fi rst-generation cephalosporins can result in overgrowth of opportunistic pathogens. 20 ampicillin administered subcutaneously at doses of 8 and 10mg/kg three times a day resulted in mortality rates of 20% and 30%, respectively, in a group of guinea pigs. at necropsy, clostridium diffi cile was cultured from the ceca of all fatalities. 42 twenty-fi ve percent of guinea pigs administered 100 mg/kg of cefazolin, a fi rst-generation cephalosporin, intramuscularly died of enterocolitis after several injections. 43 all antibiotics should be used with caution in guinea pigs because of the possibility of disruption of the gi fl ora (table 17 -2). guinea pigs can be induced using isofl urane or sevofl urane administered by mask or induction chamber (figure 17-25) . for lengthy or potentially painful procedures, preanesthetic medications may be used before induction of the inhalant anesthetic agent. as a preanesthetic, an injectable combination, midazolam (0.2-0.5 mg/kg) and butorphanol (0.2-0.5 mg/kg), has been used with success. the administration of premedications will also decrease the stress of induction. isofl urane and sevofl urane are both acceptable inhalant anesthetics for guinea pigs. the advantage of sevofl urane is that it does not appear to have as noxious a scent as isofl urane, thereby reducing breath holding during induction and producing a smoother anesthetic episode. after induction, the inhalant anesthetic may be switched to isofl urane if cost is an issue. guinea pigs should not be fasted more than 2 to 3 hours before anesthesia. because these animals are hindgut fermenters, withholding food for longer periods of time may disrupt gi fl ora. careful monitoring during anesthesia is essential. the number of respirations must be visually monitored for depth and character. heart rate and rhythm should also be monitored continuously using a pediatric stethoscope or doppler unit, which can be placed on a peripheral artery. changes in heart rate or respiratory rate can occur rapidly, so it is advantageous to have precalculated doses of emergency drugs (e.g., glycopyrrolate, epinephrine, atropine, dopram) drawn and available for use before anesthetic induction. intubation of guinea pigs is generally considered to be very diffi cult. the long, narrow oral cavity makes visualization of the glottis diffi cult. the soft tissues of the tongue and soft palate are continuous in the caudal oropharynx, leaving only a small aperture, the palatal ostium. intubation must be performed through this opening in the mucosal tissues. several techniques have been described for visualizing the glottis to assist with intubation. these usually involve alterations of laryngoscope blades so that they may be introduced into the oral cavity without traumatizing the delicate buccal mucosa. 47, 48 another possible intubation technique utilizes a stethoscope altered so that an appropriately sized endotracheal tube can be affi xed to the end. the endotracheal tube is then inserted into the caudal oropharynx. the anesthetist will then listen through the ear pieces of the stethoscope for expiration. the tube is then gently introduced into the trachea. this technique requires practice to perfect but, once the procedure has been performed several times, is a reliable way to intubate guinea pigs. application of a small amount of lidocaine to the rima glottis can ease intubation. rigid endoscopes and otoscopes, when available, are also very helpful in visualizing the glottis for intubation. indications for an ovariohysterectomy procedure in guinea pigs include routine sterilization, dystocia, and reproductive tract disease (e.g., neoplasia, muco/hydro/pyometra, ovarian cysts). in general, the surgical approach and technique are similar to those used with domestic species. the patient is placed in dorsal recumbency, and a ventral midline incision is made through the linea alba. care must be taken upon entering the abdomen to avoid incising the large cecum, which is often situated along the ventral body wall. the body of the uterus can be located dorsal to the bladder. the ovaries lie caudally and laterally to the kidneys and are 6 to 8 mm in length. 49 the abdomen of the guinea pig is deep, and the ovaries can be diffi cult to exteriorize. care must be taken to avoid tearing the short ovarian vessels. once the ovaries are removed, the uterus is ligated and transected in a manner similar to that used with other mammals. another method of sterilization that is reported in guinea pigs is ovariectomy. the incidence of uterine disease compared with ovarian disease in guinea pigs is quite low. the approach for removal of the ovaries is through small incisions of the dorsal-lateral body wall caudal to the last rib. the advantage of this approach is that the sensitive gi tract is not manipulated to reach the structures to be excised. orchiectomy is typically performed to prevent reproduction, to decrease undesirable sexual behavior, and to treat reproductive tract disease. the orchiectomy procedure in guinea pigs is performed by making an incision through the scrotum over each testicle. reactions to suture material are relatively common in guinea pigs after surgical procedures. these reactions can vary in severity from mild local irritation to abscessation. suture materials that cause a large amount of infl ammation, such as chromic gut, should never be used in guinea pigs. in general, monofi lament suture materials that are degraded by hydrolysis are preferred. advances in diagnosis and treatment of small exotic mammal dental disease practitioner's guide to domestic rodents biology, husbandry, and clinical techniques hematologic and serum biochemical values of rodents mammalian hematology: laboratory animals and miscellaneous species natural cytotoxicity in the guinea-pig: the natural killer (nk) cell activity of the kurloff cell the kurloff cell radiology equipment and positioning techniques cryptosporidiosis in guinea pigs: a retrospective study tyzzer's disease subclinical infection and transmission of tyzzer's disease in rats disease problems of guinea pigs coronavirus-like virions associated with a wasting syndrome in guinea pigs natural infections of guinea pigs salmonellosis in laboratory animals the guinea pig or cavy, the ufaw handbook on the care and management of laboratory animals macrophage function in vitamin c-defi cient guinea pigs ureterolithiasis and papilloma formation in the ureter of a guinea pig guinea pigs reproductive medicine of rabbits and rodents spontaneous tumors of small mammals airborne-induced experimental bordetella bronchiseptica pneumonia in strain 13 guinea pigs interlaboratory comparison of enzyme-linked immunosorbent assay (elisa) and indirect immunofl uorescence (iif) for detection of bordetella bronchoseptica antibodies in guinea pigs effi cacy of commercial vaccines for protecting guinea pigs against bordetella bronchiseptica pneumonia serodiagnosis of streptococcus pneumoniae infection in guinea pigs by an enzyme-linked immunosorbent assay isolation of streptobacillus moniliformis from a guinea pig with granulomatous pneumonia polymerase chain reaction for detection of guinea pig adenovirus pathogenesis of guinea pig adenovirus infection adenoviral bronchopneumonia of guinea pigs anorexia and chirodiscoides caviae infection in a guinea pig (cavia porcellus) ilar: a guide to infectious diseases in guinea pigs, gerbils, hamsters, and rabbits, part ii, diseases outlines thyroid papillary adenoma in a guinea pig with signs of cervical lymphadenitis cutaneous vascular malformation in a guinea pig (cavia porcellus) thyroid carcinoma of a guinea pig: a case report abdominal mesothelioma in an aged guinea pig guinea pig inclusion conjunctivitis (gpic) in a commercial colony verlauf einer chlamydienbedingten "meerschweinchen-einschluß körperchen-könjunktivitis" in einer versuchstierhaltung, zeitschrift für versuchsteirkunde proliferative urocystica and adenoma in a guinea pig preliminary studies on christella dentate (forssk) fern toxicity in guinea pigs rabies virus infection in a pet guinea pig and seven pet rabbits an evaluation of ampicillin pharmacokinetics and toxicity in guinea pigs pharmacokinetics of cefazolin in guinea pigs exotic animal formulary ferrets, rabbits, and rodents: clinical medicine and surgery plumb dc: veterinary drug handbook endotracheal intubation in guinea pigs by direct laryngoscopy an improved method for direct laryngeal intubation in the guinea pig biology of the guinea pig key: cord-302306-fudeixy2 authors: xu, kui; zhou, yanrong; mu, yulian; liu, zhiguo; hou, shaohua; xiong, yujian; fang, liurong; ge, changli; wei, yinghui; zhang, xiuling; xu, changjiang; che, jingjing; fan, ziyao; xiang, guangming; guo, jiankang; shang, haitao; li, hua; xiao, shaobo; li, julang; li, kui title: cd163 and papn double-knockout pigs are resistant to prrsv and tgev and exhibit decreased susceptibility to pdcov while maintaining normal production performance date: 2020-09-02 journal: elife doi: 10.7554/elife.57132 sha: doc_id: 302306 cord_uid: fudeixy2 porcine reproductive and respiratory syndrome virus (prrsv) and transmissible gastroenteritis virus (tgev) are two highly infectious and lethal viruses causing major economic losses to pig production. here, we report generation of double-gene-knockout (dko) pigs harboring edited knockout alleles for known receptor proteins cd163 and papn and show that dko pigs are completely resistant to genotype 2 prrsv and tgev. we found no differences in meat-production or reproductive-performance traits between wild-type and dko pigs, but detected increased iron in dko muscle. additional infection challenge experiments showed that dko pigs exhibited decreased susceptibility to porcine deltacoronavirus (pdcov), thus offering unprecedented in vivo evidence of papn as one of pdcov receptors. beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit papn or cd163 for entry. porcine reproductive and respiratory syndrome (prrs) is a highly infectious viral disease characterized by reproductive disorders including premature birth, late abortion, stillbirth, weak and mummy fetuses, and respiratory dysfunction in piglets and in growing pigs (wensvoort et al., 1991) . since its discovery in the united states in 1987, prrs has rapidly spread worldwide, with frequent outbreaks causing large economic losses (holtkamp et al., 2013) . three surface receptors on porcine alveolar macrophages (pams) have been shown to function in prrsv invasion in vivo: heparin sulphate (hs), sialoadhesin (sn), and cd163 (calvert et al., 2007; crocker and gordon, 1986; jusa et al., 1997) . multiple studies have reported that cd163 is an essential receptor for prrsv infection, with scavenger receptor cysteine-rich domain 5 (srcr5) serving as the core domain for virus recognition (calvert et al., 2007; van gorp et al., 2010; patton et al., 2009) . gene editing technology has been emerging as an important approach of livestock animal and plant germplasm improvement. the technology makes possible for precise modification of more than one gene simultaneously, which is particularly desirable for obtaining important economic traits that are controlled by multiple genes. in 2016, prather's group was the first to use crispr/cas9 technology to generate srcr5 domain-targeted cd163 knockout pigs. they demonstrated that a cd163 knockout line was completely resistant to genotype 2 prrsv infection (whitworth et al., 2016) . subsequently, several laboratories have generated anti-prrsv pigs targeting cd163. for example, the cd163 srcr5 domain was replaced with human cd163-like srcr8 domain to generate prrsv genotype 1 resistance (wells et al., 2017) . wei et al., 2018 reported homozygous geneedited large white pigs with a 50 bp deletion in exon 7 of the cd163 gene (wei et al., 2018) that are fully resistant to genotype 2 prrsv. there are also examples of deletion of the srcr5 domain seeking resistance to both prrsv genotypes (burkard et al., 2017) , or introducing a premature termination in the cd163 srcr5 domain to generate hp-prrsv (highly pathogenic prrsv)-resistant duroc pigs . deleting the srcr5 lbp region has also been reported to generate a prrsv genotype 2 resistant pigs . all these studies demonstrate that prrsvresistant pig breeds can be generated by editing the cd163 gene, enabling alleviation of the severity of prrsv. in addition to prrsv, transmissible gastroenteritis virus (tgev), an acute high-contact infectious virus, is known to frequently occur to co-infect with other porcine diarrhea-associated viruses such as porcine epidemic diarrhea virus (pedv), porcine rotavirus (porv) (zhang et al., 2013) . tgev is globally distributed and causes tremendous economic losses in pork production (gerdts and zakhartchouk, 2017) . characterized by vomiting, severe diarrhea, and dehydration, the mortality rate of tgev-infected piglets under the age of 14 days approaches 100%. tgev is a single-stranded, positive-sense rna coronavirus which targets pig intestinal epithelium for infection (brierley et al., 1989; wesley and lager, 2003) . studies have shown that the papn protein acts as a receptor in mediating tgev infection. the viral glycoproteins bind to papn receptors on the surface of small intestinal epithelial cells and mediate membrane fusion, thus resulting in the virus entering into elife digest pig epidemics are the biggest threat to the pork industry. in 2019 alone, hundreds of billions of dollars worldwide were lost due to various pig diseases, many of them caused by viruses. the porcine reproductive and respiratory virus (prrs virus for short), for instance, leads to reproductive disorders such as stillbirths and premature labor. two coronaviruses -the transmissible gastroenteritis virus (or tgev) and the porcine delta coronavirus -cause deadly diarrhea and could potentially cross over into humans. unfortunately, there are still no safe and effective methods to prevent or control these pig illnesses, but growing disease-resistant pigs could reduce both financial and animal losses. traditionally, breeding pigs to have a particular trait is a slow process that can take many years. but with gene editing technology, it is possible to change or remove specific genes in a single generation of animals. when viruses infect a host, they use certain proteins on the surface of the host's cells to find their inside: the prrs virus relies a protein called cd163, and tgev uses papn. xu, zhou, mu et al. used gene editing technology to delete the genes that encode the cd163 and papn proteins in pigs. when the animals were infected with prrs virus or tgev, the nonedited pigs got sick but the gene-edited animals remained healthy. unexpectedly, pigs without cd163 and papn also coped better with porcine delta coronavirus infections, suggesting that cd163 and papn may also help this coronavirus infect cells. finally, the gene-edited pigs reproduced and produced meat as well as the control pigs. these experiments show that gene editing can be a powerful technology for producing animals with desirable traits. the gene-edited pigs also provide new knowledge about how porcine viruses infect pigs, and may offer a starting point to breed disease-resistant animals on a larger scale. epithelial cells (delmas et al., 1992; hansen et al., 1998) . inhibition or direct knockout of papn in small intestinal epithelial cells can mitigate tgev infection zhu et al., 2018) . papn knockout pigs are resistant to tgev (luo et al., 2019; whitworth et al., 2019) . pdcov is a highly virulent porcine coronavirus discovered in 2012 that causes watery diarrhea and vomiting in sows and piglets, with piglet mortality rates of 30% to 40% woo et al., 2012) . there is controversy about whether or not papn is a functional receptor for pdcov. wang et al., 2018 showed that papn functions as a receptor to promote pdcov entry into cells , while zhu et al., 2018 confirmed its involvement but showed that papn was an unnecessary important functional receptor for pdcov infection (zhu et al., 2018) . li et al., 2018 suggested that pdcov infection may require a co-receptor, in addition to papn . using cells isolated from papn knockout pigs, however, stoian et al., 2020 showed that these pig cells were still susceptible to pdcov infection in vitro. it was suggested that papn may be one of the receptors for pdcov, and an unknown receptor or factor may compensate for papn function in the absence of papn (stoian et al., 2020) . however, whether papn knockout pigs may be resistant to pdcov infection in vivo remains unknown. although gene-edited cd163 knockout (prrv resistant) pigs and papn knockout (tgev resistant) pigs have been previously generated, respectively, pigs that are resistant to the infection of both viruses are lacking. our objectives in the present study were (1) to knockout cd163 and papn simultaneously using a gene editing approach; (2) to verify if the resultant dko pigs are simultaneously resistant to infection by prrsv and tgev; (3) to use the dko pigs as an in vivo experimental model to test for potential papn-mediated resistance to pdcov infection. we report successfully generated gene-edited large white pigs with both cd163 and papn gene knockouts using crispr/cas9 and somatic cell nuclear transfer (scnt). through viral challenge experiments, we found that these dko pigs exhibit complete resistance to genotype 2 prrsv and tgev, and exhibit decreased susceptibility to pdcov infection. in addition, with the exception of meat color score and iron content, no differences in the production performance, reproductive performance, or pork nutrient content were observed between dko pigs and wt pigs. thus, in addition to demonstrating that our dko pigs are robustly resistant to both prrsv and tgev without suffering deleterious effects for production performance, our study also provides insights into ongoing controversy about the papn protein as a potential receptor for pdcov infection of pigs. in order to generate cd163 and papn dko cloned pigs, we constructed sgrna delivery plasmids targeting these genes, and selected successful dko pig fetal fibroblasts (pefs) as nuclear transfer donors ( figure 1a ). for cd163, the srcr5 domain-binding site for prrsv in exon 7 (van gorp et al., 2010; ma et al., 2017) was selected as the sgrna recognition site. to inactivate the papn protein, a sgrna target site in exon two immediately downstream of the atg start codon was selected ( figure 1b) . successful dko colonies were cultured as donor cells for scnt (supplementary file 1). the cloned pigs generated in this experiment were obtained via both primary and secondary clonings. for primary cloning, the selected dko cells are used as donors for nuclear transplantation. for secondary cloning, the ear-derived fibroblasts of the primary cloned pigs are re-cloned, which rapidly provided a large number of high-quality dko donor cells, thus improving cloning efficiency and resulting in many genotypically identical pigs. in our primary cloning, a total of 3780 reconstructed embryos were transplanted into 11 surrogate sows, of which two were pregnant and gave birth to eight live piglets. of these piglets, four survived after weaning ( figure 1c and supplementary file 2). we determined the cd163 and papn genotypes of the four surviving piglets using pcr and sanger sequencing. the genotypes of the three piglets (#1143, #1144, and #1145) matched that of cell colony #25, which had an 8 bp deletion on both copies of cd163 near the target site, and a copy of papn carrying a 5 bp deletion on one copy and a 26 bp deletion on the other, both resulting in frameshift mutations or premature termination after the target site ( figure 1d ). papn protospacer pam in order to generate more dko pigs for viral challenge experiments, we collected ear tissue samples from three piglets (#1143, #1144, and #1145) and isolated ear-derived fibroblasts. a total of 2270 reconstructed embryos generated from ear-derived fibroblasts of #1145 were transplanted into nine surrogates. four sows successful gave birth to a total of 20 live piglets, among which 12 survived post-weaning (supplementary file 2). the genotypes of these 12 piglets matched that of #1145, and the three dko primary clones used for subsequent experiments. we used flow cytometry and western blotting for cd163, immunohistochemistry (ihc) and western blotting for papn, and confirmed that expression of both proteins was undetectable in dko pigs but detectable in wt pigs of the same age and breed ( figure 1e -g). we designed multiple pairs of amplification primers for the px330 vector backbone to confirm that no random integration of px330 vector fragments were in cloned pigs (figure 1-figure supplement 1). we also tested for off-target modifications in dko pigs using 10 potential off-target sites for each of the two sgrnas and found no alteration in any of these 20 predicted sites in the cloned pigs (supplementary file 3). this data demonstrates that clones of sus scrofa line with multiple gene-edited can be generated through primary and secondary cloning with high efficiency and no off-target detected. for testing of prrsv resistance in pams derived from dko pigs, we selected the highly pathogenic genotype 2 prrsv strain wuh3 to challenge dko and wt pams at a multiplicity of infection (moi) of 0.1. qrt-pcr and western blot analyses were used to assess prrsv proliferation in pams. at 12 hr post-infection (hpi), dko pams carried a significantly lower prrsv load compared with wt pams, and no viral rna or prrsv-n protein was detected thereafter in dko pams (figure 2a and b). the low level of prrsv rna that was initially detectable in the dko line at 12 hpi is likely attributable to the adsorbed prrsv independent of the existence of cd163, as cd163 is thought to be primarily responsible for the uncoating and viral rna release processes of prrsv infection van gorp et al., 2008) . we next sought to examine if dko pigs are resistant to prrsv in vivo. four 45-day-old dko pigs and six wt control pigs of the same age were challenged with the prrsv strain wuh3. nasal intubation drip (2 ml: 10 6 tcid 50 /ml) and intramuscular injection (2 ml: 10 6 tcid 50 /ml) were used to infect both experimental groups. the phenotypic data of body temperature, feed intake, respiration, defecation, and mental condition were recorded daily after infection. as shown in figure 2c , while fever (over 40˚c) began at 1 day post-infection (dpi) and persisted throughout the remainder of the experimental period in the wt group, the body temperature of the dko pigs stayed normal throughout the 14 days of the post-viral challenge observation period. scoring for other clinical symptoms of prrsv at 1 dpi showed that wt pigs exhibited decreased appetite, shortness of breath, cough, malaise, drowsiness, and difficulty walking, whereas the dko group displayed no abnormalities except for a brief cough and diarrhea in two pigs at 4 dpi and 9 dpi, respectively ( figure 2d ). the body weight of the dko pigs increased, throughout the 14 day post viral challenge observation period: the detected body weights of the wt pigs were all lower than dko pigs after 0 dpi ( figure 2e ). of the six challenged wt pigs, one was slaughtered at 10 dpi to harvest pams, and the five remaining wt pigs died within 11 dpi. in sharp contrast, all four pigs in the dko group remained healthy, and survived for the entire duration of the 14-day experiment ( figure 2f ). among the dead and slaughtered wt pigs, the lungs were swollen, with severe bleeding, and obvious lesions, while the lung tissues of dissected dko pigs did not exhibit lesions or any other distinct symptoms associated with prrsv ( figure 2g ). hematoxylin and eosin (h and e) staining showed thickening of the alveolar walls and infiltration of a large number of inflammatory examination of prrsv antigens in lung tissue via ihc, it was revealed that the viral antigens were present in the lungs of the wt group, but not that of the dko pigs ( figure 2h , lower panel). moreover, we measured the prrsv viral load in the serum of both groups at 0, 3, 7, 10, and 14 dpi and found that in the wt group, the prrsv load increased rapidly and significantly by 7 dpi, reaching its maximum at 7 dpi. in agreement with other experiments showing viral resistance, the prrsv viremia in the dko group remained negative throughout the challenge ( figure 2i ). we also tested the prrsv viral load in pams, lung tissues, and tonsil tissues of the two groups of pigs after viral challenge. whereas a high titer of prrsv was detected in all tissues examined in the wt group, prrsv was almost undetectable in dko pigs ( figure 2j ). from 3 dpi, the amount of prrsv-specific elisa antibodies in the serum of wt pigs increased significantly, and antibody levels were positive (s/ p!0.4) at 7 and 10 dpi, while such antibodies in dko pigs remained consistently negative (s/p<0.4) ( figure 2k ). taken together, these results provide compelling in vitro and in vivo evidence that the dko pigs are resistant to prrsv infection. following characterization of prrsv resistance, we next sought to determine if double knockout of cd163 and papn also conferred resistance against tgev. four 45-day-old dko pigs and six wt control pigs of the same age and breed were fed under the same conditions and infected with tgev. a total of 10 ml of tgev (7 â 10 5 tcid 50 /ml) were orally administered to each pig in two doses (day 0 and day 1, 5 ml/day). at 3 dpi, one dko pig and one wt pig were slaughtered to collect intestinal tissues for pathological examination, and the remaining pigs were housed under regular husbandry conditions until slaughter, and tissues were sampled at 14 dpi. body temperature was recorded daily beginning at day 0, prior to inoculation, and piglet weighing and blood sampling for serum separation were conducted at 0, 7, and 14 dpi. during the viral challenge period, no abnormalities were observed among the pigs, with the exception of two wt pigs that had diarrhea. there was no significant difference in weight gain between the two groups (data not shown). detection of tgev-specific neutralizing antibodies in serum showed no neutralizing antibodies in the dko pigs throughout the experiment, while two of the wt pigs were positive for neutralizing antibodies at 7 dpi, and all wt pigs were positive by 14 dpi ( figure 3a) . all slaughtered pigs from both wt and dko groups (sampled at 3 dpi and 14 dpi) were dissected to examine potential lesions in small intestine tissues. for the dko group, no lesions were found in the small intestine samples collected at either 3 dpi or 14 dpi ( figure 3b ). in marked contrast, wt group tissues collected at 3 dpi demonstrated a thin and yellowing small intestine wall, with hemorrhages typical of tgev clinical symptoms, and by 14 dpi there were notable duodenum, jejunum, and ileum hemorrhages, accompanied by intestinal wall thinning and enlarged mesenteric lymph nodes ( figure 3b ). pathological examination of small intestine tissue sections revealed pathological changes, including necrosis and shedding of intestinal mucosal epithelial cells, intestinal villi fusion, plasma cells accumulating in the lamina propria, and infiltration of eosinophils in the duodenum, figure 2 continued in serum. wt: 0 to 3 dpi, n = 6; 7 dpi, n = 5; 10 dpi, n = 2. dko: 0 to 14 dpi, n = 4. data are expressed as means ± sd. statistical significance was determined by student's t test; ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001. the online version of this article includes the following source data for figure 2: source data 1. the qrt-pcr detection of prrsv load in pams. source data 2. rectal temperatures of pigs. source data 3. clinical symptoms scores of pigs. source data 4. body weights of pigs. source data 5. the survival rate of pigs. source data 6. the qrt-pcr detection of prrsv load in serum. source data 7. the qrt-pcr detection of prrsv load in pams, lung tissues and tonsil tissues. source data 8. prrsv-specific antibodies in serum (s/p ration). jejunum, and ileum of wt pigs at 3 dpi and 14 dpi, while the same small intestine tissues in dko pigs appeared healthy ( figure 3c ). we also analyzed the ratio of intestinal villus height (vh) to the crypt depth (cd). the smaller the ratio, the more severe the intestinal villi atrophy. we found that compared with the mock group, the three intestinal segments of the wt group had significant intestinal villous atrophy, and the intestinal villi of these intestinal segments in the dko group did not show atrophy; that is, the degree of intestinal villous atrophy in the three intestine segments in the wt group was significantly higher than that in the dko group ( figure 3d) . these results consistently demonstrate that our cd163/papn dko pigs exhibit strong resistance to tgev infection. pdcov is a highly pathogenic virus that has recently been shown to cause diarrhea in newborn piglets, although the functional receptors for pdcov have not yet been confirmed stoian et al., 2020; zhu et al., 2018) . whether papn functions as a receptor or co-receptor in pdcov infection of pigs remains controversial. to test the hypothesis that papn may functionally mediate pdcov infection, we tested the susceptibility of our dko pigs to this virus. two 45-day-old dko pigs and four wt pigs of the same age and breed were challenged with pdcov. a total of 16 ml of pdcov (2.5 â 10 8 tcid 50 /ml) was orally administered to each pig in two doses (day 0 and day 1, 8 ml/day). during the 14 days of pdcov challenge study, both the dko and wt pigs appeared normal, with no distinct differences in body temperature or weight (data not shown). blood was collected at 0, 7, and 14 dpi to assay for levels of virus-specific antibodies. at 7 and 14 dpi, wt pigs were all antibody-positive, while the dko pigs were all antibody-negative at 7 dpi, but carried antibody levels comparable to that of the wt group by 14 dpi ( figure 4a ). this suggests that the double-gene knockout led to a delayed onset of humoral immunity in pigs, possibly due to delayed-immune system exposure to the virus. all pigs were slaughtered at 14 dpi, and the small intestine tissues were collected to evaluate disease severity. it was found that the intestinal wall of the wt had become thinner, with watery fluid in the small intestine, and mesenteric hyperemia, none of which was observed in the small intestine of the dko pigs ( figure 4b ). pathological examination of small intestine tissue sections revealed significant lesions in the small intestine tissues of both of the wt and dko groups, which included intestinal villi fusion, infiltration of lymphocytes in the intestinal mucosa, with many lesions in the intrinsic membrane in the duodenum and jejunum tissues. in the ileum, there were signs of necrosis and shedding of intestinal mucosal intraepithelial cells and naked lamina propria. the extent of lesions in the wt pigs was more severe than that of the dko pigs ( figure 4c ). we also detected the ratio of intestinal villus height to the crypt depth, and found that compared with the mock group, the three intestinal segments of both of the wt group and the dko group had intestinal villous atrophy, but the degree of villous atrophy in the ileal tissue in the dko group was lower than that of the wt group ( figure 4d) . in addition, we tested the resistance of pams derived from dko pigs to pdcov. dko and wt pams were infected with pdcov, and indirect immunofluorescence assays (ifa), tissue culture infectious dose 50 (tcid 50 ) assays, qrt-pcr, and western blot analyses to assess pdcov proliferation in pams all indicated that dko pams exhibit significantly decreased susceptibility of pdcov infection compared to wt pams (figure 4-figure supplement 1) . these data suggest that although the dko line is still susceptible to pdcov infection, the viral invasion and damage to the small intestines was partially inhibited compared to that of the wt line. we next evaluated the growth and performance indices of dko pigs. three 11-month-old dko large white boars and three wt large white boars of the same age were selected for slaughter testing. the live weight at slaughter, carcass weight and length, dressing percentage, ham percentage, lean rate, loin eye area, average backfat thickness, muscle ph, marbling, and drip loss were determined. as shown in table 1 , with the exception of meat coloring score, dko pigs showed no difference in comparison with wt pigs for these indices. in addition, there was no significant difference in birth weight or in the average daily gain between wt and dko pigs (supplementary file 8). most notably, the meat color score in the dko pigs (4.667 ± 0.1667 n=3) was significantly higher than that of wt pigs (3.833 ± 0.1667 n=3), although both were within the normal range of 2 to 5 according to the guideline of 'rules for performance testing of breeding pigs' document published by the ministry of agriculture and rural affairs of pr china (ny/t 821-2004) ( table 1 and figure 5a -b). since the cd163 protein is known to play a role in the degradation of haemoglobinhaptoglobin (hb-hp), and considering that fe is an important component of haemoglobin, we reasoned that the increased meat color score (redness) may be due to the decreased hb metabolism as a consequence of cd163 knockout, and subsequently mild accumulation of fe containing hb in the meat. to test this hypothesis, the meat fe level was analyzed, it was found that the concentration of fe was significantly higher in dko pigs compared to wt pigs ( figure 5c ). we also tested the serum haptoglobin (hp) content and found that the hp content in dko pigs was significantly higher than that of wt control pigs ( figure 5d ). evaluation of the nutritional components of pork such as total protein, total fat, ash, moisture, specific minerals, and amino acid content was also performed. as shown in table 2 and supplementary file 4, no differences in these indices were observed between the two groups. in order to test the reproductive performance of the dko boars, semen from dko male pigs (n = 3) and that of wt pigs (n = 4) of the same age and breed were analyzed. it was revealed that the concentration, motility, and velocity distribution of the sperm from dko boars did not differ from wt boars (table 3) . furthermore, there was no difference in the litter size between the two genotypes: dko litters were 10.67 ± 1.202 (n = 3, litter size from 9 to 13) and the wt litters were 12.05 ± 0.6496 (n = 22, litter size from 7 to 17). in addition, these three dko pigs did not show any growth abnormalities or disease phenomena during the 11-month rearing process, and no abnormalities were observed in the main tissues and organs after slaughter (data not shown). taken together, with the exception of slight meat coloring score increase, these results show that the simultaneous, editing-based disruption of the cd163 and papn loci, does not affect the normal growth and reproductive performance of the resultant dko pigs. conventional breeding for complex traits using molecular marker-assisted selection is a lengthy process, requiring multiple rounds of crosses and backcrosses to introgress each individual gene. crispr/cas9 gene editing not only allows bypassing of this long process, but also provides a possible means to obtain multiple beneficial genotypes in a single generation while also avoiding gene penetration from donor species, thus maintaining the desirable qualities of the original species. zhou et al., 2015 first used crispr/cas9 in combination with scnt to generate knockout of park2 and pink1 genes, whose dysfunction are known to contribute to the early onset of parkinson's disease in humans . huang et al., 2017 got the pig model with metabolic disorder successfully by editing apolipoprotein e and low density lipoprotein receptor genes simultaneously . our study is the first report on how multiple gene edits can be combined in livestock animal to offer simultaneous resistance to two major viral infection. similar to the previous reports above, double knockout efficiency using crispr/cas9-mediated dual gene editing method without any drug or flow cytometry screening was high in our study, reaching 6.30% (17 dko cell colonies out of 270 cell colonies). in this experiment, we quickly generated a large number of dko pigs by re-cloning. we found that the re-cloning efficiency (0.9%, 20/2270) was much higher than the primary cloning efficiency (0.2%, 8/3780). a possible reason for this elevated efficiency could be that the monoclonal cells used for the primary cloning must be cultured in vitro for a long time, which has been reported to inhibit cloning efficiency (li et al., 2003; magnani et al., 2008; mastromonaco et al., 2006) . the donor cells used in re-cloning were ear-derived fibroblasts isolated directly from dko pigs, eliminating the requirement for a long-term, in vitro screening process. our findings support the notion that the efficiency of this approach is not gene specific, and may be applicable to the knockout of other genes that allow improving disease resistance or animal production. in 2007, calvert et al. first discovered that cd163 functions as a prrsv receptor protein during pams infection, which has since been confirmed by several studies (calvert et al., 2007; van gorp et al., 2008; guo et al., 2014; patton et al., 2009) . structural studies of cd163 revealed that the srcr5 domain corresponding to cd163 exon seven is necessary to mediate prrsv infection (van gorp et al., 2010) . in recent years, several groups have successfully generated prrsv-resistant gene-edited pigs by targeting exon 7 of the pig cd163 gene (burkard et al., 2017; the online version of this article includes the following source data for the online version of this article includes the following source data for table 3 : source data 1. comparison of the concentration, motility, and velocity distribution of the sperm between dko and wt large white pigs. 2019; whitworth et al., 2016; yang et al., 2018) . in the present study, we used a single sgrna targeting exon 7 of cd163, generated an 8 bp double-stranded deletion that terminated protein translation near the target site. our finding on the complete resistance to prrsv genotype 2 in our knockout line is consistent with those previous reports. cd163 is known to play a role in promoting the clearance of plasma free haemoglobin (kristiansen et al., 2001) . our finding that the dko pigs have higher meat fe content and have elevated serum hp levels is consistent with this idea, and may explain the observed darker red color in our dko meat. interestingly, and consistent to our finding, wells et al., 2017 also reported that the serum hp levels are elevated in cd163 knockout pigs (wells et al., 2017) . despite the slight color score increase, no abnormal growth or reproductive performance was observed in our dko pigs, and the meat color of both dko pigs and wt pigs were within the normal range. production performance evaluations and identification of pork nutritional components showed that our dko pigs were indistinguishable from that of the wt pigs in growth rate and reproductive performances, except for the meat color score and iron content. however, the number of dko pigs tested by us is still small, and the production performance of dko pigs still needs to be verified in large populations in the future. apn is known to be a receptor for many coronaviruses, and studies have shown that separate domains function in virus recognition vs. hydrolase catalytic activity (reguera et al., 2012) . two research groups have recently demonstrated that papn knockout pigs block tgev but not pedv infection (luo et al., 2019; whitworth et al., 2019) . our data showing that papn knockout can completely prevent tgev virus infection are consistent with these recently published findings. in addition to tgev and prrsv, we also determined if papn deletion conferred protection against pdcov. apn is a receptor for multiple coronaviruses and is abundantly expressed on small intestinal epithelial cells, which has led to the speculation that papn may also be a receptor for pdcov. wang et al., 2018 and li et al., 2018 proposed that papn functions as a receptor in mediating pdcov infection wang et al., 2018) . however, another study found that knockout of papn in ipi-2i cells inhibited but did not completely block pdcov infection, suggesting that papn was not essential for viral recognition (zhu et al., 2018) . taken together, these studies suggest that papn may be involved in pdcov infection, but pdcov may also be able to enter cell through other pathway(s). our results on the delayed pdcov-specific neutralizing antibodies production, and a reduced extent of gross and histopathological lesions on small intestine in dko pigs compared to wt pigs are consistent with this previous suggestion that papn may play a role but is not the only path for pdcov cell entry. interestingly, a recent study showed that pams, but not lung fibroblast-like cells, from papn knockout pigs showed resistance to pdcov infection (stoian et al., 2020) , a finding consistent with our in vitro experiments showing that dko pams exhibit decreased susceptibility to pdcov infection. in addition, papn knockout pigs are susceptible to pdcov when virus levels were detected using qrt-pcr, and virus neutralization activity was measured, although the extent of tissue lesions between the ko and wt groups was not compared (stoian et al., 2020) . our findings are in line with this study reporting that papn knockout pigs are still susceptible to pdcov. however, as reflected by the delay in neutralizing antibody response, and much lighter intestine damage in the dko pigs, the susceptibility of the papn knockout group to the virus is reduced compared that of the wt pigs, indicating the potential role of papn in mediating pdcov infection. additionally, the effect of cd163 knockout in the delayed adaptive immune response cannot be ignored. despite the important role of cd163 in innate immunity, an inhibiting effect of soluble cd163 on the adaptive immune system has also been reported (frings et al., 2002; o'connell et al., 2017) . it is thus possible that the delayed adaptive immune response we observed in pdcov-infected dko pigs may be associated with cd163 knockout-induced immunosuppression. in summary, the dko pigs generated in this study are simultaneously resistant to prrsv and tegv, and exhibit decreased susceptibility to pdcov, while maintaining the same growth and reproductive production traits when compared to wt animals. these pigs may offer breeding starting points for disease-resistant pig colony generation and will be a valuable model to help deepen our understanding of the role and mechanisms of these receptor proteins in the infection mechanisms of multiple viruses. the cd163 and papn genotypes of colonies and piglets born after nuclear transfer were detected by pcr and sanger sequencing. one third of the cells in the 48-well plate and the ear tissue were used to extract the genomic dna. the primer pairs cd163-f/cd163-r and papn-f/papn-r were used to amplify the sequences near the sgrna target sites in the cd163 and papn genes, respectively. the primer sequences are shown in supplementary file 5. the pcr products were genotyped by sanger sequencing. potential off-target sites were predicted using an online software: crispor (http://crispor.tefor.net/ ). we identified the 10 potential off-target sites for each of the two sgrnas. twenty pairs of primers were designed to amplify the potential off-target sites from the genomic dna isolated from the 3 dko pigs (1143#, 1144#, 1145#). sanger sequencing was performed to determine whether any mutations occurred. the primer sequences are shown in supplementary file 7. the total protein extracted from lung tissue, liver tissue, and spleen tissue of non-challenged wt pigs and dko pigs was used to detect cd163, and protein extracted from duodenal, jejunal, and ileal tissues were used to detect papn expression. whole cell lysates of prrsv-infected pams and pdcov-infected pams were used to quantify the expression levels of prrsv nucleocapsid (n) protein and pdcov nucleocapsid (n) protein, respectively. the protein samples were separated by 8% or 12% sds-page and transferred to a polyvinylidene fluoride membrane (millipore). the membrane was blocked with 5% skim milk for 2 hr, and then incubated with primary antibody at 4˚c overnight and secondary antibody at room temperature for 2 hr. chemiluminescent signals were developed with supersignal west pico plus chemiluninescent substrate (thermos scientific) and captured with a tanon-520 (tanon). cd163 rabbit polyclonal antibody (16646-1-ap; proteintech) was used to detect porcine cd163. apn polyclonal antibody (a5662; abclonal) was used to detect papn, anti-prrsv-n antibody (made in our laboratory) was used to detect prrsv-n protein, anti-pdcov-n antibody (made in our laboratory) was used to detect pdcov-n protein, gapdh rabbit antibody (3683; cell signaling) or b-actin rabbit antibody (ac026; abclonal technology) was used to stain gapdh or b-actin as a loading control. hrp-conjugated affinipure goat anti-rabbit igg(h+l) (sa00001-2; proteintech) and hrp-conjugated affinipure goat anti-mouse igg(h+l) (sa00001-1; proteintech) were used as the secondary antibody. pams were isolated from dko piglets and wt piglets. the lungs were obtained from the euthanized piglets. the lung surfaces were rinsed with pbs, and pams were subsequently obtained by bronchoalveolar lavage with prmi-1640 medium (gibco, usa). the collected lavage solution was dispensed into a 50 ml centrifuge tube, centrifuged at 300 g for 10 min, and the supernatant was discarded. pams were washed again with prmi-1640 medium and then frozen in cryopreservation solution containing 90% fbs and 10% dmso. for further in vitro infection experiments, pams were cultured in rpmi-1640 medium with 10% fbs and 1 â antibiotic antimycotic (15240062; invitrogen) at 37˚c/5% co 2 , and then infected with a highly pathogenic prrsv (hp-rrsv) strain wuh3 (gen-bank accession number hm853673) (li et al., 2009 ) at a dose of moi = 0.1 and pdcov strain chn-hn-2014 (genbank accession number kt336560) at a dose of moi = 10. the production of progeny prrsv was evaluated through western blot, and qrt-pcr assays, and the production of progeny pdcov was evaluted through ifa, tcid 50 , qrt-pcr and western blot assays. pams were fixed in 4% formaldehyde for 15 min at room temperature. the cells were then blocked with 2% bsa overnight at 4˚c and incubated with mouse anti-pig cd163 mabs (mca2311pe; bio-rad) at 37˚c for 1 hr in the dark. after washing with pbs three times, pams were resuspended in pbs and immediately analyzed using a bd facsverse flow cytometer (bd biosciences, ca) and flowjo software (treestar, ca). all wt pigs used in the infection experiment were born from natural breeding, and they were matched by age and breed with the dko pigs. the four dko and six wt pigs used for prrsv wuh3 viral challenge were both about 45 days old. viral inoculation was conducted by nasal intubation drip (2 ml: 10 6 tcid 50 /ml) and intramuscular injection (2 ml: 10 6 tcid 50 /ml). during the 14 days of prrsv challenge, piglet rectal temperature and clinical symptoms data (feeding, breathing, defecation, mental state) were collected every morning. at the same time, piglet survival rate was recorded, blood was collected, and the piglets were weighed regularly. if any pigs died during the course of the prrsv challenge, pictures were immediately taken and samples were collected. all surviving pigs were slaughtered at 14 days post-infection (dpi) and lung tissue was examined for disease symptoms. four dko pigs and six wt pigs were used for tgev challenge. pigs were inoculated with a total of 10 ml of tgev strain wh-1 (genbank accession number hq462571) (7 â 10 5 tcid 50 /ml) that were orally administered to each pig in two doses (day 0 and day 1, 5 ml/day). for the pdcov challenge, two dko pigs and four wt pigs were orally administered a total of 16 ml of pdcov strain chn-hn-2014 (2.5 â 10 8 tcid 50 /ml) divided into two doses delivered on day 0 and day 1 (8 ml/day). for both the tgev and pdcov groups, the rectal temperature of the pigs was measured daily for the full 14 day experiment and the diarrhea of the piglets was observed. blood was collected and the piglets were weighed regularly. in the tgev group, a dko pig and a wt pig were slaughtered on day 3, and the remaining pigs in the tgev group and all pigs in the pdcov group were slaughtered at 14 dpi. after slaughter, the pigs were dissected to observe the gross lesions in small intestine tissue, to collect small intestine tissue samples, and to detect any pathological changes by h and e staining. meanwhile, during these 14 days, 4 wt pigs and 2 dko pigs were reared under the same conditions without any virus infection, and these pigs were used as the mock group. lung tissues of pigs in the prrsv challenge group, and duodenum, jejunum, and ileum tissues in the tgev and pdcov groups were collected. the tissues were fixed in 4% paraformaldehyde fixative, dehydrated, embedded, and cut into 3~8 mm-thick sections. for histopathology, the sections were stained by h and e. for ihc, tissue sections were stained with antibodies specific to the corresponding protein antigens. tissue sections were then observed and photographed with a fluorescence microscope. the antibodies used to detect papn protein were purchased from abcam (ab108310); the antibody used to detect prrsv-n protein was made by our laboratory. the blood tissues of three experimental groups of pigs were collected at different times after viral challenge and the sera were separated. for the prrsv group, the sera from all samples were subjected to prrs antibody detection by commercially available enzyme-linked immunosorbent assay (elisa) kit (idexx, me). the antibody level was determined to be negative or positive according to the s/p value. if s/p<0.4, the antibody is negative, and if s/p!0.4, the antibody is positive. in order to detect tgev-specific and pdcov-specific antibody levels in serum, we used a serum neutralization test (snt). briefly, sera were heat inactivated by 30 min of incubation in a 56˚c water bath. then serial 2-fold dilutions of serum samples in four replicates were mixed with 200 tcid 50 of tgev strain wh-1 in a 1:1 ration. after incubation, 100 ml of the mixture was added into st cells (a swine testicular cell line permissive of tgev infection; atcc crl-1746) at a confluence of~90%, seeded in 96well cell culture plates. appropriate serum, virus (200 tcid 50 , 20 tcid 50 , 2 tcid 50 , and 0.2 tcid 50 ), and cell controls were included in this test. for about 72 hr after incubation, the cells were monitored for tgev-specific cytopathic effects. neutralization titers were calculated as the reciprocal of the highest dilution resulting in complete neutralization. similarly, sera were diluted mixed with 200 tcid 50 of pdcov strain chn-hn-2014. in contrast, pdcov titers were assessed using llc-pk1 cells (a porcine kidney cell line permissive of pdcov infection; atcc cl-101) that were washed twice with dulbecco's modified eagle's medium (dmem) (invitrogen, ca), and supplemented with 7.5 mg/ ml trypsin (gibco, usa) prior to and after 1 hr incubation with these mixtures. cells were then cultured in dmem supplemented with 7.5 mg/ml trypsin for approximately 72 hr, and the neutralization titers of sera from pdcov group were calculated. to quantify the copies of prrsv and pdcov in the infected experimental group, we extracted prrsv rna from pams, serum, lung tissue, and tonsil tissue from both the challenge and the mockinoculated group, and extracted pdcov rna from dko pams and wt pams after infected with pdcov. rna extraction was performed using trizol reagent (omega bio-tek). the rna was reverse transcribed into cdna according to the instructions for a transcriptor first strand cdna synthesis kit (roche). the cdna was then amplified with sybr green real-time pcr master mix (applied biosystems) in an abi 7500 real-time pcr system (applied biosystems). rna copy numbers were calculated from a standard curve drawn from positive standards at different dilutions. the primers used for qrt-pcr are listed as follows: 5'-gcaattgtgtctgtcgtc-3' and 5'-cttatcctccctgaatc tgac-3' for prrsv; 5'-gccctcggtggttctatctt-3' and 5'-tccttagcttgccccaaata-3' for pdcov. dko pams and wt pams in 24-well cell culture plates were infected or mock-infected with pdcov at a multiplicity of infection (moi) of 10. at 24 hpi, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with methanol for 10 min at room temperature. the cells were then blocked with bovine serum albumin (5%) diluted in phosphate-buffered saline (pbs) for 1 hr, and incubated with a pdcov-n-protein-specific monoclonal antibody for 1 hr and an alexa fluor 488-conjugated donkey anti-mouse igg for 1 hr. the cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (dapi) for 15 min at room temperature. after three washes with pbs, the stained cells were observed with an inverted fluorescence microscope (olympus ix73, japan). pdcov-infected pams were frozen and thawed repeatedly to completely release viruses. next, llc-pk1 cells (a pig kidney cell line known to be highly permissive to pdcov infection) were seeded in 96-well plates and were infected with 10-fold serial dilutions of virus samples in eight replicates. at 72 hpi, pdcov titers were calculated based on cytopathic effects and expressed as the tcid 50 value per milliliter, using the reed-muench method. the amount of hp in serum was measured using an enzyme-linked immunosorbent assay (elisa) kit (6250-40, alpha diagnostic) specific to pig hp, as previously described . assays were performed in triplicate for each sample. the quality and performance of pigs related to slaughter were determined by a third-party testing center (the national breeding swine quality supervision and testing center (chongqing), ministry of agriculture and rural affairs of china). all testing followed the guidelines stipulated in the 'rules for performance testing of breeding pigs' document published by the ministry of agriculture and rural affairs of pr china (ny/t 822-2004) . briefly, dko pigs and control wt pigs were weighed before slaughter, euthanized after fasting for 24 hr, and hairs, heads, hoofs, and internal organs were removed after carcass dissection. the weight of carcass, length of carcass, loin eye areas, thickness of skin, and backfat thickness of carcass were all measured. ham, skin, bone, lean, and fat were dissected from the left side of the carcass and their individual weights were determined. to evaluate meat quality, we measured muscle ph, meat color score, intramuscular fat, marbling, and drip loss of longissimus dorsi. for analysis of pork nutrition, total protein, total fat, ash, moisture, amino acid, and individual minerals, amino acids were analyzed for the longissimus dorsi. the nutritional content of the pork was tested by the beijing institute of nutritional sources. semen from dko pigs and wt control pigs were collected and returned to the laboratory in a 17˚c incubator for testing their quality. the detection system was hamilton-thorne research ivos ii computer-assisted sperm analyzer to measure the concentration, motility, and velocity distribution of the sperm. all data are presented as the mean ± standard error of mean (sem). data from each of the two groups of pigs were compared with an unpaired t-test when a normal distribution was not obtained. the significance levels were set at 0.05, 0.01, and 0.001, as indicated by *, **, ***, respectively. the data was analyzed with graphpad prism 6.0.0 for windows (graphpad software, la jolla, california). animal experimentation: all experimental protocols related to animal work described in this study were reviewed and approved by the institutional animal care and use committee (iacuc) at the institute of animal sciences, chinese academy of agricultural sciences. all experiments were performed in accordance with the approved guidelines for animal care and management of research projects. (ias2018-12 ). decision letter and author response decision letter https://doi.org/10.7554/elife.57132.sa1 author response https://doi.org/10.7554/elife.57132.sa2 supplementary files . source data 1. amino acid content of dko lean meat and wt lean meat. . source data 2. birth weights and average daily gains of wt pigs and dko pigs from birth weight to slaughtering weight. table 1, table 2 and table 3 . quantitative proteomic analysis reveals that transmissible gastroenteritis virus activates the jak-stat1 signaling pathway characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd163 srcr5 domain are fully resistant to both prrsv genotypes while maintaining biological function cd163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses generation of pigs resistant to highly pathogenic-porcine reproductive and respiratory syndrome virus through gene editing of cd163 properties and distribution of a lectin-like hemagglutinin differentially expressed by murine stromal tissue macrophages aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev isolation, genomic characterization, and pathogenicity of a chinese porcine deltacoronavirus strain chn-hn-2014 only the soluble form of the scavenger receptor cd163 acts inhibitory on phorbol ester-activated t-lymphocytes, whereas membrane-bound protein has no effect vaccines for porcine epidemic diarrhea virus and other swine coronaviruses modulation of cd163 expression by metalloprotease adam17 regulates porcine reproductive and respiratory syndrome virus entry highly efficient generation of pigs harboring a partial deletion of the cd163 srcr5 domain, which are fully resistant to porcine reproductive and respiratory syndrome virus 2 infection the coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers crispr/cas9-mediated apoe-/-and ldlr-/-double gene knockout in pigs elevates serum ldl-c and tc levels aminopeptidase-n-independent entry of porcine epidemic diarrhea virus into vero or porcine small intestine epithelial cells n-acetylpenicillamine inhibits the replication of porcine reproductive and respiratory syndrome virus in vitro effect of heparin on infection of cells by porcine reproductive and respiratory syndrome virus identification of the haemoglobin scavenger receptor effect of the number of passages of fetal and adult fibroblasts on nuclear remodelling and first embryonic division in reconstructed horse oocytes after nuclear transfer recombination in vaccine and circulating strains of porcine reproductive and respiratory syndrome viruses broad receptor engagement of an emerging global coronavirus may potentiate its diverse crossspecies transmissibility porcine deltacoronavirus (pdcov) infection suppresses rig-i-mediated interferon-b production aminopeptidase n-null neonatal piglets are protected from transmissible gastroenteritis virus but not porcine epidemic diarrhea virus the crystal structure of the fifth scavenger receptor cysteine-rich domain of porcine cd163 reveals an important residue involved in porcine reproductive and respiratory syndrome virus infection developmental capacity of porcine nuclear transfer embryos correlate with levels of chromatin-remodeling transcripts in donor cells role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer monocytelymphocyte cross-communication via soluble cd163 directly links innate immune system activation and adaptive immune system suppression following ischemic stroke modulation of cd163 receptor expression and replication of porcine reproductive and respiratory syndrome virus in porcine macrophages structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies highly efficient crispr/cas9-mediated transgene knockin at the h11 locus in pigs the use of cells from anpep knockout pigs to evaluate the role of aminopeptidase n (apn) as a receptor for porcine deltacoronavirus (pdcov) sialoadhesin and cd163 join forces during entry of the porcine reproductive and respiratory syndrome virus identification of the cd163 protein domains involved in infection of the porcine reproductive and respiratory syndrome virus porcine coronavirus hku15 detected in 9 us states porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase n for infectious cellular entry generation and propagation of cluster of differentiation 163 biallelic gene edit ing pigs replacement of porcine cd163 scavenger receptor cysteine-rich domain 5 with a cd163-like homolog confers resistance of pigs to genotype 1 but not genotype 2 porcine reproductive and respiratory syndrome virus mystery swine disease in the netherlands: the isolation of lelystad virus increased litter survival rates, reduced clinical illness and better lactogenic immunity against tgev in gilts that were primed as neonates with porcine respiratory coronavirus (prcv) gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus resistance to coronavirus infection in amino peptidase n-deficient pigs discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus cd163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus occurrence and investigation of enteric viral infections in pigs with diarrhea in china generation of crispr/cas9-mediated gene-targeted pigs via somatic cell nuclear transfer contribution of porcine aminopeptidase n to porcine deltacoronavirus infection the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. for the cd163 gene, the sgrna was designed to target exon 7, and for the papn gene, the sgrna was designed to target exon 2. the sequences of the two sgrnas are as follows: ggaaacc-caggctggttggaggg (cd163-sgrna) and gcatcctcctcggcgtggcgg (papn-sgrna). the pam is indicated in bold font. the two sgrna sequences were cloned into the px330 vector (addgene plasmid # 42230) and named px330-cd163 and px330-papn, respectively. two plasmids were extracted (tiangen, dp117) in large quantities and used to transfect the fetal fibroblasts of large white pigs. the fetuses of large white pigs at 35-day-old were used to isolate pefs, which were then cultured in dmem medium containing 20% fbs. when the cells grew to 80% confluence, approximately 10 6 cells were transfected with px330-cd163 (2.5 ug) and px330-papn (2.5 ug) plasmids. a lonza 2b nuclear transfection system was used for transfection with nucleofector program t-016. the entire transfection process was performed according to the kit instructions (lonza, vpi-1002). cells were cultured for 48 hr after transfection and then seeded into 10 cm dishes at a density of 150 cells/dish. the culture medium was changed every 3 days, and cells were cultured for 10 days to form singlecell colonies. single-cell colonies were transferred to 48-well plates for expansion culture. when cells in the 48-well plates reached confluence, 1/3 of the cells were taken for genotype identification, and the remaining cells continued to expand. cells with genotypes identified as double-gene mutations were cultured and frozen for scnt. the oocytes for scnt were derived from a nearby slaughterhouse, and the nuclear donor cells were the dko fibroblasts. the nuclear transfer donor cells were transferred into enucleated oocytes, and the reconstructed embryos were activated and cultured to develop into blastocysts. we then selected well-developed recombinant embryo clones to be surgically transferred into the oviduct of recipient gilts on the day after estrus was observed. after the embryo transfer, the technicians observed the estrus of the sow, and regularly checked the pregnancy by b-ultrasound. changli ge: is affiliated with shandong landsee genetics co., ltd. the author has no financial interests to declare. key: cord-260840-tudl9k1g authors: opriessnig, tanja; gauger, phillip c.; faaberg, kay s.; shen, huigang; beach, nathan m.; meng, xiang-jin; wang, chong; halbur, patrick g. title: effect of porcine circovirus type 2a or 2b on infection kinetics and pathogenicity of two genetically divergent strains of porcine reproductive and respiratory syndrome virus in the conventional pig model date: 2012-07-06 journal: vet microbiol doi: 10.1016/j.vetmic.2012.02.010 sha: doc_id: 260840 cord_uid: tudl9k1g to determine differences in infection kinetics of two temporally and genetically different type 2 porcine reproductive and respiratory syndrome virus (prrsv) isolates in vivo with and without concurrent porcine circovirus (pcv) type 2a or 2b infection, 62 pigs were randomly assigned to one of seven groups: negative controls (n = 8); pigs coinfected with a 1992 prrsv strain (vr-2385) and pcv2a (coi-92-2a; n = 9), pigs coinfected with vr-2385 and pcv2b (coi-92-2b; n = 9), pigs coinfected with a 2006 prrsv strain (nc16845b) and pcv2a (coi-06-2a; n = 9), pigs coinfected with nc16845b and pcv2b (coi-06-2b; n = 9), pigs infected with vr-2385 (n = 9), and pigs infected with nc16845b (n = 9). blood samples were collected before inoculation and at day post-inoculation (dpi) 3, 6, 9 and 12 and tested for the presence of prrsv antibody and rna, pcv2 antibody and dna, complete blood counts, and interferon gamma (ifn-γ) levels. regardless of concurrent pcv2 infection, vr-2385 initially replicated at higher levels and reached peak replication levels at dpi 6. pigs infected with vr-2385 had significantly higher amounts of viral rna in serum on both dpi 3 and dpi 6, compared to pigs infected with nc16845b. the peak of nc16845b virus replication occurred between dpi 9 and dpi 12 and was associated with a delayed anti-prrsv antibody response in these pigs. pcv2 coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-prrsv igg response compared to pigs infected with prrsv alone. this work further emphasizes in vivo replication differences among prrsv strains and the importance of coinfecting pathogens. porcine reproductive and respiratory syndrome virus (prrsv), a single-stranded, positive-sense rna virus, is characterized by a high mutation rate with the potential of genetically diverse strains evolving over time (forsberg et al., 2001; hanada et al., 2005; pirzadeh et al., 1998; rowland et al., 1999) . in the past, prrsv isolates have emerged within the swine population with varying degrees of virulence (fang et al., 2007; han et al., 2006; nelsen et al., 1999) possibly due to a high degree of mutation and recombination (yuan et al., 1999 (yuan et al., , 2000 (yuan et al., , 2001 (yuan et al., , 2004 . more recently, attention has focused on the occurrence of high mortality in chinese swine herds which veterinary microbiology 158 (2012) [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] to determine differences in infection kinetics of two temporally and genetically different type 2 porcine reproductive and respiratory syndrome virus (prrsv) isolates in vivo with and without concurrent porcine circovirus (pcv) type 2a or 2b infection, 62 pigs were randomly assigned to one of seven groups: negative controls (n = 8); pigs coinfected with a 1992 prrsv strain (vr-2385) and pcv2a (coi-92-2a; n = 9), pigs coinfected with vr-2385 and pcv2b (coi-92-2b; n = 9), pigs coinfected with a 2006 prrsv strain (nc16845b) and pcv2a (coi-06-2a; n = 9), pigs coinfected with nc16845b and pcv2b (coi-06-2b; n = 9), pigs infected with vr-2385 (n = 9), and pigs infected with nc16845b (n = 9). blood samples were collected before inoculation and at day post-inoculation (dpi) 3, 6, 9 and 12 and tested for the presence of prrsv antibody and rna, pcv2 antibody and dna, complete blood counts, and interferon gamma (ifn-g) levels. regardless of concurrent pcv2 infection, vr-2385 initially replicated at higher levels and reached peak replication levels at dpi 6. pigs infected with vr-2385 had significantly higher amounts of viral rna in serum on both dpi 3 and dpi 6, compared to pigs infected with nc16845b. the peak of nc16845b virus replication occurred between dpi 9 and dpi 12 and was associated with a delayed anti-prrsv antibody response in these pigs. pcv2 coinfection resulted in significantly more severe macroscopic and microscopic lung lesions and a stronger anti-prrsv igg response compared to pigs infected with prrsv alone. this work further emphasizes in vivo replication differences among prrsv strains and the importance of coinfecting pathogens. ß 2012 elsevier b.v. all rights reserved. was associated with novel prrsv isolates and described as porcine high fever disease in 2006 tong et al., 2007; wu et al., 2009) . the prrsv isolates involved in porcine high fever disease contained unique nucleotide differences compared to other isolates. specifically, a discontinuous, 30 amino acid deletion was identified within the nsp2 region which was initially suggested to be correlated with the pathogenicity of the virus wu et al., 2009 ). however, more recent reports have concluded that this deletion is unrelated to virulence (zhou et al., 2009) in spite of the high mortality that was initially associated with this prrsv variant tong et al., 2007) . interestingly, analysis of 582 samples from affected pigs resulted in the identification of prrsv, porcine circovirus type 2 (pcv2) and classical swine fever virus as the most common co-infection pathogens, suggesting that a potential synergistic interaction among these viruses may account for the unusually high mortality (lv et al., 2008; wu et al., 2009 ). pcv2 is a small, circular, non-enveloped dna virus belonging to the circoviridae family in the genus circovirus. pcv2 can be further divided into several subtypes of which pcv2a and pcv2b are prevalent worldwide (patterson and opriessnig, 2010) . to date, experimental infections comparing pcv2a and pcv2b in gnotobiotic and conventional pigs have not demonstrated major differences in virulence (beach et al., 2010; fort et al., 2008; lager et al., 2007; opriessnig et al., 2008b) . pcv2 is the cause of porcine circovirus associated disease (pcvad) with multiple clinical manifestations including respiratory disease . prrsv has become an important component of the porcine respiratory disease complex (prdc) with major economic impact on the swine industry (chae, 2005) . retrospective studies identified prrsv as the most common cofactor in cases of pcvad (pallaré s et al., 2002) . experimental coinfection with prrsv and pcv2 has yielded mixed results. one study completed in 2002 reported minimal clinical disease or death loss in conventional pigs coinfected with pcv2 and prrsv (rovira et al., 2002) . in contrast, in another study, severe clinical disease and death in 10 of 11 pigs between 10 and 21 days postinfection (dpi) was reported in dually infected, caesarianderived and colostrum-deprived (cdcd) pigs (harms et al., 2001) . despite differences in severity of clinical presentation, experimental coinfection of pigs with pcv2 and prrsv has consistently resulted in up-regulation of pcv2 replication (enhanced viremia and pcv2 tissue load) and increased severity of prrsv-induced lesions in lung tissues (allan et al., 2000; harms et al., 2001; rovira et al., 2002) . in north america, both prrsv and pcv2b have been identified in pcvad outbreaks characterized by excessive mortality suggesting a synergistic relationship between these two viruses gagnon et al., 2007; horlen et al., 2007) . the objective of this study was to characterize the infection dynamics and pathogenicity of two different type 2 prrsv isolates in a conventional pig model under the influence of concurrent pcv2a or pcv2b infection. the severity of clinical disease, macroscopic and microscopic lesions, amount of prrsv and pcv2 antibodies and nucleic acids in sera, amount of prrsv and pcv2 antigen associated with lesions, and interferon gamma (ifn-g) concentrations in serum were measured and compared between groups. fifty-three colostrum-fed, crossbred pigs were derived from sows known to be free of pcv2, prrsv and mycoplasma hyopneumoniae in two separate batches, 44 pigs in batch 1 (b1) and 9 pigs in batch 2 (b2). in addition, batch 3 (b3) consisted of 9 colostrum-fed crossbred pigs derived from sows free of prrsv and m. hyopneumoniae but seropositive for pcv2. b2 and b3 pigs were challenged at the same age as b1 pigs but the experiment was conducted approximately 24 months after b1 pigs. insufficient numbers of pigs were available from the source herd for singularly prrsv-infected groups to be included with the original experiment. the experimental design and group designations are summarized in table 1 . all pigs were housed under the same conditions and treated in a similar way. all pigs were weaned at three weeks of age and transported to the livestock infectious disease isolation facility at iowa state university, ames, iowa. on the day of arrival, all b1 pigs were comingled and randomly assigned to one of five rooms each containing 8 or 9 pigs: negative controls (n = 8); pigs coinfected with a 1992 isolate of prrsv (vr-2385) and pcv2a (coi-92-2a; n = 9), pigs coinfected with prrsv vr-2385 and pcv2b (coi-92-2b; n = 9), pigs coinfected with a 2006 isolate of prrsv (nc16845b) and pcv2a (coi-06-2a; n = 9) and pigs coi-06-2b 1 9 pcv2b nc16845b prrsv-i-92 2 4 none vr-2385 prrsv-i-06 2 5 none nc16845b b3-prrsv-i-92 3 5 none vr-2385 b3-prrsv-i-06 3 4 none nc16845b a batch 1 and 2 pigs were derived from the same source herd free of prrsv and pcv2 whereas batch 3 pigs were derived from a different source herd seropositive for pcv2. coinfected with prrsv nc16845b and pcv2b (coi-06-2b; n = 9). b2 pigs were randomly assigned to one of two rooms each containing 4 or 5 pigs which were infected with prrsv vr-2385 (prrsv-i-92) and prrsv nc16845b (prrsv-i-06), respectively. similarly, b3 pigs were comingled and randomly assigned to one of two rooms each containing 4 or 5 pigs that were infected with prrsv vr-2385 (b3-prrsv-i-92) and prrsv nc16845b (b3-prrsv-i-06), respectively. the pigs from the different batches were kept in different but identical rooms. each room had 18 m 2 of solid concrete floor space, separate ventilation systems and one nipple drinker. inoculation was conducted at approximately 23 days of age. blood samples were collected from all pigs prior to inoculation and at dpi 3, 6, 9 and 12 in serum separator tubes (8.5 ml bd vacutainer, benton dixon, franklin lakes, nj, usa). the blood was centrifuged at 2000 â g for 10 min at 4 8c and serum was stored at à80 8c until testing. serum samples were analyzed for levels of anti-pcv2 igg antibody, anti-prrsv-igg antibody, ifn-g, pcv2 dna, and prrsv rna. in addition, edta tubes (8.5 ml monoject tm 15% edta liquid, tyco healthcare group lp, mansfield, ma, usa) were collected at dpi 3, 6, 9, and 12, stored at room temperature and used within 12 h after collection to determine blood cell counts. all pigs were necropsied on dpi 12 and tissues collected during necropsy were analyzed by immunohistochemistry (ihc) for the presence of pcv2 and prrsv antigens. the experimental protocol was approved by iowa state university institutional animal care and use committee (iacuc approval # 7-08-6595-s). prrsv isolate vr-2385 with a rflp pattern 1-3-4 was recovered in 1992 from pig tissues obtained from a 160 sow herd in southwestern iowa affected by severe respiratory disease in 3-16-week-old pigs and high numbers of late term abortions (halbur et al., 1995b; meng et al., 1994) . the passage 5 virus of the original vr-2385 isolate was used to inoculate pigs in 2001 as described previously . serum from the pigs infected with vr-2385 in 2001 was used to re-isolate the virus followed by two subsequent passages in marc-145 cells to produce the virus stock of vr-2385 for this study. prrsv isolate nc16845b with a rflp pattern 1-18-2 was isolated in 2006 from a clinically affected 9-week-old pig with systemic pcvad from a group of pigs from north carolina with a history of severe respiratory disease in 50% of the pigs and approximatley 20% mortality in the group (gauger et al., 2012) . the passage 2 virus of the original isolate was used to experimentally infect a set of prrsv-free conventional pigs (data not shown) and the lung tissues from these pigs collected two weeks after infection were used for reisolation of the nc16845b virus followed by two subsequent passages in marc-145 cells to produce the nc16845b virus stock for this study. the two inocula were separated in different aliquots, stored at à80 8c, and virus of the same lot was used for all batches of pigs. on dpi 0, coi-92-2a, coi-92-2b, prrsv-i-92, and b3-prrsv-i-92 groups received 2 ml of prrsv isolate vr-2385 at a dose of 10 5.0 median tissue culture infective dose (tcid 50 ) per ml. all pigs in groups coi-06-2a, coi-06-2b, prrsv-i-06, and b3-prrsv-i-06 received 2 ml of prrsv isolate nc16845b at a dose of 10 5.0 tcid 50 per ml. inoculation was intranasal by holding the pig in the upright position and slowly dripping 1 ml of the inoculum into each nostril using a 3 ml syringe (fisher scientific, inc.). two different pcv2 subtypes were used for the inoculation of pigs. pigs in groups coi-92-2a and coi-06-2a were inoculated with the pcv2a isolate 40895, which was recovered from an iowa farm in 1998 (fenaux et al., 2000) and has been well characterized genetically (fenaux et al., 2000) and in the conventional specific pathogen free (spf) pig model (opriessnig et al., 2003 (opriessnig et al., , 2004a . both pcv2a and pcv2b viruses were produced as described previously (opriessnig et al., 2008b) and used for inoculation in this study at a titer of 10 4.0 tcid 50 per ml . pigs in groups coi-92-2b and coi-06-2b were inoculated with pcv2b isolate nc16845 which was isolated in 2006 from a pig farm in north carolina (opriessnig et al., 2008b) . both, pcv2b nc16845 and prrsv nc16845b originated from the same tissues. the pcv2 groups were inoculated intranasally (3 ml) and intramuscularly (2 ml) with their respective pcv2 subtype by injecting 2 ml of the inoculum intramuscularly into the right neck area and 3 ml (1.5 ml per nostril) intranasally by holding the pig in the upright position and slowly dripping 1.5 ml of the inoculum into each nostril using a 3 ml syringe (fisher scientific, inc.). edta-treated blood samples were analyzed for white blood cells using a multispecies hematology instrument (hemavet hv950fs, drew scientific, inc.). the white blood cell (wbc) count was reported as actual numbers of neutrophils, lymphocytes and total wbc per ml of whole blood. in addition to wbc, a ratio was determined between the total neutrophil count and the total lymphocyte count reported as the n/l ratio. values from negative control pigs were considered as baseline for the infected pigs on each dpi. serum samples from all pigs were also tested for the presence of anti-prrsv antibodies by a commercial prrsv elisa (herdchek prrs virus antibody test kit 2xr, idexx laboratories inc. westbrook, ma, usa), according to the instructions of the manufacturer. samples were considered positive if the calculated s/p ratio was equal to 0.4 or greater. all serum samples were tested for the presence of anti-pcv2 igg antibodies based on an open reading frame 2 (orf2) elisa (nawagitgul et al., 2002) . samples were considered positive if the calculated sample-to-positive (s/ p) ratio was equal to 0.2 or greater. on dpi 12, three samples were randomly chosen from each group and room and tested for the presence of swine influenza virus (siv) antibodies by an in house nucleoprotein ns1 elisa (richt et al., 2006) and for the presence of antibodies to porcine parvovirus (ppv) by hemagglutination inhibition (hi) assay (mengeling et al., 1988) . a commercial elisa kit (swine ifn-g; invitrogen, camarillo, ca, usa) was used to detect and quantify ifn-g concentrations in serum according to the instructions of the manufacturer. following prrsv/pcv2 coinfection, the pigs were monitored daily for respiratory disease (dyspnea, sneezing, coughing, nasal discharge). rectal temperatures and behavioral changes such as lethargy and inappetence/ anorexia were also recorded daily. the observers were aware (not blinded) to the treatment status. rna extraction on serum collected at dpi 0, 3, 6, 9, and 12 was performed using a qiaamp viral rna mini kit (qiagen, valencia, ca, usa). the agpath-id prrsv multiplex reagent kit (applied biosystems, foster city, ca, usa) was used for the real-time, reverse transcriptase pcr (rt-pcr) on each extracted rna sample. all samples were run in duplicate. each pcr consisted of 8 ml template rna and 17 ml of pcr master mix. the pcr master mix contained 12.5 ml of 2â rt-pcr buffer, 2.5 ml 10â prrsv primer probe mix, 1.25 ml 20â multiplex rt-pcr enzyme mix, 0.25 ml of zenorna-01 internal control rna and 0.5 ml nuclease-free water. each reaction included eight progressive 1:10 dilutions of a known copy number of prrsv to generate a standard curve for quantification. each plate was run in the sequence detection system (geneamp 5700 sequence detection system, applied biosystems) using the agpath-id company specific conditions (10 min at 45 8c, 10 min at 95 8c, followed by 40 cycles of 2 s at 97 8c and 40 s at 60 8c). samples were considered negative when no signal was observed within the 40 amplification cycles. dna extraction on serum collected on dpi 0, 3, 6, 9, and 12 days was performed using the qiaamp dna blood mini kit (qiagen, valencia, ca, usa) and subsequently used for detection of pcv2 dna by quantitative real-time pcr utilizing primers and a probe designed for pcv2 orf1 as described (opriessnig et al., 2003) . the real-time pcr reaction consisted of a 25 ml pcr mixture containing 12.5 ml commercially available master mix (taqman 1 universal pcr master mix, applied biosystems by life technologies), 2.5 ml dna, 1 ml (0.4 mm) of each primer, and 0.5 ml (0.2 mm) probe. the reaction was run in a 7500 fast real-time pcr system (abi, foster city, ca, usa) under the following conditions: 50 8c for 2 min, 95 8c for 10 min, followed by 40 cycles of 95 8c for 15 s and 60 8c for 1 min. all samples were run in duplicate. serial dilutions of a recombinant pcv2 dna clone were included on each plate to generate a standard curve. viral concentrations were expressed as the dna copy numbers per ml of sample. samples were considered negative when no signal was observed within the 40 amplification cycles. all dna extracts were also tested for presence of pcv2a and pcv2b dna by utilizing a forward primer (5 0 -gcagggccagaattcaacc-3 0 ), a reverse primer (5 0 -ggcggtggacatgatgaga-3 0 ), a probe specific for pcv2a (5 0 -cal fluor orange 560-ggggaccaacaaaatctcta-tacccttt-bhq-3 0 ), and a probe specific for pcv2b (5 0 -quasar 670-ctcaaacccccgctctgtgccc-bhq-3 0 ), which were designed in the pcv2 orf2 as described . the multiplex real-time pcr reaction consisted of a total volume of 25 ml containing 12.5 ml of the commercially available master mix (applied biosystems), 5 ml dna, 0.4 mm of each primer, and 0.2 mm of each probe. all samples were run in duplicate. the reactions were carried out under the following conditions: 50 8c for 2 min, 95 8c for 10 min, followed by 40 cycles of 95 8c for 15 s and 60 8c for 1 min. the sensitivity and specificity of the real-time pcr reaction was evaluated using known pcv2a and pcv2b isolates as well as ppv, prrsv, and pcv type 1 (pcv1) isolates. samples were considered negative when no signal was observed within the 40 amplification cycles. open reading frame (orf) 5 of one prrsv rt-pcr positive pig in each group was sequenced on dpi 12 as previously described (gauger et al., 2012) . on dpi 12, all pigs were humanely euthanized by intravenous pentobarbital overdose (fatal-plus 1 vortech pharmaceutical, ltd., dearborn, mi, usa). macroscopic lung lesions were estimated based on the percentage of the lung surface affected by pneumonia ranging from 0 to 100% (halbur et al., 1995b ). the scoring system was based on the approximate volume that each lung lobe contributes to the entire lung: the right cranial lobe, cranial part of the left cranial lobe, and the caudal part of the left cranial lobe contribute 10% each to the total lung volume, the accessory lobe contributes 5%, and the right and left caudal lobes contribute 27.5% each (halbur et al., 1995b) . additionally, lymph node size was scored ranging from 0 (normal) to 3 (four times the normal size) (opriessnig et al., 2006) . lungs were insufflated with fixative as previously described (halbur et al., 1995b) . sections of lymph nodes (tracheobronchial, mesenteric, mediastinal, superficial inguinal, and external iliac), tonsil, thymus, ileum, kidney, colon, spleen, heart, liver, and brain were collected at necropsy and fixed in 10% neutral-buffered formalin and routinely processed for histological examination. microscopic lesions were evaluated independently by two veterinary pathologists (to, pcg) blinded to the treatment status. sections of lung were scored for the presence and severity of interstitial pneumonia ranging from 0 (normal) to 6 (severe, diffuse) (halbur et al., 1995b) . sections of heart, liver, kidney, ileum, colon and brain were evaluated for the presence of lymphohistiocytic inflammation and scored from 0 (none) to 3 (severe). lymphoid tissues including lymph nodes (trachea-bronchial, mediastinal, mesenteric, external iliac and superficial inguinal), tonsil, spleen and thymus were evaluated for the presence of lymphoid depletion ranging from 0 (normal) to 3 (severe) and histiocytic inflammation and replacement of follicles ranging from 0 (normal) to 3 (severe) (opriessnig et al., 2006) . 2.11. immunohistochemistry 2.11.1. prrsv detection of prrsv-specific antigen was performed by ihc staining on lung sections as previously described (halbur et al., 1995a) . sections were scored for presence of prrsv antigen independently by two veterinary pathologists (to, pcg) blinded to the treatment groups. ihc for detection of pcv2-specific antigen was performed on sections of lung, lymph nodes (tracheobronchial, mediastinal, mesenteric, superficial inguinal and external iliac), tonsil, spleen, thymus and small intestine using a rabbit polyclonal antiserum (sorden et al., 1999) . sections were scored for presence and amount of pcv2 antigen independently by two veterinary pathologists (to, pcg) blinded to the treatment groups. if the results obtained by the two pathologists on a certain tissue differed, the mean of the two scores was used. pcv2 scores ranged from 0 (no antigen) to 3 (more than 50% of the lymphoid follicles contain cells with pcv2-antigen staining) (opriessnig et al., 2006) . any tissue or tissue pool with detectable staining was given at least a score of 1. for the purpose of determining prevalence rates, a score of 0 was considered negative and scores of 1, 2 and 3 were considered positive. for data analysis, jmp 1 software version 8.0.1and sas 1 software version 9.2.0 (both sas institute, cary, nc, usa) were used. summary statistics were calculated for groups to assess the distributional property and data that were not distributed normally (pcr data) were log transformed prior to analysis. as log transformation can only be applied to numbers above 0, a constant number (1) was added to each number in the data set prior to log transformation. a linear mixed model with the random effects ''source'' (source a: b1 and b2 and source b: b3) and ''batch'' (b1, b2, b3, nested within ''source'') and the fixed effects ''prrsv strain'' (none, vr-2385, nc16845b) and ''pcv2 subtype'' (none, pcv2a, pcv2b) was used first on all outcomes. from this, it was determined that the random effect ''source'' contributed to the overall variation whereas ''batch'' did not. to decrease the heterogeneity of the animals in the analysis, all data obtained from the second source, b3, were removed from the analysis but were provided as supplemental information throughout the ''results'' and tables. the final model to analyze continuous data collected over time (rectal temperatures, blood cell counts, log transformed pcv2 and prrsv genomic copies, and elisa s/p ratios) was a repeated measures analysis of variance (anova), where prrsv strain, pcv2 subtype, dpi and their interactions were the fixed effects and pig was the subject of repeated measures. compound symmetry variancecovariance structure was used to model the within pig correlation. a one-way anova was used to analyze crosssectional data (macroscopic and microscopic lung lesions) where prrsv strain, pcv2 subtype, and their interaction were the fixed effects. differences among the interacting groups (prrsv strain â pcv2 subtype) in the repeated measures anova or the one-way anova were assessed using tukey's t-test. a p-value of less than 0.05 was considered significant. differences in prevalence of prrsv and pcv2 antigen between groups (ihc staining) were determined by fisher's exact test. mild, transient lethargy and inappetence were observed in all inoculated groups, although coughing or sneezing was not a feature. pigs in all inoculated groups regardless of coinfection status developed a transient to persistent fever ranging from 40.0 8c to 41.8 8c between dpi 3 and dpi 12. the mean rectal temperature time by group interaction after inoculation was significant (p < 0.05). all six inoculated groups had rectal temperatures significantly higher than the negative controls at dpi 6 and dpi 9. by dpi 12, the mean group rectal temperatures in the prrsv-i-92, prrsv-i-06, coi-92-2a, coi-92-2b and coi-06-2a groups were significantly (p < 0.05) higher compared to the negative controls. when the effect of ''prrsv strain'' was evaluated across groups, no differences were found. compared to pigs infected with prrsv alone, coinfected pigs had higher mean rectal temperatures at dpi 3, 6 and 9. when the effect ''pcv2 subtype'' was evaluated among coinfected groups, pcv2a pigs had significantly (p < 0.01) higher rectal temperatures on dpi 9 compared to pcv2b pigs (data not shown). b3 pigs (b3-prrsv-i-92 and b3-prrsv-i-06) had similar rectal temperatures as b2 (prrsv-i-92 and prrsv-i-06) pigs. hematology results are summarized in table 2 . there was an effect of ''prrsv strain'' on white blood cell counts at dpi 3 with pigs infected with nc16845b having significantly (p = 0.03) higher levels of white blood cells compared to pigs infected with vr-2385 (10.5 ae 0.9 versus 8.1 ae 0.5). also, there was a significant effect of ''pcv2'' (p < 0.05): pcv2-infected pigs had higher levels of white blood cells at dpi 9 and 12 compared to non-pcv2-infected pigs (17.1 ae 0.9 versus 9.4 ae 0.6 and 20.3 ae 1.0 versus 14.5 ae 1.2). there was no effect of ''prrsv strain'' on numbers of neutrophils; however, there was a significant effect of ''pcv2'' on mean group neutrophil counts at dpi 6, 9, and 12 with pcv2-infected pigs having elevated levels compared to pigs not infected with pcv2 (5.3 ae 0.4 versus 3.1 ae 0.3, 10.0 ae 0.6 versus 3.4 ae 0.3, and 11.9 ae 6.1 versus 6.1 ae 0.9, respectively). differences in mean group lymphocyte counts were only observed on dpi 9 (table 2) and there was an effect of prrsv strain (p = 0.048): pigs infected with nc16845b had lower levels of lymphocytes compared to pigs infected with vr-2385 (3.9 ae 0.3 versus 5.1 ae 0.5). additionally, pigs coinfected with pcv2 had higher levels of lymphocytes (p = 0.016) compared to pigs infected with prrsv alone (4.8 ae 0.3 versus 3.0 ae 0.3) suggesting an effect of ''pcv2''. all pigs in all groups were negative for prrsv-specific antibodies at 0 dpi and negative control pigs remained negative for anti-prrsv antibody throughout the study. prevalence and group mean s/p ratios are summarized in table 3 . overall, there was a significant effect of ''prrsv strain'' and ''pcv2'' on the anti-prrsv antibody response at dpi 9. specifically, pigs infected with vr-2385 had a significantly (p = 0.017) higher anti-prrsv antibody response compared to those infected with nc16845b. similarly, coinfected pigs had significantly (p = 0.028) higher anti-prrsv s/p ratios compared to pigs singularly infected with prrsv. there was no effect of ''pcv2 subtype'' on the magnitude of the anti-prrsv-antibody response among coinfected groups. all pigs in b1 and b2 were negative for pcv2-specific anti-igg antibodies at 0 dpi and the negative controls and b2 pigs remained pcv2 seronegative throughout the trial. in the pcv2 coinfected groups, seroconversion was observed at dpi 9. the prevalence and mean group anti-pcv2-igg s/p ratios table 2 mean group leukocyte values (â1000/ml of whole blood except for ratios) in the different treatment groups on days post-inoculation (dpi) 0, 3, 6, 9 and 12. data obtained from b3-prrsv-i-92 and b3-prrsv-i-06 pigs (gray shaded area) were not included in the analysis. hematology a 0 3 6 9 12 negative controls (n = 8) wbc 8.6 ae 1.0 6.0 ae 0. are summarized in table 4 . there was no effect of ''prrsv strain'' or ''pcv2 subtype'' on the magnitude of the anti-pcv2-antibody response. b3 pigs were seropositive for pcv2 at the time of arrival (mean pcv2 elisa s/p ratio: 1.58 ae 0.09) and the s/p ratios remained at a similar level for the duration of the study (data not shown). at termination of the study, pigs randomly selected from each batch tested negative for antibodies against ppv, siv h1n1 and siv h3n2 (data not shown). prevalence of ifn-g positive samples and mean group ifn-g concentrations are summarized in table 5 . there was no effect of ''prrsv strain'' or ''pcv2'' on the ifn-g levels and no differences were found among groups; however, analysis of an effect of ''pcv2 subtype'' among coinfected groups revealed that on dpi 6, pcv2b-inoculated pigs had significantly (p = 0.028) higher levels of ifng compared to pcv2a-inoculated pigs (0.46 ae 0.17 pg/ml versus 0.06 ae 0.05 pg/ml). all pigs were negative for prrsv-rna in serum at 0 dpi and the negative controls remained negative for prrsv rna throughout the study. the prevalence of prrsv rna positive pigs and group mean genomic copy numbers/ml are summarized in table 6 . sequencing of the orf5 gene of prrsv and comparison with the original inocula confirmed that the correct prrsv isolates were present in the table 3 prevalence of anti-prrsv antibodies and mean group sample-to-positive (s/p) ratios in the different treatment groups on days post-inoculation (dpi) 0, 3, 6, 9 and 12. data presented as prevalence (mean s/p ratio ae se). data obtained from b3-prrsv-i-92 and b3-prrsv-i-06 pigs (gray shaded area) were not included in the analysis. different superscripts (a, b, c) within columns indicate significant (p < 0.05) differences in mean group s/p ratios among groups. prevalence of anti-pcv2 igg antibodies and mean group sample-to-positive (s/p) ratios in the different treatment groups except prrsv-i-92 and prrsv-i-06 on day post-inoculation (dpi) 0, 3, 6, 9 and 12. data presented as prevalence (mean s/p ratio ae se). grey shaded areas indicate the presence of pcv2 seropositive pigs (s/p ratio > 0.2) within a treatment group. different superscripts (a, b) within columns indicate significant (p < 0.05) differences in mean group s/p ratios among groups. prevalence of ifn-g and mean group concentration (pg/ml) in the different treatment groups on day post-inoculation (dpi) 0, 3, 6, 9 and 12. data presented as prevalence (mean log 10 group concentration ae se). data obtained from b3-prrsv-i-92 and b3-prrsv-i-06 pigs (gray shaded area) were not included in the analysis. respective groups and rooms. when results were analyzed for a possible effect of ''prrsv strain'', a significantly (p < 0.05) higher amount of prrsv rna was detected in pigs infected with vr-2385 at dpi 3 and 6 compared to pigs infected with nc16845b ( fig. 1) . when the pigs infected with prrsv alone were removed from the analysis, coinfected pigs with nc16845b had significantly higher amounts of prrsv rna in serum compared to pigs infected with vr-2385 on dpi 9 (6.49 ae 0.10 versus 5.99 ae 0.11) and dpi 12 (6.24 ae 0.08 versus 5.55 ae 0.12), respectively. an effect of ''pcv2'' or ''pcv2 subtype'' on prrsv replication was not evident. all pigs were negative for pcv2-dna in serum at 0 dpi and the negative controls and b2 and b3 pigs remained pcv2 dna negative throughout the study (data not shown). at dpi 3, 28/36 pcv2-inoculated pigs were positive for pcv2-dna, and all pcv2-inoculated pigs were positive for pcv2-dna by 6 dpi and remained positive until dpi 12. the log 10 group mean pcv2 dna amounts are summarized in fig. 2 . when results were analyzed for a possible effect of ''prrsv strain'' it was found that there was a significantly higher amount of pcv2 dna in pigs infected with vr-2385 (7.99 ae 0.19) compared to pigs infected with nc16845b (7.01 ae 0.21) on dpi 12. there was a significant effect of ''pcv2 subtype'' on dpi 3; groups infected with pcv2b had significantly higher amounts of pcv2 dna in serum compared to groups infected with pcv2a (4.63 ae 0.40 versus 2.96 ae 0.51, respectively). an effect of ''pcv2 subtype'' was not evident in the later stages of infection. all pigs in the pcv2a or pcv2b groups were correctly infected with their respective subtype as determined by multiplex real-time pcr (data not shown) and crosscontamination between groups and rooms was not detected. table 6 prevalence of prrsv and group mean log 10 prrsv genomic copies per ml in the different treatment groups on days post-inoculation (dpi) 3, 6, 9 and 12. data presented as prevalence (log 10 prrsv rna ae se). data obtained from b3-prrsv-i-92 and b3-prrsv-i-06 pigs (gray shaded area) were not included into the analysis. group 3 6 9 12 negative controls 0/8 (0.00 ae 0.00) a 0/8 (0.00 ae 0.00) a 0/8 (0.00 ae 0.00) a 0/8 (0.00 ae 0.00) a coi-92-2a 9/9 (5.44 ae 0.23) b,d 9/9 (6.74 ae 0.14) b 9/9 (6.63 ae 0.05) b 9/9 (6.17 ae 0.08) b coi-92-2b 9/9 (5.42 ae 0.22) b,d 9/9 (6.93 ae 0.16) b 9/9 (6.23 ae 0.18) b 9/9 (5.76 ae 0.22) b coi-06-2a 9/9 (4.66 ae 0.16) b,c 9/9 (6.35 ae 0.17) b,c 9/9 (6.93 ae 0.12) b 9/9 (6.56 ae 0.08) b coi-06-2b 9/9 (5.35 ae 0.36) b,d 9/9 (6.53 ae 0.28) b 9/9 (6.90 ae 0.16) b 9/9 (6.76 ae 0.14) b prrsv-i-92 4/4 (6.36 ae 0.25) d 4/4 (7.45 ae 0.18) b 4/4 (7.11 ae 0.09) b 4/4 (7.11 ae 0.16) b prrsv-i-06 3/5 (2.99 ae 1.26) c 4/5 (4.82 ae 1.47) c 5/5 (6.00 ae 0.92) b 5/5 (6.25 ae 0.02) b b3-prrsv-i-92 4/5 (4.52 ae 1.14) 5/5 (7.47 ae 0.12) 5/5 (7.43 ae 0.14) 5/5 (7.28 ae 0.09) b3-prrsv-i-06 4/4 (3.84 ae 0.63) 3/4 (4.57 ae 1.53) 4/4 (6.12 ae 1.33) 4/4 (7.13 ae 0.44) different superscripts (a,b,c,d) within columns indicate significant (p < 0.05) differences in mean group s/p ratios among groups. fig. 1 . log 10 transformed mean prrsv rna genomic copies (aese) in vr-2385 and nc16845b infected pigs regardless of coinfection status on day postinoculation (dpi) 0, 3, 6, 9 and 12. significant (p < 0.05) differences between groups within a dpi are indicated by asterisks. the lines indicate the linear trend for pigs infected with vr-2385 (gray, dashed) or nc16845b (black, solid). macroscopic lesions were characterized by mild-tomoderate enlargement of lymph nodes (especially tracheobrochiolar lymph nodes and mediastinal lymph nodes) and mottled-tan lungs with varying degrees of the lung surface affected by visible pneumonia lesions. the group mean lung lesion severity scores are summarized in table 7 and were significantly (p < 0.05) lower for negative controls compared to all coinfected groups. there were no significant differences in lung lesions severity between the negative controls and the pigs infected with prrsv alone. there was an effect of ''pcv2'' on the mean group macroscopic lung lesion scores as evidenced by the coinfected pigs having more severe macroscopic lung lesions compared to pigs infected with prrsv alone. however, there was no effect of ''prrsv strain'' or ''pcv2 subtype'' on the severity of the observed macroscopic lung lesions. lung tissues had multifocal-to-diffuse, mild-to-severe, lymphohistiocytic interstitial pneumonia. the mean microscopic lung lesion scores, which are summarized in table 7 , were significantly (p < 0.0001) lower in the negative controls compared to the four coinfected groups; however, the scores in the negative controls were not significantly lower than observed in the groups singularly infected with prrsv. there was a significant effect of ''pcv2'' (p < 0.001) on microscopic lung lesions but there was no effect of ''prrsv strain'' or ''pcv2 subtype'' on the severity of the observed microscopic lung lesions. lymphoid lesions were either not observed or were characterized by mild depletion of follicles and minimal granulomatous lymphadenitis in all coinfected groups. significant differences in lymphoid lesion scores were not observed among the groups (data not shown). 3.8.1. prrsv all control pigs were negative for prrsv antigen by ihc on sections of lung. the prevalence of prrsv antigen in lung sections was 16/23 pigs in the nc16845b-inoculated group (b3: 4/4 pigs) compared to 21/22 pigs in the vr-2385-inoculated group (b3: 5/5 pigs). there were no significant differences in the prevalence rates of prrsv fig. 2 . log 10 transformed group mean pcv2 dna amounts (aese) in the pcv2-prrsv coinfected groups on day post-inoculation (dpi) 0, 3, 6, 9 and 12. significant (p < 0.05) differences between groups within a dpi are indicated by different superscripts (a, b). mean group macroscopic (percentage of lung surface affected by lesions) and microscopic (interstitial pneumonia ranging from 0 = normal to 6 = severe, diffuse) lung lesions (mean group amount ae se). data obtained from b3-prrsv-i-92 and b3-prrsv-i-06 pigs (gray shaded area) were not included in the analysis. significant (p < 0.05) differences between groups are indicated by different superscripts (a, b, c). macroscopic lung lesions (0-100%) antigen in lungs between the virus-inoculated groups. the prevalence of prrsv antigen in lungs was independent of ''prrsv strain'' or ''pcv2 subtype''. all control pigs and all b2 and b3 pigs were negative for pcv2 antigen by ihc. low-to-high amounts of pcv2antigen in lung sections were detected in 8/9 coi-92-2a pigs, 7/9 coi-92-2b pigs, 8/9 coi-06-2a pigs and in 4/9 coi-06-2b pigs which corresponds to16/18 pcv2a-inoculated pigs and 11/18 pcv2b-inoculated pigs. moreover, pcv2 antigen was detected in 15/18 vr-2385-inoculated pigs and in 12/18 nc16845b-inoculated pigs. the prevalence of pcv2 antigen in lung tissues was independent of ''prrsv strain'' or ''pcv2 subtype''. in lymphoid tissues, low-tohigh amounts of pcv2 antigen were detected in 8/9 coi-92-2a pigs, 7/9 coi-92-2b pigs, 8/9 coi-06-2a pigs and in 5/9 coi-06-2b pigs which corresponds to 16/18 pigs inoculated with pcv2a and 12/18 pigs inoculated with pcv2b, as well as 13/18 pigs inoculated with nc16845b and 15/18 pigs inoculated with vr-2385. the prevalence of pcv2 antigen in lymphoid tissues was independent of ''prrsv strain'' or ''pcv2 subtype''. the objective of this study was to characterize the infection dynamics and pathogenicity of two different type 2 prrsv isolates recovered from pigs in 1992 and 2006 in a conventional pig model. to mimic field conditions where coinfections frequently occur, the pigs were concurrently infected with either pcv2a or pcv2b. the effect of each prrsv isolate was also evaluated in singularly inoculated pigs. however, due to limitations in numbers of available pigs from the source herd, the experiments with singularly prrsv-inoculated groups were conducted separately but under the same study conditions, using the same inocula and assays to analyze the samples. the prrsv isolate vr-2385 used in this experiment has been well-characterized in the cdcd and the conventional pig models and is considered a relatively highly pathogenic prrsv isolate from the 1990s (halbur et al., 1995b meng et al., 1996) . in contrast, nc16845b represents a more recent prrsv isolated from an outbreak of respiratory disease on a farm characterized by high morbidity and mortality in 2006 (gauger et al., 2012) . the orf2-7 sequence homology between vr-2385 (genbank accession pru20788 and pru03040) and nc16845b (genbank accession hq699067) was approximately 90.4%. the orf5 region demonstrated the least nucleotide and amino acid homology at 87.4% and 87.1%, respectively (gauger et al., 2012) . in the past, dual infections with prrsv and porcine respiratory coronavirus (prcv) or prrsv and siv were studied using conventional pigs (van reeth et al., 1996) and gnotobiotic pigs (van reeth and nauwynck, 2000) and in general disease was found to be more pronounced in dually inoculated pigs. interestingly, in gnotobiotic pigs the effect of the coinfection appeared additive rather than synergistic (van reeth and nauwynck, 2000) . more recent studies have shown that prrsv modifies the innate immune response, induces immunosuppression and enhances the inflammatory response to prcv in pigs (jung et al., 2009; renukaradhya et al., 2010) . in another study, dual infection of specific pathogen-free pigs with prrsv and pseudorabies virus (prv) resulted in more severe clinical signs and increased pneumonia in pigs inoculated with both viruses compared to pigs infected with prrsv or prv alone (shibata et al., 2003) . it is also well recognized that pcv2 replication is enhanced by concurrent prrsv infection in both cd and conventional pigs compared to singularly inoculated pigs (allan et al., 2000; rovira et al., 2002) . to the authors' knowledge, the pathogenicity of genetically different prrsv isolates in the presence of concurrent viral infection has not been evaluated in vivo. the combination of prrsv and pcv2 is one of the most common coinfections associated with swine respiratory disease under field conditions (dorr et al., 2007; pallaré s et al., 2002) . both prrsv isolates used in the current study were isolated from field cases of high mortality and experimental infection of pigs with prrsv vr-2385 has resulted in severe lesions and high levels of viremia (halbur et al., 1995b . the two pcv2 isolates were initially recovered from typical field cases of pcvad in iowa and north carolina and have been characterized in the conventional pig model side by side without identifiable differences between pcv2 subtypes (opriessnig et al., 2008b; sinha et al., 2011) . in the current study, clinical disease in the treatment groups was characterized by variable numbers of infected pigs experiencing transient, mild lethargy, mild respiratory disease and inappetence. coinfected groups had significantly higher mean rectal temperatures compared to pigs infected with prrsv alone and the negative controls. interestingly, when organized by coinfection status and analyzed by pcv2 subtype, pigs inoculated with pcv2a had significantly higher mean group rectal temperatures compared to pigs inoculated with pcv2b on dpi 9 which was associated with an anti-pcv2-antibody response in 38.9% (7/18) of the pcv2ainoculated pigs on dpi 9 whereas a delayed antibody response was seen in pcv2b-inoculated pigs (11.1%; 2/18). it is well documented that pathogenic differences between type 2 prrsv isolates exist (halbur et al., 1995b . the uniqueness of the current study is the utilization of two temporally and genetically different prrsv isolates both from cases of high morbidity and mortality in the field but isolated 15 years apart. in a separate in vitro study comparing phenotypic traits of the two prrs viruses, nc16845b demonstrated reduced growth characteristics compared to vr-2385 (gauger et al., 2012) . nc16845b plaque sizes were slightly smaller than vr-2385 and the peak viral titer demonstrated by nc16845b was approximately 13-fold lower than the vr-2385 peak titer. this is in contrast to the in vivo growth characteristics demonstrated in this report. there were clear differences in initial replication between the two prrsv isolates used in this study. the vr-2385-inoculated pigs had significantly higher levels of prrsv rna in serum on dpi 3 and 6. moreover, nc16845b replicated at higher levels at dpi 9 and dpi 12 compared to vr-2385 which was associated with significantly lower levels of lymphocytes at dpi 9 and a significantly lower n/l ratio at dpi 12. these results suggest that highly pathogenic prrsvs may replicate more efficiently in vivo in contrast to their decreased growth properties in vitro as previously suggested (johnson et al., 2004; wang et al., 2008) . this is further supported by the data obtained from the pigs infected with prrsv alone (b2 and b3) which clearly show an increase in replication in pigs infected with nc16845b in the later stages of the in vivo study. similar to other pcv2-prrsv coinfection studies (allan et al., 2000; harms et al., 2001) , macroscopic and microscopic lesions in coinfected groups were enhanced compared to pigs singularly infected with prrsv. recently, it has been shown that pigs infected with vr-2385 had significantly prolonged (until 70 dpi) pcv2 viremia and shedding in prrsv-pcv2 coinfected pigs (sinha et al., 2011) . a similar approach using prrsv nc16845b, which replicated differently from vr-2385 in the early stages of infection, could potentially offer new insights on viral interactions in pigs. in the current study, pcv2b replication was significantly up-regulated shortly after initiation of the study at dpi 3 compared to pcv2a. furthermore, the coi-92-2b group had significantly higher quantities of pcv2b in the serum compared to coi-06-2a (dpi 3 and 12) and coi-06-2b (dpi 12) which was associated with a higher prevalence of pcv2 antigen in tissues (93.8% versus 75.0%) indicating a synergistic relationship between prrsv-1992 (vr-2385) and pcv2. unlike previous studies where the average trial length ranged from 21 to 32 days (allan et al., 2000; rovira et al., 2002) , this trial was terminated at dpi 12 to evaluate prrsv-induced lung lesions which tend to be most severe between dpi 10 and dpi 14. it remains unknown if the observed trend would have resulted in a difference in clinical disease in the later stages of infection. as expected, and similar to a previous study , the pathological lesions associated with pcv2 were either not present or they were mild; however, pcv2 antigen was detected in most tissues in coinfected pigs. in this study, pcv2 naïve pigs were utilized, thus the relevance of the model to actual field situations is limited considering the majority of young pigs have high levels of passively acquired anti-pcv2 antibodies (opriessnig et al., 2004b) . therefore, the impact of anti-pcv2 immunity on the pcv2 infection could not be ascertained in the experiment; however, this was not a major concern as we know from several experiments that pigs with passively derived antibodies, although protected from clinical pcv2 associated disease, can still be infected with pcv2 (mckeown et al., 2005; opriessnig et al., 2008a) . therefore, we believe that a pcv2 naïve pig model increases the ability to identify trends and associations between prrsv and pcv2. in the current study, prrsv-pcv2 coinfection was administered intranasally on the same day. this model of simultaneous dual inoculation does not fully mimic the population dynamics due to the variability in timing of exposure to these two pathogens within and between herds in field situations. on many conventional farms, endemic exposure and seroconversion to prrsv often occurs earlier than exposure to pcv2. infection of pigs with prrsv prior to pcv2 may contribute to the manifestation of more severe pcv2-induced clinical disease and lesions. prrsv is immunosuppressive, primarily infecting porcine alveolar macrophages (drew, 2000) , which decreases the pig's ability to clear subsequent infections. in contrast, prior prrsv infection may induce an immunostimulatory effect on the host immune response that serves to enhance pcv2 replication and lesions (krakowka et al., 2001) . it is possible that amino acid mutations acquired during serial passaging of prrsv on marc-145 cells could result in attenuation as reported previously (allende et al., 2000; an et al., 2011) . while this is also applicable to the current study, we attempted to minimize this risk, by using a relatively low passage of both viruses with a pig passage followed by only two in vitro passages in marc-145 cells. inoculation was completed two days after weaning and transport of the pigs to the research facility. it is also possible that the stress from weaning, transport, new socialization, and adjusting to a new environment may have affected the ability of the pigs to respond to concurrent prrsv-pcv2 infection and influenced the level of prrsv replication in the pigs. however, the data obtained from pigs infected with prrsv alone indicate that this was not the case and that the ability of the pigs to develop a humoral immune response was normal. overall, the data indicate no significant differences between the two prrsv isolates based on clinical signs, gross pathology, histology or hematology even though the prrsv isolates we utilized in this study were isolated from geographically separated herds (vr-2385 from iowa and nc16845b from north carolina) over a period of 15 years. differences in in vivo replication kinetics were identified. vr-2385 initially replicated more quickly and to higher levels and peaked at dpi 6 and the amount of vr-2385 rna steadily declined thereafter. in contrast, pigs infected with nc16845b had lower levels of prrsv rna in serum initially and this steadily increased through termination of the study at dpi 12. concurrent pcv2 viremia was enhanced by prrsv vr-2385 infection but not by concurrent prrsv nc16845b infection. a higher prevalence of pcv2 antigen was demonstrated in the lungs of pigs coinfected with vr-2385 (83.3%) compared to pigs coinfected with prrsv nc16845b (66.7%). this work further emphasizes in vivo replication differences among prrsv strains and the importance of coinfecting pathogens on prrsv kinetics. additional investigations are necessary to further elucidate the specific mechanisms of the pcv2-prrsv interaction in pigs. comparative genomic analysis of five pairs of virulent parental/attenuated vaccine strains of prrsv experimental infection of colostrum deprived piglets with porcine circovirus 2 (pcv2) and porcine reproductive and respiratory syndrome virus (prrsv) potentiates pcv2 replication mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype novel chimeric porcine circovirus (pcv) with the capsid gene of the emerging pcv2b subtype cloned in the genomic backbone of the nonpathogenic pcv1 is attenuated in vivo and induces protective and cross-protective immunity against pcv2b and pcv2a subtypes in pigs a review of porcine circovirus 2-associated syndromes and diseases detection of two porcine circovirus type 2 genotypic groups in united states swine herds epidemiologic assessment of porcine circovirus type 2 coinfection with other pathogens in swine a review of evidence for immunosuppression due to porcine reproductive and respiratory syndrome virus diversity and evolution of a newly emerged north american type 1 porcine arterivirus: analysis of isolates collected between genetic characterization of type 2 porcine circovirus (pcv-2) from pigs with postweaning multisystemic wasting syndrome in different geographic regions of north america and development of a differential pcr-restriction fragment length polymorphism assay to detect and differentiate between infections with pcv-1 and pcv-2 a molecular clock dates the common ancestor of european-type porcine reproductive and respiratory syndrome virus at more than 10 years before the emergence of disease porcine circovirus type 2 (pcv2) vaccination of conventional pigs prevents viremia against pcv2 isolates of different genotypes and geographic origins the emergence of porcine circovirus 2b genotype (pcv-2b) in swine in canada genetic and phenotypic characterization of a 2006 united states porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field immunohistochemical identification of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model replication and expression analysis of prrsv defective rna the origin and evolution of porcine reproductive and respiratory syndrome viruses three cases of porcine respiratory disease complex associated with porcine circovirus type 2 infection experimental reproduction of severe disease in cd/cd pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus a cluster of farms experiencing severe porcine circovirus associated disease: clinical features and association with the pcv2b genotype pathogenic and humoral immune responses to porcine reproductive and respiratory syndrome virus (prrsv) are related to viral load in acute infection porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (pcv-2) mortality in pigs given porcine circovirus type 2 subgroup 1 and 2 viruses derived from dna clones an infectious cdna clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome effects of porcine circovirus type 2 (pcv2) maternal antibodies on experimental infection of piglets with pcv2 molecular cloning and nucleotide sequencing of the 3 0 -terminal genomic rna of the porcine reproductive and respiratory syndrome virus characterization of a high-virulence us isolate of porcine reproductive and respiratory syndrome virus in a continuous cell line, atcc crl11171 size and antigenic comparisons among the structural proteins of selected autonomous parvoviruses modified indirect porcine circovirus (pcv) type 2-based and recombinant capsid protein (orf2)-based enzyme-linked immunosorbent assays for detection of antibodies to pcv porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents comparison of molecular and biological characteristics of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine (ingelvac prrs mlv), the parent strain of the vaccine (atcc vr2332), atcc vr2385, and two recent field isolates of prrsv effects of the timing of the administration of mycoplasma hyopneumoniae bacterin on the development of lesions associated with porcine circovirus type 2 influence of maternal antibodies on efficacy of porcine circovirus type 2 (pcv2) vaccination to protect pigs from experimental infection with pcv2 porcine circovirus type 2 (pcv2)-infection and re-inoculation with homologous or heterologous strains: virological, serological, pathological and clinical effects in growing pigs differences in virulence among porcine circovirus type 2 isolates are unrelated to cluster type 2a or 2b and prior infection provides heterologous protection experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with mycoplasma hyopneumoniae and porcine circovirus type 2 effect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus derivation of porcine circovirus type 2-negative pigs from positive breeding herds porcine circovirus type 2 (pcv-2) coinfections in us field cases of postweaning multisystemic wasting syndrome (pmws) epidemiology and horizontal transmission of porcine circovirus type 2 (pcv2) genomic and antigenic variations of porcine reproductive and respiratory syndrome virus major envelope gp5 glycoprotein porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs vaccination of pigs against swine influenza viruses by using an ns1-truncated modified live-virus vaccine experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2 the evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with vr-2332 experimental dual infection of specific pathogen-free pigs with porcine reproductive and respiratory syndrome virus and pseudorabies virus porcine reproductive and respiratory syndrome virus (prrsv) influences infection dynamics of porcine circovirus type 2 (pcv2) subtypes pcv2a and pcv2b by prolonging pcv2 viremia and shedding development of a polyclonal-antibody-based immunohistochemical method for the detection of type 2 porcine circovirus in formalinfixed, paraffin-embedded tissue emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark highly pathogenic porcine reproductive and respiratory syndrome proinflammatory cytokines and viral respiratory disease in pigs dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study attenuation of porcine reproductive and respiratory syndrome virus strain mn184 using chimeric construction with vaccine sequence genetic variation and pathogenicity of highly virulent porcine reproductive and respiratory syndrome virus emerging in china porcine circovirus type 2 (pcv2) distribution and replication in tissues and immune cells in early infected pigs complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains heteroclite subgenomic rnas are produced in porcine reproductive and respiratory syndrome virus infection characterization of heteroclite subgenomic rnas associated with prrsv infection recombination between north american strains of porcine reproductive and respiratory syndrome virus the 30-amino-acid deletion in the nsp2 of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china is not related to its virulence the authors thank the iowa livestock health advisory council for funding of this study. key: cord-310844-7i92mk4x authors: hryhorowicz, magdalena; lipiński, daniel; hryhorowicz, szymon; nowak-terpiłowska, agnieszka; ryczek, natalia; zeyland, joanna title: application of genetically engineered pigs in biomedical research date: 2020-06-19 journal: genes (basel) doi: 10.3390/genes11060670 sha: doc_id: 310844 cord_uid: 7i92mk4x progress in genetic engineering over the past few decades has made it possible to develop methods that have led to the production of transgenic animals. the development of transgenesis has created new directions in research and possibilities for its practical application. generating transgenic animal species is not only aimed towards accelerating traditional breeding programs and improving animal health and the quality of animal products for consumption but can also be used in biomedicine. animal studies are conducted to develop models used in gene function and regulation research and the genetic determinants of certain human diseases. another direction of research, described in this review, focuses on the use of transgenic animals as a source of high-quality biopharmaceuticals, such as recombinant proteins. the further aspect discussed is the use of genetically modified animals as a source of cells, tissues, and organs for transplantation into human recipients, i.e., xenotransplantation. numerous studies have shown that the pig (sus scrofa domestica) is the most suitable species both as a research model for human diseases and as an optimal organ donor for xenotransplantation. short pregnancy, short generation interval, and high litter size make the production of transgenic pigs less time-consuming in comparison with other livestock species this review describes genetically modified pigs used for biomedical research and the future challenges and perspectives for the use of the swine animal models. pigs have been extensively used in biomedical research due to anatomical and physiological similarities to humans. moreover, progress in gene editing platforms and construct delivery methods allow efficiently, targeted modifications of the porcine genome and significantly broadened the application of pig models in biopharming and biomedicine. target editing is possible through site-specific nucleases, of which the following are most commonly used: zinc finger nucleases (zfns), transcription activator-like effectors (tales), and nucleases from the crispr/cas (clustered regularly interspaced short palindromic repeats/crispr associated) system. introducing modifications in a specific site of the genome is possible due to cellular processes of repairing double-strand breaks induced by site-specific nucleases. double-strand breaks may be repaired in two ways: by non-homologous end joining (nhej) or by homologous recombination (hr). repair provided by nhej may lead to the formation of indel (insertion/deletion) mutations in the table 1 . summary of the most important advantages and disadvantages of the methods for obtaining genetically modified animals. advantages increased efficiency of the transgene integration precise transformation and selection of modified cells used in cloning the number of damaged zygotes do not exceed 10% the obtained animals do not exhibit mosaicism modification is also revealed in germ cells-transgenic offspring disadvantages low process efficiency (2%-3% in pigs) very low efficiency the possibility of random integration of the transgene early fetal mortality high process invasiveness the possibility of genetic defects genetically modified animals as research models for human diseases are a very important tool in searching for and developing new methods of therapy. a suitable model organism should be characterized by rapid growth, a high number of offspring, easy and inexpensive breeding, ability to be easily manipulated, and having a sequenced genome. initially, only rodents were used as a model in biomedical research. experiments on mice contributed to understanding the genetic background of numerous diseases. however, not every genetic disease induced in mice has the same clinical manifestation as in humans. furthermore, the short life span, together with a higher metabolic rate, makes the analysis of some hereditary diseases challenging. currently, pigs are one of the most important large animal models for biomedical research. many human diseases, such as cardiovascular diseases, obesity, and diabetes, have their counterparts in this species. the use of model organisms makes it possible to analyze diseases that occur naturally in animals with specific mutations and those deliberately induced. introduced animal genome changes may reflect mutations occurring in people suffering from specific genetic disorders. moreover, accurate and efficient genome editing can be used in the treatment of monogenic diseases. a slightly different direction of research is the use of genetically modified animal models in toxicological studies for testing drugs. genetically modified pigs are used as model organisms in research into various diseases, including cardiovascular and neurodegenerative diseases, neoplasms, and diabetes. cystic fibrosis (cf) is an autosomal recessive disorder manifested by bronchopulmonary failure and pancreatic enzyme insufficiency. cf is caused by a mutation in the gene responsible for the synthesis of the cftr (cystic fibrosis transmembrane conductance regulator) chloride channel, altering the mucosal function in the respiratory epithelium, pancreatic ducts, intestines, and sweat glands. the most common cystic fibrosis-causing mutation is the deletion of a phenylalanine at amino acid position 508 (df508). cf lung disease is the main cause of morbidity and mortality in cf patients. porcine lungs share many anatomical and histological similarities with humans. it has been shown that pigs in which the cftr gene was inactivated develop all symptoms of the disease occurring in humans, such as meconium ileus, defective chloride transport, pancreatic destruction, and focal biliary cirrhosis. this makes them a very good model species for this disease [14] [15] [16] . cystic fibrosis is a monogenic disease, and the insertion of the functional cftr gene into cf patient cells should theoretically restore the cftr channel function. therefore, pigs have also been used in gene therapy. the treatment with viral vectors successfully improved anion transport and inhibited bacterial growth [17, 18] . currently, the research focuses on improving the cf gene therapy with the use of the crispr/cas9 system. these efforts are focused on increasing the delivery efficiency of crispr/cas9 elements to target locus and obtaining sustained expression of the cftr transgene [19, 20] . it was demonstrated that precise integration of the human cftr gene at a porcine safe harbor locus through crispr/cas9-induced hdr-mediated knock-in allowed the achievement of persistent in vitro expression of the transgene in transduced cells. these results can help design effective gene therapy to treat cf patients [20] . duchenne muscular dystrophy (dmd) is a progressive, monogenic, x-linked lethal disease characterized by degenerative changes in muscle fibers and the connective tissue. it involves the degeneration of subsequent muscles-skeletal, respiratory, and cardiac-and progressive muscular dystrophy. muscular dystrophy is caused by a frameshift mutation in the dmd gene, which encodes dystrophin, a protein in muscle cells that connects the cytoskeleton with the cell membrane. the dystrophin gene contains 79 exons, with exons 3-7 and 45-55 being the most susceptible to mutations. dmd gene mutations are usually large deletions or duplications of one or several exons, as well as point mutations, leading to a change in the reading frame, the appearance of a premature stop codon, and failure to produce a stable protein. muscular dystrophy is most often diagnosed in early childhood, and patients become wheelchair dependent by 12 years of age. untreated boys die of cardiorespiratory complications around their 20 years. the rapid progress in gene editing gives hope for effective targeted therapies for dmd. moreover, the use of an animal model can facilitate the development of personalized treatment approaches. pigs with a dmd gene mutation (exon 52 deletion) develop human disease symptoms, such as lack of dystrophin in skeletal muscles, increased serum creatine kinase levels, progressive muscle dystrophy, and impaired mobility [21] . however, these animals died prematurely (up to 3 months old at most) what precluded natural breeding. the histological evaluation of skeletal muscles and diaphragm confirmed the presence of excessive fiber size variation, hypercontracted fibers, and segmentally necrotic fibers, resembled that of human dmd patients [21] . moreover, proteome analysis of biceps femoris muscle was performed. an increased amount of muscle repair-related proteins and reduced amount of respiratory chain proteins was found in tissue from 3-month-old dmd pigs. this indicated severe disturbances in aerobic energy production and a decrease in functional muscle tissue [22] . as the deletion of exon 52 in the human dmd gene is a common cause of duchenne muscular dystrophy, pigs can make an accurate research model for gene therapy. another porcine dmd model is genetically modified miniature pigs with a mutation in exon 27 in the dmd gene obtained by the crispr/cas9 system. in addition, these animals have shown symptoms of skeletal and heart muscle degeneration, characteristic of human patients with duchenne muscular dystrophy. reduced thickness of smooth muscle in the stomach and intestine was also observed in the pigs studied. however, founder pigs died of unreported causes [23] . although mutations in exon 27 are not reported in human dmd patients, pigs with this deletion constitute another useful animal dmd model. recently, moretti et al. demonstrated the restoration of dystrophin by intramuscular injection of crispr/cas9 components with the use of adeno-associated viral vectors in a pig model. in this study, pigs with dmd carrying a deletion of dmd exon 52 (d52dmd), resulting in a complete loss of dystrophin expression, were used. the restoration of dystrophin expression was possible due to the excision of exon 51 and the restoration of the dmd reading frame. the internally truncated d51-52dmd sufficed to improve skeletal muscle function, prevent malignant arrhythmias as well as prolonging lifespan of dmd pigs [24] . in the future, this strategy may prove useful in the clinical treatment of patients with d52dmd. alzheimer's disease (ad) is an age-related, progressive neurodegenerative disorder characterized by memory dysfunction followed by cognitive decline and disorientation. ad accounts for 50%-80% of human dementia cases. familial forms of ad are caused by autosomal mutations in the genes encoding presenilin 1 (psen1) and presenilin 2 (psen2) and amyloid precursor protein (app). these mutations are associated with the accumulation of amyloid β (aβ) peptide in senile plaques and phosphorylated tau protein in neurofibrillary tangles (nfts), which leads to synaptic damage and neuronal dysfunction [25] . the first ad model with the use of transgenic pigs was generated in 2009 by kragh et al. they produced göttingen mini pigs that carried a randomly integrated construct containing the cdna of the human app gene with ad causing a dominant mutation known as the swedish mutation (appsw) and a human pdgfβ promoter fragment [26] . although the transgene was specifically expressed in brain tissue at a high level, no ad phenotype was observed in mutant pigs. the same group also obtained göttingen minipigs with the human psen1 gene carrying the ad-causing met146ile mutation (psen1 m146i) and driven by a cytomegalovirus (cmv)-enhanced human ubic promoter. pigs were generated with the use of a site-specific integration system-recombinase-mediated cassette exchange (rmce) [27] . the psen1 m146i protein was expressed and tolerated well in the porcine brain, but also in this case, no symptoms of the ad disease were noticed. therefore, this group generated double transgenic göttingen minipigs with both appsw and psen1 m146i mutations. such a solution allowed the increase in intraneuronal accumulation of aβ [28] . in turn, another group obtained ad transgenic pigs using a retroviral multi-cistronic vector containing three ad-related human genes: app, tau, and psen1, with a total of six well-characterized mutations under the control of a fusion promoter: cmve+ hpdgfβ promoter region. they confirmed that transgenes were expressed at high levels in brain tissue and demonstrated a two-fold increase in aβ levels in the brains of transgenic pigs compared to wild-type [29] . cancer is a genetic disease involving uncontrolled, abnormal cell growth in the blood or solid organs resulting from acquired or inherited mutations. pigs represent a useful animal for the development and validation of new medicines and procedures in human tumor models. there are many resemblances in cancer biology between pigs and humans. these animals can correctly mimic human tumors and show similar pharmacokinetic responses to humans. adam et al. demonstrated that autologous transplantation of primary porcine cells transformed with retroviral oncogenic vectors caused tumorigenesis akin to those found in humans [30] . in turn, schook et al. induced tumor formation in pigs by introducing random transgenes that encode cre-dependent kras (kirsten rat sarcoma viral oncogene homolog) g12d and tp53 (tumor protein 53) r167h oncogenic mutations (orthologous to human tp53 r175h) [31] . moreover, saalfrank et al. reported that porcine mesenchymal stem cells (mscs) resemble human mscs requiring disturbance of p53, kras, and myc signaling pathways to become a fully transformed phenotype [32] . at present, pig models commonly used in cancer research include the tp53 knock-out model of osteosarcoma and apc (adenomatous polyposis coli) mutations model of familial adenomatous polyposis (fap). tp53 is a known tumor suppressor gene, and a germline mutation within this gene leads to li-fraumeni syndrome, a rare, autosomal dominant disorder that predisposes carriers to cancer development. the first model of li-fraumeni syndrome using genetically modified pigs has been described by leuchs et al. they generated pigs carrying a latent tp53 r167h mutation that can be activated by the cre-lox recombinase system [33] . after several years of observation, it was noted that both pigs with homozygous tp53 knock-out and pigs with heterozygous knock-out of tp53 showed osteosarcoma development. the heterozygous knock-out caused the development of spontaneous osteosarcoma in older animals, while homozygous tp53 knock-out resulted in multiple large osteosarcomas in 7 to 8-month-old pigs [32] . moreover, sieren and colleagues generated genetically modified yucatan minipigs that carried the tp53 r167h mutation. animals heterozygous for this mutant allele showed no tumorigenesis process, whereas homozygotes that reached sexual maturity developed lymphomas, osteogenic tumors, and renal tumors at varying rates. the tumor formations were validated by computed tomography, histopathological evaluation, and magnetic resonance imaging [34] . familial adenomatous polyposis is an inherited disorder characterized by the development of numerous adenomatous polyps in the colon and rectum which greatly increases the risk of colorectal cancer. the mutations in the apc tumor-suppressor gene are responsible for fap and may result in a hereditary predisposition to colorectal cancer. flisikowska et al. generated gene-targeted cloned pigs with translational stop signals at codon 1311 in porcine apc (apc 1311), orthologous to common germline apc 1309 mutations in human fap. evaluation of one-year-old pigs carrying the apc 1311 mutation showed aberrant crypt foci and adenomatous polyps with low-to high-grade intraepithelial dysplasia, similar to tumor progression as in human fap [35] . the apc 1311 pig model resulting in the development of polyposis in the colon and rectum can be useful in the diagnosis and therapy of colorectal cancer. cardiovascular diseases (cvds) are the major cause of morbidity and mortality worldwide. cvd is a group of disorders of the heart and blood vessels that involve coronary heart disease (such as angina and myocardial infarction), deep vein thrombosis and pulmonary embolism, peripheral arterial disease, cerebrovascular disease, and rheumatic heart disease. the dominant cause of cvd is atherosclerosis, which is characterized by the narrowing of arteries due to the accumulation of lipid and plaque formation. the plaque buildup restricts blood flow, and plaque burst can entail blood clots. similarities in heart anatomy and physiology, vessel size, blood parameters, coronary artery system anatomy, and lipoprotein metabolism make pigs a suitable model for the human cardiovascular system. atherosclerosis starts with the buildup of serum low-density lipoprotein (ldl), and mutations in the ldl receptor (ldlr) gene may cause familial hypercholesterolemia (fh). a porcine fh model has been generated in yucatan miniature pigs through recombinant adeno-associated virus-mediated targeted disruptions of the endogenous ldlr gene. ldlr+/− heterozygous pigs exhibited mild hypercholesterolemia, while ldlr−/− homozygotes animals were born with severe hypercholesterolemia and developed atherosclerotic lesions in the coronary arteries. these phenotypes were accelerated by high fat and high cholesterol diets [36] . the utilization of ldlr-deficient yucatan minipigs in the preclinical evaluation of therapeutics was also demonstrated. ldlr+/− and ldlr−/− pigs were used to assess the ability of novel drug-bempedoic acid (bema)-to reduce cholesterol biosynthesis. long-term treatment with bema decreased ldl cholesterol and attenuated aortic and coronary atherosclerosis in this fa model [37] . moreover, a model of fa and atherosclerosis was created by using the yucatan miniature pigs with liver-specific expression of a human proprotein convertase subtilisin/kexin type 9 (pcsk9) carrying the gain-of-function mutation d374y. pcsk9 plays important functions in cholesterol homeostasis by reducing ldlr levels on the plasma membrane. gain-of-function mutations in this protein cause increased levels of plasma ldl cholesterol, which in turn may result in more susceptibility to coronary heart disease. pcsk9 d374y transgenic pigs exhibited decreased hepatic ldlr levels, severe hypercholesterolemia on high-fat, high-cholesterol diets, and atherosclerotic lesions [38] . it is also considered that hypertriglyceridemia is an independent risk factor for coronary heart disease, in which apolipoprotein (apo)ciii is associated with plasma triglyceride levels. the hypertriglyceridemic apociii transgenic miniature pig model was generated for the examination of the correlation between hyperlipidemia and atherosclerosis. transgenic pigs expressing human apociii exhibited increased plasma triglyceride levels with their delayed clearance and reduced lipoprotein lipase activity compared to non-transgenic controls [39] . diabetes mellitus (dm) is a group of metabolic disorders characterized by hyperglycemia (elevated levels of blood sugar over a prolonged period), which results from deficiency or ineffectiveness of insulin. dm may lead over time to cardiovascular disease, chronic kidney disease, damage to the nerves and eyes. there are two main types of diabetes mellitus, called type 1 and type 2. type 1 dm, also referred to as juvenile diabetes or insulin-dependent diabetes mellitus, is caused by the pancreas's failure to produce enough insulin. the most common is type 2 diabetes, which is characterized by insulin resistance (reduced tissue sensitivity to insulin) that may be combined with relative insulin deficiency. the anatomical and physiological resemblance to the human pancreas and islets makes pigs excellent animals for metabolic diseases modeling. moreover, the structure of porcine and human insulin is also very similar (differs by only one amino acid). a transgenic pig model for type 2 dm was generated to evaluate the role of impaired glucose-dependent insulinotropic poly-peptide (gip). the main function of incretin hormones gip and glucagon-like peptide-1 (glp1) is stimulated insulin secretion from pancreatic beta cells in a glucose-dependent manner. in type 2 dm, the insulinotropic action of gip is impaired, which may suggest its association with early disease pathogenesis. the transgenic pigs expressing a dominant negative gip receptor (giprdn) in pancreatic cells were produced by lentiviral vectors. a significant reduction in oral glucose tolerance due to delayed insulin secretion as well as in β-cell mass caused by diminished cell proliferation was observed in giprdn animals [40] . these observations resemble the characteristic features of human type 2 diabetes, which makes the porcine giprdn model useful for testing incretin-based therapeutic strategies. further analyses revealed characteristic changes in plasma concentrations of seven amino acids (phe, orn, val, xleu, his, arg, and tyr) and specific lipids (sphingomyelins, diacylglycerols, and ether phospholipids) in the plasma of 5-month-old giprdn transgenic pigs that correlate significantly with β-cell mass [41] . these metabolites represent possible biomarkers for the early stages of prediabetes. moreover, the porcine giprdn model has been used to test liraglutide, glp1 receptor agonist, which improve glycemic control in type 2 diabetic patients. ninety-day liraglutide treatment of adolescent transgenic pigs resulted in improved glycemic control and insulin sensitivity as well as reduction in body weight gain and food intake compared to placebo-treated animals. however, the use of liraglutide did not stimulate beta-cell proliferation in the endocrine pancreas [42] . another type of diabetes, type 3 dm, is maturity-onset diabetes of the young (mody3). mody3 is a noninsulin-dependent type of diabetes with an autosomal dominant inheritance and is caused by mutations in the human hepatocyte nuclear factor 1 α (hnf1α) gene. mutation in hnf1α gene leads to pancreatic β-cell dysfunction and impaired insulin secretion. a pig model for mody3 was generated by expressing a mutant human hnf1α gene (hnf1α p291fsinsc) using intracytoplasmic sperm injection-mediated gene transfer and somatic cell nuclear transfer. the transgenic piglets exhibited the pathophysiological characteristics of diabetes, including high glucose level and reduced insulin secretion from the small and irregularly formed langerhans islets [43] . furthermore, hnf1α p291fsinsc pigs revealed nodular lesions in the renal glomeruli, diabetic retinopathy, and cataract, complications similar to those in patients with dm [44] . mutations in the insulin (ins) gene may result in permanent neonatal diabetes mellitus (pndm) in humans. a pndm large animal model was establish by generated pigs expressing a mutant porcine ins gene (ins c94y), orthologous to human ins c96y. transgenic animals showed signs of pndm, such as lower fasting insulin levels, decreased β-cell mass, reduced body weight, and cataract development. in addition, ins c94y pigs exhibited significant β-cell impairment, including the reduction in insulin secretory granules and dilation of the endoplasmic reticulum [45] . the porcine ins c94y model was further used to perform analysis of pathological changes in retinas and evaluation of the liver of transgenic pigs. the studies revealed several features of diabetic retinopathy, such as intraretinal microvascular abnormalities or central retinal edema [46] . moreover, the multi-omics analysis of the liver demonstrated higher activities in amino acid metabolism, oxidation of fatty acids, gluconeogenesis, and ketogenesis, characteristic of insulin-deficient diabetes mellitus [47] . the genetically modified pig models for human diseases described in this review are summarized in table 2 . human-derived proteins have long been used as therapeutics in the treatment of numerous diseases. however, their quantities are limited by the availability of human tissues. thanks to the development of biotechnology and genetic engineering, modified animals can be used as "bioreactors" to produce recombinant proteins for pharmaceutical use. by using adequate regulatory sequences, promoters, the expression of transgenes can be directed to selected cells and organs. the therapeutic proteins can be obtained from milk, blood, urine, seminal plasma, egg white, or salivary gland that can be collected, purified, and used at an industrial scale. moreover, it is possible to generate multi-transgenic animals that produce many biopharmaceuticals or vaccines in a single organism. the use of an animal platform allows for the relatively low-cost production of pharmacologically valuable preparations in high quantity and quality. the mammary gland is considered to be an excellent bioreactor system for pharmaceutical protein production. the advantage of milk is that it contains large amounts of foreign proteins that do not affect the animal's health during lactation as well as the ease of product collection and purification. while cows are the best species for obtaining large amounts of pharmaceuticals in milk, the cost and time necessary to carry out successful transgenesis make rabbits, sheep, goats, and pigs more popular species. although the pig is not a typical dairy animal, a lactating sow can give about 300 l of milk per year. velander et al. generated transgenic pigs that synthesized human protein c in the mammary gland. protein c plays an important role in human blood clotting, which makes it a potentially attractive drug. the collected milk contained 1 g/l of this protein [48] . other recombinant human proteins involved in the coagulation process, such as factor viii [49] , factor ix [50, 51] , von willebrand factor [52] , were also successfully obtained in the porcine mammary gland. furthermore, the line of transgenic pigs producing functional recombinant human erythropoietin in their milk was demonstrated. erythropoietin regulates red blood cell production (erythropoiesis) in the bone marrow by binding to a specific membrane receptor and has been used in the treatment of anemia. this bioreactor system generates active recombinant human erythropoietin at concentrations of approximately 877.9 ± 92.8 iu/1 ml [53] . in turn, lu et al. generated transgenic cloned pigs expressing large quantities of recombinant human lysozyme in milk. lysozyme is a natural broad-spectrum antimicrobial enzyme which constitutes part of the innate immune system. the authors demonstrated that the highest concentration of recombinant human lysozyme with in vitro bioactivity was 2759.6 ± 265.0 mg/l [54] . biopharmaceuticals can also be synthesized in pigs with the use of alternative systems, such as blood, urine, and semen. the blood of transgenic animals can be a source of human blood proteins, such as hemoglobin. swanson et al. and sharma et al. obtained transgenic pigs that produced recombinant human hemoglobin in their blood cells at a high level, with the ability to bind oxygen identical to that of human blood hemoglobin [55, 56] . there remains, however, the issue of obtaining large amounts of animal-generated therapeutics easily and inexpensively, without killing the animal. moreover, blood cannot store high levels of recombinant proteins for a long time, which are innately unstable, and bioactive proteins in the blood may affect the metabolism of the animals [57] . for this reason, research is being conducted into the production of recombinant proteins secreted into the urine or semen. the advantage of semen is that it is easily obtained and produced in high amounts in species such as pigs (boars can produce 200-300 ml of semen 2-3 times a week), while the advantage of urine is that proteins can be obtained from animals of both sexes throughout their lives. in addition, urine contains few proteins, which facilitates the purification of the protein product, and the urine-based systems pose a low risk to the animal's health. however, the limitation of protein production in the bladder is low yield [58] . the recombinant pharmaceutical proteins produced from transgenic pigs are listed in table 3 . genetically modified pigs can also be used as a source of cells, tissues, and organs for transplantation into human recipients. despite the growing knowledge and ability to perform transplants, the shortage of organs means that the number of patients awaiting a transplant is constantly increasing. xenotransplantation is any procedure involving the transplantation, implantation, or infusion of cells, tissues or animal donor organs, and also body fluids, cells, tissues, and human organs (or their fragments), which had ex vivo contact with animal cells, tissues, or organs into a human recipient. organ xenotransplantation would give us an unlimited and predictable source of organs and enable careful planning of the surgery and preoperative drug treatment of the donor. the animal that best meets the criteria for xenotransplantation is the domestic pig (sus scrofa domestica). pig and human organs show great anatomical and physiological similarities. however, the significant phylogenetic distance results in serious immunological problems after transplantation. despite major difficulties, the pig is currently the focus of all research aimed towards eliminating the problem of organ shortage for human transplantation in the future. thus, the challenge now is to overcome interspecies differences that cause xenograft rejection by the human immune system. the solution, therefore, is to modify pigs in such a way that their organs are not rejected as belonging to another species. advances in genetic engineering have brought scientists closer to obtaining modified animals that would be useful for pig to human transplants. a number of studies have reached the preclinical stage, using primates as model organisms. pig organs transplanted into human recipients are immediately rejected as a result of the so-called hyperacute immunological reaction. xenograft rejection is mainly caused by the gal antigen found on the donor's cell surface, which is synthesized by the ggta-1 enzyme. humans lack both the gal antigen and the ggta-1 enzyme, but have xenoreactive antibodies directed against the porcine gal antigen, which leads to the so-called enzymatic complement cascade in the recipient. the sequence of reactions results in a formation membrane attack complex, lysis, and destruction of the graft cells. the best possible solution to the problem of hyperacute rejection is to inactivate the gene encoding the ggta-1 enzyme responsible for the formation of the gal antigen. in 2001, the first heterozygous ggta1 knock-out pigs were produced [59] , and one year later, the first piglets with two knock-out alleles of the ggta1 gene were born [60] . a series of ggta1 knock-out pigs has also been generated by using zfns [61] , talens [62] , and the crispr/cas9 system [63] . moreover, other carbohydrate xenoantigens present on pig cells but absent in humans have been identified and include neu5gc antigen (n-glycolylneuraminic acid) catalyzed by cytidine monophosphate-n-acetylneuraminic acid hydroxylase (cmah) and the sda antigen produced by beta-1,4-n-acetyl-galactosaminyltransferase 2 (β4galnt2). pigs with ggta1/cmah/β4galnt2 triple gene knock-out were generated using the crispr/cas9 system. cells from these genetically modified animals exhibited a reduced level of human igm and igg binding resulting in diminished porcine xenoantigenicity [64] . to prevent hyperacute rejection, it is possible to introduce human genes regulating the enzymatic complement cascade into the porcine genome. as the complement system may undergo spontaneous autoactivation and attack the body's own cells, defense mechanisms have developed in the course of evolution. they regulate complement activity through a family of structurally and functionally similar proteins blocking complement activation and preventing the formation of a membrane attack complex (mac). introduction of human genes encoding complement inhibitors, such as cd55 (daf, decay-accelerating factor), cd46 (mcp, membrane cofactor protein), cd59 (membrane inhibitor of reactive lysis), into the porcine genome may overcome xenogeneic hyperacute organ rejection [65] . it was demonstrated that the expression of human complement-regulatory proteins can prevent complement-mediated xenograft injury and prolong the survival time of the xenotransplant [66] [67] [68] . studies have shown that the absence of ggta1 and additional human cd55, cd59, or cd46 expression has greater survival rates than just ggta1 knock-out [69, 70] . many genetically modified pigs with human complement inhibitors and other modifications important for xenotransplantation were also generated [71] [72] [73] . the modifications of the porcine genome described above largely resolved the problem of hyperacute rejection. however, xenogenic transplant becomes subject to less severe rejection mechanisms resulting from coagulation dysregulation, natural killer (nk) cells-mediated cytotoxicity, macrophage-mediated cytotoxicity as well as t-cell response. the coagulative disorders result from incompatibilities between pig anticoagulants and human coagulation factors. overcoming coagulation dysregulation in xenotransplantation will require the introduction of human gene encoding coagulation-regulatory proteins into the porcine genome, for example, thrombomodulin (tbm), endothelial cell protein c receptor (epcr), tissue factor pathway inhibitor (tfpi), and ectonucleoside triphosphate diphosphohydrolase 1 (cd39). thrombomodulin binds thrombin and functions as a cofactor for the activation of protein c, which is strongly anticoagulative. porcine tbm binds human thrombin less strongly and cannot effectively activate protein c. it was demonstrated that expressing human tbm (htbm) in porcine aortic endothelial cells (paecs) suppresses prothrombinase activity and delays clotting time [71] . the endothelial protein c receptor enhances the activation of protein c and decreases proinflammatory cytokine synthesis. in vitro studies revealed the correlation between human epcr (hepcr) expression in paecs and reduced human platelet aggregation [74] . a meta-analysis of multiple genetic modifications on pig lung xenotransplant showed that hepcr was one of the modifications that had a positive effect on xenograft survival prolongation in the ex vivo organ perfusion model with human blood [75] . further study demonstrated that kidneys from genetically-engineered pigs (carrying six modifications) functioned in baboons for 237 and 260 days. the authors suggested that prolonged survival time was associated, among others, with the expression of the human epcr gene [76] . tissue factor pathway inhibitor is the primary physiological regulator of the early stage of coagulation. tfpi binds to factor xa, and then xa/tfpi inhibits the procoagulant activity of the tissue factor (tf)/factor viia complex. it was demonstrated that the expression of human tfpi in paecs can inhibit tf activity, suggesting potential for controlling the tf-dependent pathway of blood coagulation in xenotransplantation [77] . more recently, multi-modified pigs carrying human tfpi transgene were produced [76, 78] . cd39 is an ectoenzyme that plays a key role in reducing platelet activation. cd39 converts adenosine triphosphate (atp) and adenosine diphosphate (adp) to adenosine monophosphate (amp), which in turn is further degraded by ecto-5 -nucleotidase (cd73) to antithrombotic adenosine. transgenic pigs with human cd39 (hcd39) gene were generated. the study showed that hcd39 expression protects against myocardial injury in a model of myocardial acute ischemia-reperfusion injury [79] . another approach to xenograft protection may be introducing a human gene that protects against the inflammatory response into the porcine genome. transgenic pigs expressing antiapoptotic and anti-inflammatory proteins, such as human heme oxygenase-1 (ho-1) and human tumor necrosis factor-alpha-induced protein 3 (a20), were produced [80, 81] . porcine aortic endothelial cells derived from pigs carrying human a20 transgene were protected against tnf-α (tumor necrosis factor alpha)-mediated apoptosis and less susceptible to cell death induced by cd95 (fas) ligands [81] . similarly, overexpression of human ho-1 ensured prolonged porcine kidney survival in an ex vivo perfusion model with human blood and paecs protection from tnf-α-mediated apoptosis [80] . furthermore, pigs with a combined expression of human a20 and ho-1 on a ggta1 knock-out background were generated. that transgenic approach alleviated rejection and ischemia-reperfusion damage during ex vivo kidney perfusion [82] . the cellular immune response is another barrier to xenotransplantation. human nk cells can activate the endothelium and lyse porcine cells through direct nk cytotoxicity and by antibody-dependent cellular mechanisms. direct nk cytotoxicity is regulated by activating and inhibitory receptor-ligand interactions. to prevent nk-mediated lysis through the inhibitory cd94/nkg2a receptor, pigs with human leukocyte antigens-e (hla-e) were obtained [83, 84] . the study showed that the expression of hla-e in endothelial cells from transgenic pigs markedly reduces xenogeneic human nk responses. in addition, it was demonstrated that the introduction of the hla-e gene into the porcine genome may also protect pig cells from macrophage-mediated cytotoxicity [85] . more recently, ggta1 knock-out pigs with hcd46 and hla-e/human β2-microglobulin transgenes were produced. the study showed that multiple genetically modified porcine hearts were protected from complement activation and myocardial natural killer cell infiltration in an ex vivo perfusion model with human blood [86] . another approach to inhibit direct xenogeneic nk cytotoxicity is the elimination of porcine ul-16-binding protein 1 (ulbp1), which binds to nkg2d activating nk receptors. crispr technology was adapted to create genetically modified pigs with a disrupted ul16-binding protein 1 gene. in vitro studies confirmed that porcine aortic endothelial cells derived from ulbp1 knock-out pigs were less susceptible to nk-cells' cytotoxic effects [87] . macrophages also play an important role in xenograft rejection and can be activated by direct interactions between receptors present on their surface and donor endothelial antigens as well as by xenoreactive t lymphocytes. the binding cd47 antigen to macrophage surface signaling regulatory protein (sirp-α) delivers a signal to prevent phagocytosis. however, the interaction between porcine cd47 and human sirp-α does not supply the inhibitory effect on macrophages [88] . therefore, the introduction of human cd47 (hcd47) into the porcine genome can overcome macrophage-mediated responses in xenotransplantation. the overexpression of hcd47 in porcine endothelial cells suppressed the phagocytic and cytotoxic activity of macrophages, decreased inflammatory cytokine (tnf-α, il-6, il-1β) secretion and inhibited the infiltration of human t cells [89] . the pigs with ggta1 knock-out and hcd47 were obtained [90] . it was demonstrated that the expression of human cd47 markedly prolonged survival of donor porcine skin xenografts on baboons in the absence of immunosuppression [91] . another challenge in xenotransplantation is the prevention of t cell-mediated rejection. t cells can be induced directly by swine leukocyte antigen (sla) class i and class ii on porcine antigen-presenting cells (apcs) or by swine donor peptides presented on recipient apcs. the main co-stimulatory signals regulating t cell function include cd40-cd154 and cd28-cd80/86 pathways. the cytotoxic t-lymphocyte antigen 4-immunoglobulin (ctla4) can inhibit the cd28-cd80/86 co-stimulatory pathway. therefore, the introduction of human ctla4-ig (hctla4-ig) into the porcine genome may alleviate t cell response in xenografts. it was shown that neuronal expression of hctla4-ig in pigs reduced human t lymphocyte proliferation [92] . moreover, transgenic hctla4-ig protein in pigs extended the survival time of porcine skin grafts in a xenogeneic rat transplantation model [93] . another approach to inhibit t-cell immune response may be the deletion of swine leukocyte antigen class i. reyes et al. created sla class i knock-out pigs using grna and the cas9 endonuclease. the obtained animals revealed decreased levels of cd4−cd8+ t cells in peripheral blood [94] . recently, pigs carrying functional knock-outs of ggta1, cmah, b4galnt2, and sla class i with multi-transgenic background (hcd46, hcd55, hcd59, hho1, ha20) were produced. in vitro study presented that the four-fold knock-out reduced the binding of human igg and igm to porcine kidney cells [95] . beyond immune barriers in xenotransplantation, there is also concern about the risk of cross-species pathogens infection. the main problem constitutes porcine endogenous retroviruses (perv), which are integrated into multiple locations in the pig genome. utilizing the crispr/cas9 technology gives great hopes for the complete elimination of the risk of perv transmission. niu et al. using the crispr/cas9 system inactivated all 25 copies of functional pervs in a porcine primary cell line and successfully generated healthy perv-inactivated pigs via somatic cell nuclear transfer. what is more, no reinfection was observed in the obtained pigs [96] . advances in genetic engineering and immunosuppressive therapies prolong organ survival time in preclinical pig-to-non-human primate (nhp) xenotransplantation models. the first xenotransplantation using pig hearts with eliminated gal antigen into immunosuppressed baboons was performed in 2005. the longest surviving heterotopic graft functioned in the recipient for 179 days [97] , in comparison to 4-6 hours of survival time with the use of wild-type pig hearts [98] . introducing additional modifications extended the xenograft survival time even more. the longest survival was obtained for heterotopic cardiac xenotransplantation-up to 945 days. the authors used hearts derived from genetically multimodified pigs (ggta1 knock-out, hcd46, htbm) and chimeric 2c10r4 anti-cd40 antibody therapy [99] . additional expression of htbm in ggta1 knock-out, hcd46 genetically modified pigs prevented early dysregulation of coagulation and prolonged the cardiac xenografts survival time [99, 100] . using the same genetic background, orthotopic heart xenotransplantation was performed, resulting in a maximum survival of 195 days [101] . however, xenograft survival time depends on the types of transplanted organs. in the case of kidneys in pig-to-nhp transplantation models, the longest survival of a life-sustaining xenograft was 499 days. ggta1 knock-out pigs carrying hcd55 gene as well as immunosuppression with transient pan-t cell depletion and an anti-cd154-based regimen were used in the experiments. moreover, the selection of recipients with low-titer anti-pig antibodies improved the long-term survival of pig-to-rhesus macaque renal xenotransplants [102] . the success of porcine liver and lung xenotransplantation remains limited, which is mainly associated with the occurrence of coagulation disorders [103] . the longest survival time for orthotopic liver xenografts (29 days) was achieved using ggta1 knock-out pigs, exogenous human coagulation factors, and immunosuppression, including co-stimulation blockade [104] . in turn, watanabe et al. demonstrated prolonged survival time of lung xenotransplants (14 days) from ggta1 knock-out, hcd47/hcd55 donor pigs in immunosuppressed baboons [105] . the authors indicated the important role of hcd47 expression in reducing immunologic damages and extending lung graft survival in the pig-to-nhp model. however, additional genetic modifications of the porcine genome and immunosuppressive regimen strategy are necessary for the clinical application of xenotransplantation. table 4 summarizes the most important genetic modifications of the porcine genome for xenotransplantation purposes. the anatomical and physiological similarity between pigs and humans makes this species very interesting for biomedical research [110] . the rapid development of genetic engineering in recent years has allowed for precise and efficient modification of the animal genome using site-specific nucleases. the nuclease-mediated editing of the porcine genome, as well as potential applications of genetically modified pigs in biomedicine, are shown in figure 1 . certainly, the driving force for development is the human mind and ideas that arise in it. one of the factors limiting the possibilities of using the potential of our ideas is the technical aspect. the transfer of new technologies, tested on smaller animal models, for example, is often limited and requires optimization for a large animal model. in the case of crispr/cas9 technology, a lot of emphasis should be placed on the possibility of reducing the risk of so-called off-targets by improving this system. paired nicking has the potential to reduce off-target activity in mice from 50-1000 times without compromising on-target performance [111] . another strategy to limit the number of undesirable off-targets is to increase the specificity of the system. in this situation, one can focus on enhancing or improving cas9 protein or sgrna modifications. protein cas9 properties can be modified, or their lifespan can be changed [112, 113] . for the future clinical success, it is also important to improve the efficiency of hr-mediated gene correction, especially in the situation of treating disease in which a template sequence is delivered to replace the mutated variant. another important goal to achieve is the possibility of applying hr not only for dividing cells but also for cells in the post-mitotic stage. hopes are placed in the fusion of the crispr/cas9 technique and aav (adeno-associated virus) as a donor template provider [114] . considering the low immunogenicity of the aav virus, the ability to transduce a wide spectrum of cells in terms of both type and developmental stage and strong limiting factor-capacity, it should be considered to minimize the crispr/cas9 system or use more than one separate virus to simultaneously exploit the potential of both technologies. the use of other delivery systems, e.g., nanoparticles, is also worth considering [115] . the different site-specific nucleases (zfn, talen, crispr/cas9) used for genome editing and two techniques (somatic nuclear transfer and microinjection) to produce genetically modified pigs are shown. biomedical applications for which genetically engineered pigs are generated include modeling human diseases, production of pharmaceutical proteins, and xenotransplantation. genetically modified pigs serve as an important large animal model for studying the genetic background of human diseases, testing novel drugs and therapy methods as well as developing models for gene therapy [116] [117] [118] . pigs can be used as anatomical (e.g., endovascular), surgical, behavioral, and cytotoxic models. ideas for new models of large animals are provided by the reality that shapes current demand. a lot has been done (pig model for influenza a infection), but still there is a need for pig models of other human viral diseases (hepatitis b; human immunodeficiency virus, hiv; severe acute respiratory syndrome coronavirus 2, sars-cov-2) [119] . hiv has been modeled in mice, filoviruses (ebolavirus, marburgvirus) have been modeled in small animals (i.e., mice, hamsters), but still, we need large models to investigate vaccines and antiviral drugs [120, 121] . transgenic pigs can also be a promising source of recombinant proteins used as pharmacological preparations. actually, the possibility of using pigs for the production of biopharmaceuticals has been slowed in recent years. some studies demonstrated that the pig mammary gland can be used as a complex recombinant protein source with appropriate post-translational modifications [122] . despite the advantages of pig animal platform (natural secretion, correct posttranslational modifications, constant production), some ethical doubts are probably limiting the boost. finally, the use of genetically engineered pigs for xenotransplantation is becoming an increasingly feasible alternative to standard allogeneic transplants and a potential solution to the problem of organ shortage. the combination of various multi-modified pigs and immunosuppressive therapies is required for overcoming immune rejection and effective xenotransplantation of different solid organs [123] [124] [125] . when it comes to treating end-stage organ failure, biomedical research could go a step further and try to create chimeric genetically modified pigs that would be carriers of human organs [126] . the publication was co-financed within the framework of a ministry of science and higher education program as "regional initiative excellence" in the years 2019-2022, project no. 005/rid/2018/19. the authors declare no conflict of interest. genetically engineered pigs as models for human disease trends in recombinant protein use in animal production. microb. cell fact genetically modified pigs as organ donors for xenotransplantation genetic transformation of mouse embryos by microinjection of purified dna dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes pronuclear microinjection transgenic technology in farm animals-progress and perspectives increased efficiency of transgenic livestock production increased transgene integration efficiency upon microinjection of dna into both pronuclei of rabbit embryos a simple and highly efficient transgenesis method in mice with the tol2 transposon system and cytoplasmic microinjection efficient generation of gene-modified pigs via injection of zygote with cas9/sgrna sheep cloned by nuclear transfer from a cultured cell line a background to nuclear transfer and its applications in agriculture and human therapeutic medicine disruption of the cftr gene produces a model of cystic fibrosis in newborn pigs the deltaf508 mutation causes cftr misprocessing and cystic fibrosis-like disease in pigs sequential targeting of cftr by bac vectors generates a novel pig model of cystic fibrosis lentiviral-mediated phenotypic correction of cystic fibrosis pigs cftr gene transfer with aav improves early cystic fibrosis pig phenotypes efficient gene editing at major cftr mutation loci in vitro validation of a crispr-mediated cftr correction strategy for preclinical translation in pigs dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle progressive muscle proteome changes in a clinically relevant pig model of duchenne muscular dystrophy porcine zygote injection with cas9/sgrna results in dmd-modified pig with muscle dystrophy somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of duchenne muscular dystrophy the role of cell-derived oligomers of abeta in alzheimer's disease and avenues for therapeutic intervention hemizygous minipigs produced by random gene insertion and handmade cloning express the alzheimer's disease-causing dominant mutation appsw generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (rmce) and somatic cell nuclear transfer (scnt) expression of the alzheimer's disease mutations aβpp695sw and psen1m146i in double-transgenic göttingen minipigs production of transgenic pig as an alzheimer's disease model using a multi-cistronic vector system genetic induction of tumorigenesis in swine a genetic porcine model of cancer a porcine model of osteosarcoma inactivation and inducible oncogenic mutation of p53 in gene targeted pigs development and translational imaging of a tp53 porcine tumorigenesis model a porcine model of familial adenomatous polyposis targeted disruption of ldlr causes hypercholesterolemia and atherosclerosis in yucatan miniature pigs bempedoic acid lowers low-density lipoprotein cholesterol and attenuates atherosclerosis in low-density lipoprotein receptor-deficient (ldlr+/− and ldlr−/−) yucatan miniature pigs familial hypercholesterolemia and atherosclerosis in cloned minipigs created by dna transposition of a human pcsk9 gain-of-function mutant characterization of a hypertriglyceridemic transgenic miniature pig model expressing human apolipoprotein ciii glucose intolerance and reduced proliferation of pancreatic beta-cells in transgenic pigs with impaired glucose-dependent insulinotropic polypeptide function changing metabolic signatures of amino acids and lipids during the prediabetic period in a pig model with impaired incretin function and reduced β-cell mass effects of the glucagon-like peptide-1 receptor agonist liraglutide in juvenile transgenic pigs modeling a pre-diabetic condition dominant-negative mutant hepatocyte nuclear factor 1α induces diabetes in transgenic-cloned pigs diabetic phenotype of transgenic pigs introduced by dominant-negative mutant hepatocyte nuclear factor 1α permanent neonatal diabetes in ins(c94y) transgenic pigs retinopathy with central oedema in an ins c94y transgenic pig model of long-term diabetes multi-omics insights into functional alterations of the liver in insulin-deficient diabetes mellitus high-level expression of a heterologous protein in the milk of transgenic swine using the cdna encoding human protein c transgenic pigs produce functional human factor viii in milk recombinant human factor ix produced from transgenic porcine milk engineering protein processing of the mammary gland to produce abundant hemophilia b therapy in milk production of recombinant human von willebrand factor in the milk of transgenic pigs recombinant human erythropoietin produced in milk of transgenic pigs production of transgenic-cloned pigs expressing large quantities of recombinant human lysozyme in milk production of functional human hemoglobin in transgenic swine an isologous porcine promoter permits high level expression of human hemoglobin in transgenic swine production of pharmaceutical proteins by transgenic animals expression systems and species used for transgenic animal bioreactors production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning production of alpha 1,3-galactosyltransferase-deficient pigs production of zfn-mediated ggta1 knock-out pigs by microinjection of gene constructs into pronuclei of zygotes production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology generation of ggta1 mutant pigs by direct pronuclear microinjection of crispr/cas9 plasmid vectors evaluation of human and non-human primate antibody binding to pig cells lacking ggta1/cmah/β4galnt2 genes expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection cardiac xenotransplantation: recent preclinical progress with 3-month median survival cytomegalovirus early promoter induced expression of hcd59 in porcine organs provides protection against hyperacute rejection long-term survival of nonhuman primates receiving life-supporting transgenic porcine kidney xenografts generation of gtko diannan miniature pig expressing human complementary regulator proteins hcd55 and hcd59 via t2a peptide-based bicistronic vectors and scnt b-cell depletion extends the survival of gtko.hcd46tg pig heart xenografts in baboons for up to 8 months potential value of human thrombomodulin and daf expression for coagulation control in pig-to-human xenotransplantation complement dependent early immunological responses during ex vivo xenoperfusion of hcd46/hla-e double transgenic pig forelimbs with human blood production of ulbp1-ko pigs with human cd55 expression using crispr technology regulation of human platelet aggregation by genetically modified pig endothelial cells and thrombin inhibition meta-analysis of the independent and cumulative effects of multiple genetic modifications on pig lung xenograft performance during ex vivo perfusion with human blood immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts atorvastatin or transgenic expression of tfpi inhibits coagulation initiated by anti-nongal igg binding to porcine aortic endothelial cells generation of α-1,3-galactosyltransferase knocked-out transgenic cloned pigs with knocked-in five human genes transgenic swine: expression of human cd39 protects against myocardial injury transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys transgenic expression of the human a20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli kidneys from α1,3-galactosyltransferase knockout/human heme oxygenase-1/human a20 transgenic pigs are protected from rejection during ex vivo perfusion with human blood hla-e/human beta2-microglobulin transgenic pigs: protection against xenogeneic human anti-pig natural killer cell cytotoxicity characterization of three generations of transgenic pigs expressing the hla-e gene the suppression of inflammatory macrophage-mediated cytotoxicity and proinflammatory cytokine production by transgenic expression of hla-e multiple genetically modified gtko/hcd46/hla-e/hβ2-mg porcine hearts are protected from complement activation and natural killer cell infiltration during ex vivo perfusion with human blood the production of ul16-binding protein 1 targeted pigs using crispr technology role for cd47-sirpalpha signaling in xenograft rejection by macrophages role of human cd200 overexpression in pig-to-human xenogeneic immune response compared with human cd47 overexpression transgenic expression of human cd47 markedly increases engraftment in a murine model of pig-to-human hematopoietic cell transplantation prolonged survival of pig skin on baboons after administration of pig cells expressing human cd47 transgenic expression of ctla4-ig by fetal pig neurons for xenotransplantation transgenic expression of human cytoxic t-lymphocyte associated antigen4-immunoglobulin (hctla4ig) by porcine skin for xenogeneic skin grafting creating class i mhc-null pigs using guide rna and the cas9 endonuclease viable pigs after simultaneous inactivation of porcine mhc class i and three xenoreactive antigen genes ggta1, cmah and b4galnt2 inactivation of porcine endogenous retrovirus in pigs using crispr-cas9 galactosyltransferase gene-knockout pig heart transplantation in baboons with survival approaching 6 months intravenous infusion of galα-3gal oligosaccharides in baboons delays hyperacute rejection of porcine heart xenografts chimeric 2c10r4 anti-cd40 antibody therapy is critical for long-term survival of gtko.hcd46.htbm pig-to-primate cardiac xenograft cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs consistent success in life-supporting porcine cardiac xenotransplantation long-term survival of pig-to-rhesus macaque renal xenografts is dependent on cd4 t cell depletion overcoming coagulation dysregulation in pig solid organ transplantation in nonhuman primates: recent progress prolonged survival following pig-to-primate liver xenotransplantation utilizing exogenous coagulation factors and costimulation blockade intra-bone bone marrow transplantation from hcd47 transgenic pigs to baboons prolongs chimerism to >60 days and promotes increased porcine lung transplant survival production of biallelic cmp-neu5ac hydroxylase knock-out pigs the generation of transgenic pigs as potential organ donors for humans a human cd46 transgenic pig model system for the study of discordant xenotransplantation pigs transgenic for human thrombomodulin have elevated production of activated protein c transgenic pigs as models for translational biomedical research double nicking by rna-guided crispr cas9 for enhanced genome editing specificity high-fidelity crispr-cas9 nucleases with no detectable genome-wide off-target effects enhanced homology-directed human genome engineering by controlled timing of crispr/cas9 delivery virus-mediated genome editing via homology-directed repair in mitotic and postmitotic cells in mammalian brain improved delivery of crispr/cas9 system using magnetic nanoparticles into porcine fibroblast current progress of genetically engineered pig models for biomedical research genome editing of pigs for agriculture and biomedicine genetically engineered pigs to study cancer the microminipig as an animal model for influenza a virus infection in vitro and in vivo models of hiv latency animal models for filovirus infections production of recombinant human coagulation factor ix by transgenic pig the role of genetically engineered pigs in xenotransplantation research genetically modified pigs as donors of cells, tissues, and organs for xenotransplantation xenotransplantation: current status in preclinical research interspecies chimerism with mammalian pluripotent stem cells this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-350626-ov9fy10b authors: nazki, salik; khatun, amina; jeong, chang-gi; mattoo, sameer ul salam; gu, suna; lee, sim-in; kim, seung-chai; park, ji-hyo; yang, myoun-sik; kim, bumseok; park, choi-kyu; lee, sang-myeong; kim, won-il title: evaluation of local and systemic immune responses in pigs experimentally challenged with porcine reproductive and respiratory syndrome virus date: 2020-05-13 journal: vet res doi: 10.1186/s13567-020-00789-7 sha: doc_id: 350626 cord_uid: ov9fy10b the host-associated defence system responsible for the clearance of porcine reproductive and respiratory syndrome virus (prrsv) from infected pigs is currently poorly understood. to better understand the dynamics of host–pathogen interactions, seventy-five of 100 pigs infected with prrsv-ja142 and 25 control pigs were euthanized at 3, 10, 21, 28 and 35 days post-challenge (dpc). blood, lung, bronchoalveolar lavage (bal) and bronchial lymph node (bln) samples were collected to evaluate the cellular immune responses. the humoral responses were evaluated by measuring the levels of anti-prrsv igg and serum virus-neutralizing (svn) antibodies. consequently, the highest viral loads in the sera and lungs of the infected pigs were detected between 3 and 10 dpc, and these resulted in moderate to mild interstitial pneumonia, which resolved accompanied by the clearance of most of the virus by 28 dpc. at peak viremia, the frequencies of alveolar macrophages in infected pigs were significantly decreased, whereas the monocyte-derived dc/macrophage and conventional dc frequencies were increased, and these effects coincided with the early induction of local t-cell responses and the presence of proinflammatory cytokines/chemokines in the lungs, bal, and bln as early as 10 dpc. conversely, the systemic t-cell responses measured in the peripheral blood mononuclear cells were delayed and significantly induced only after the peak viremic stage between 3 and 10 dpc. taken together, our results suggest that activation of immune responses in the lung could be the key elements for restraining prrsv through the early induction of t-cell responses at the sites of virus replication. porcine reproductive and respiratory syndrome virus (prrsv), a single-stranded positive-sense rna virus with an approximate 15.4-kb genome, belongs to the genus betaarterivirus of the family arteriviridae (ictv 2018). in pigs, prrsv causes porcine reproductive and respiratory syndrome (prrs), which is characterized by reproductive failure in breeding sows and severe respiratory distress in young and growing pigs [1] . prrs results in colossal economic losses in the swine industry worldwide, and these losses are still observed three decades after its emergence in the united states and europe. after the exposure of pigs to prrsv, the virus replicates in alveolar macrophages (am) and further spreads rapidly throughout the body via a lymphohematic route. this viral spread results in acute infection characterized by viremia that lasts for approximately 1 month [2] , and a few studies have reported a nonviremic persistent infection of secondary lymphoid tissues lasting for approximately 150 days or longer [3] . in general, the viremia peaks at approximately 7-10 days post-infection (dpi) and is almost cleared by 28 dpi depending on the viral strain and age of the pigs [4, 5] . additionally, the immune response against prrsv depends on the strain, but the virus usually has immunosuppressive properties [4, 5] , which leads to the increased susceptibility of pigs to secondary microbial infections [6] . the interactions between prrsv and host immune responses have been widely studied, but most studies investigated systemic immune responses using pbmc and/or serum [7] . previous studies have shown that interstitial pneumonia constitutes the major lung lesions in prrsv-infected pigs and that significantly decreased numbers of alveolar macrophages are found in bronchoalveolar lavage (bal) and lung parenchyma samples from prrsv-infected pigs [8] . however, to the best of our knowledge, the kinetics of local immune responses in the lungs or lymph nodes during the course of infection compared with those of peripheral immune responses have not been previously studied. this information would provide a more in-depth understanding of the sequential activation of both immune compartments and the correlation between local or peripheral immune responses and virus clearance in infected pigs. as a result, achieving a comprehensive understanding of the immune responses against prrsv infection remains an important goal in prrsv research. during prrsv infection, the pig immune system is capable of escalating an immune response to ultimately clear the virus from the body [9] . for clearance, proper stimulation of the pig innate immune system is required to direct the development of protective adaptive immunity against prrsv. interestingly, the preferential sites for prrsv replication are alveolar macrophages present in the lungs, which form the major component of the respiratory dc/macrophage network. this network is predominantly involved in sensing foreign antigens, controlling inflammation, and initiating the adaptive immune response [10] . different dc subsets with specific functional specializations exist in the respiratory dc/macrophage network in the lungs and are reportedly resistant to prrsv infection [8, 11] . however, upon activation, these dc travel to lymphoid tissues to present antigen to t lymphocytes and thereby serve as the link between innate and adaptive immunity [10, 11] . t cells, in turn, play a critical role in the development of anti-prrsv immunity due to their cytotoxic effector functions in clearing infected cells from the body and developing and regulating antigen-specific immune responses [12] . however, whether the peripheral virusspecific immune response is appropriately correlated with the local immune response during infection, which could result in the precise use of the peripheral response as a surrogate for scrutinizing the local immune response during viral clearance, remains unclear. therefore, discerning the local and peripheral immune responses during prrsv infection is important for understanding the basic mechanism of viral clearance from the host. cytokines secreted by immune cells act on their targets in an autocrine, paracrine, and/or endocrine manner to prompt local and/or systemic immune responses. in porcine respiratory diseases, proinflammatory cytokines play a key role in activating and synchronizing the adaptive immune responses to clear the virus from the body [13] . however, the tissue damage caused by excessive production of these cytokines is controlled by the secretion of anti-inflammatory cytokines, which results in the maintenance of homeostasis in the body [14] . moreover, effective instigation of the local inflammatory response in the lungs accompanied by significant changes in the proinflammatory cytokine levels in serum has been observed in pigs with respiratory diseases [15, 16] . nevertheless, whether the local or the systemic cytokine/ chemokine response plays the primary role in the clearance of prrsv from pigs during the acute phase of infection remains unclear. in this context, the present study aimed to investigate the trend of host immune responses against prrsv infection during disease progression and to elucidate the innate and adaptive immunological mediators modulated by the prrsv-ja142 strain both systemically in peripheral blood and locally in the bronchoalveolar lavage, lung parenchyma and bronchial lymph nodes (bln) of infected pigs. marc-145 cells, an african green monkey kidney cell line that is highly permissive to prrsv [17] , were used for virus propagation and assays. these cells were maintained in rpmi-1640 medium (gibco ® rpmi-1640, life technologies, carlsbad, ca, usa) supplemented with 10% heat-inactivated foetal bovine serum (fbs, life technologies), 2 mm l-glutamine, and an antibiotic-antimycotic cocktail (anti-anti, life technologies) containing 100 iu/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone ® [amphotericin b] in a humidified chamber with 5% co 2 at 37 °c. in this manuscript, this medium is designated rpmi growth medium. the north american prrsv-2 strain ja142 (genbank: ay424271.1) was used in the present study. one hundred 4-week-old piglets purchased from a prrsv-seronegative farm were randomly assigned to two groups and housed in separate animal rooms. after 3 days of acclimatization, the pigs in the infected group (n = 75) were intramuscularly inoculated with 2 ml of the prrsv-ja142 strain (1 × 10 3 tcid 50 /ml) diluted in sterile pbs. the control pigs (n = 25) remained uninfected. feed and water were provided ad libitum to all the pigs. five pigs from the control group and 12, 16, 14, 18 and 12 pigs from the infected group were humanely euthanized on days 3, 10, 21, 28 and 35 post-challenge (dpc), respectively. euthanasia was performed by electrocution after the intramuscular injection of 2 ml of azaperone (40 mg/ml, stressguard ® , dong bang inc., seoul, south korea). three infected pigs died during the course of the experiment due to high fever and reduced body growth. an overview of the animal study is presented in figure 1 . after euthanasia, the lungs, trachea and bronchi were aseptically extracted and lavaged with 75 ml of sterile pbs. the collected lavage fluid was centrifuged at 1000 × g and room temperature for 10 min to separate the bronchoalveolar lavage fluid (balf) and cells (bal), and these samples, along with the lung parenchyma and bln, were also used for immune cell analysis. these tissues and balf were collected in tubes, snap-frozen using liquid nitrogen and stored immediately at −80 °c for rna extraction and cytokine analysis, respectively. for histopathology, the lung tissues were also collected in 10% neutral-buffered formalin. blood was collected from the euthanized pigs at 3, 10, 21, 28 and 35 dpc, and the serum and pbmc were separated. in addition, blood samples from 42 pigs, including uninfected and infected pigs that were going to be euthanized on 28 and 35 dpc, were also collected at 0, 3, 7, 10, 14, 21 and 28 dpc, and the serum and pbmc were separated. the body weights of all the pigs were measured at 0 dpc, and the body weight gains of the euthanized pigs were measured at 3, 10, 21, 28 and 35 dpc. serum viremia was measured at 0, 3, 7, 10, 14, 21, 28 and 35 dpc, and the viral load in the lungs was quantified in the euthanized pigs at 3, 10, 21, 28 and 35 dpc. viral rna was extracted from serum using a magmax viral rna isolation kit (ambion; applied biosystems, life technologies, inc.) according to the manufacturer's instructions. real-time reverse transcription-polymerase chain reaction (rt-qpcr) employing the prime-q pcv2 prrsv detection kit (genet bio, daejeon, south korea) was performed for the quantification of serum viremia. one-step rt-qpcr was performed in accordance with the manufacturer's instructions. pcr amplification was performed using a model 7500 fast real-time pcr system (applied biosystems, foster city, ca, usa). the cycling conditions were as follows: (i) cdna synthesis for 20 min at 50 °c; (ii) 10-min predenaturation step at 95 °c; and (iii) 40 cycles of denaturation for 10 s at 95 °c figure 1 overview of the animal study. prrsv-negative piglets (n = 100) were purchased and acclimatized for 3 days. seventy-five pigs were then infected, and 25 pigs were used as a negative control (nc). pigs belonging to both the nc and infected (i) groups were euthanized at 3, 10, 21, 28 and 35 days post-challenge (dpc), and lung, bronchoalveolar lavage (bal) and bronchial lymph node (bln) samples were collected. blood samples for sera and peripheral blood mononuclear cells (pbmc) were collected weekly post-challenge. the body weight of the pigs was also monitored from the day of purchase to the end of the experiment. and annealing/extension for 30 s at 60 °c. to calculate the amount of prrsv in each sample, the cq values were converted to virus titres (tcid 50 /ml) by generating a standard curve through the titration of prrsv-2 strain ja142. in addition, marc-145 cells were used to quantify the virus titres in lung tissues using a microtitration infectivity assay [18] . briefly, tissue homogenates (10% [weight/ volume]) of finely chopped lung pieces were prepared in dulbecco modified eagle medium (dmem) with antibiotics, and these mixtures were vortexed vigorously for 10-15 min and then centrifuged at ~4000 × g and 4 °c for 1 h. the collected supernatant was filtered through a 0.20-μm sterile syringe filter and used as an inoculum for the measurement of virus titres. the virus titres were calculated at 5 to 6 days post-inoculation based on the cytopathic effect (cpe) and are expressed as tcid 50 /ml [19] . the lungs of the necropsied pigs in both groups were subjected to pathological evaluation on each day of necropsy. the microscopic lung lesions were given a score on a scale from 0 to 3 to reflect no lesion, mild interstitial pneumonia, moderate multifocal interstitial pneumonia, and severe interstitial pneumonia, respectively. the microscopic lesions were examined from five different lobes of the lungs, and the average value was ultimately utilized for scoring purposes. the serum samples from uninfected and challenged pigs were tested for anti-prrsv antibody (igg) using a commercially available elisa kit (prrs ab elisa 4.0; bionote inc., hwaseong-si, republic of korea) according to the manufacturer's instructions. samples with an s/p ratio (the ratio of the net optical density of the test samples to the net optical density of the positive controls) ≥ 0.4 were considered to be positive for the prrsv antibody. the serum virus-neutralizing (svn) antibodies were measured through a fluorescent focus neutralization (ffn)-based svn assay with marc-145 cells as described previously [20] , with some modifications. after heat inactivation at 56 °c for 1 h, the serum samples were serially (twofold) diluted using rpmi-1640 growth medium. two-hundred-microlitre mixtures were prepared by mixing each diluted serum sample with 10 3 fluorescent focus-forming units per ml (ffu/ml) of prrsv-ja142 at a ratio of 1:1 and were then incubated for 1 h at 37 °c in a humidified atmosphere with 5% co 2 . each mixture was transferred onto a monolayer of marc-145 cells in 96-well plates and incubated for another 1 h at 37 °c. the medium was replaced with 200 µl of fresh rpmi growth medium per well and further incubated for 20 h at 37 °c. the cells were later fixed using ice-cold 80% (v/v) acetone, air-dried, and stained with mouse anti-prrs nc mab 4a5 (median diagnostic, gangwondo, korea) and fitc-conjugated goat anti-mouse igg (h + l) (bethyl laboratories, tx, usa). subsequently, the plates were washed at least three times with pbs and observed under a fluorescence microscope to examine the prrsv-specific ffu. the svn titre is expressed as the reciprocal of the highest dilution at which a 90% or higher reduction in the number of ffu was observed. pbmc were isolated from the blood samples (6 ml) by the density gradient method using leucosep ™ centrifuge tubes (greiner bio-one north america inc., nc, usa) and leucoprep ™ lymphocyte separation media (greiner bio-one north america inc.) according to the manufacturers' instructions. the blood samples were briefly stratified on leucoprep ™ solution at a ratio of 2:1 (blood:leucoprep) and centrifuged at 1000 × g for 10 min. the purified pbmc were collected, washed twice with sterile pbs (ph 7.0) and resuspended in 0.5 ml of sterile pbs supplemented with 1% heat-inactivated fbs (gibco, carlsbad, ca, usa). contaminating red blood cells (rbc) were removed by treatment with rbc lysis buffer (ebioscience, ca, usa). for pathological evaluation, the right-sided lobes were clamped to collect specimens for rna extraction and histopathology, and the left lobes of the lungs were used for bal collection according to a previous study [21] . the lungs were lavaged with 50-75 ml of pbs containing 100 µg/ml ampicillin (usb corporation cleveland, oh, usa) and an antibiotic-antimycotic cocktail (anti-anti, life technologies), and the harvested fluid was centrifuged for 10 min at 1000 × g. the resulting supernatant was collected as bal fluid (balf), whereas the cell pellet (bal cells) was washed three times with pbs after rbc lysis. the cells were resuspended in facs buffer (3% fbs in phosphate-buffered saline and 0.02% sodium azide). the bln were passed through a 40-μm cell strainer (spl life sciences, pocheon, korea) in pbs and then washed with facs buffer according to a previous study [22] . the single cell suspension obtained was used for flow cytometric analysis. mononuclear cells from lung parenchyma were prepared based on a previous study [23] , with few modifications. briefly, lung tissue was collected, washed in sterile ice-cold pbs and suspended in serum-free rpmi media containing dnase i (25 u/ml, sigma, st. louis, mo, usa) and collagenase d (2 mg/ml, roche diagnostics, mannheim, germany). single-cell suspensions were prepared using the gentlemacs octo dissociator (miltenyi biotec, san diego, ca, usa) and incubated at 37 °c for 30 min. subsequently, the cells were passed through a 40-μm cell strainer, washed, and resuspended in facs buffer for flow cytometry analysis after rbc lysis. finally, the cells were counted with a countess ™ automated cell counter (invitrogen, carlsbad, ca, usa), and their viability was tested by trypan blue (sigma-aldrich, st. louis, mo, usa) exclusion [24] . for cell surface staining, single-cell suspensions were incubated on ice for 30 min with specific antibodies as listed in additional file 1, and the cells were then washed three times with facs buffer. when necessary, secondary antibodies conjugated with fluorochrome were used. natural killer (nk) cells, dc and macrophages required only cell surface staining, whereas the different subsets of t cells required intranuclear and intracellular staining. two subsets of nk cells have been phenotypically defined based on nkp46 marker expression: nkp46 + and nkp46 − nk cells [25, 26] . following pbmc staining, a similar gating hierarchy was followed by excluding the unstained cells, doublets and cd3 + cells, and the cd3 − lymphocytes were further analysed for cd8α and nkp46 expression. among cd3 − cells, two populations were found in the pbmc, namely, nkp46 + and nkp46 − nk cells, and both of these cells were cd8α + (additional file 2a). bal cells were subjected to cell surface staining for the cd163 surface marker, and the viability of these cells was analysed using propidium iodide (pi) staining (additional file 2b). additionally, dc and macrophages were segregated from the bal cell population based on staining and gating strategies outlined previously (additional file 2c) [10, 11] . from the mhc-ii + cell population, five phenotypically and functionally defined subpopulations were distinguished using the cd163 and cd172a (sirpα) surface markers. among the mhc-ii + cells, cd172a + / cd163 high cells were defined as am, whereas cd172a + / cd163 int , cd172a + /cd163 low , cd172a + /cd163 − and cd172a − /cd163 − cells were defined as monocytederived macrophages (momɸs), monocyte-derived dendritic cells (modc), conventional dendritic cells 2 (cdc2) and conventional dendritic cells 1 (cdc1), respectively, based on a previous study [10] . regulatory t cells (tregs), which require intranuclear staining of foxp3 after cell surface staining, were fixed with cold fixation/permeabilization buffer (ebioscience, thermo fisher scientific, seoul, korea) at 4 °c for 30 min and were then stained for foxp3 at 4 °c for 30 min. based on a previously described staining and gating strategy [27] , tregs (cd25 + foxp3 + cells) were apparent among the cd4 + cd8 − population (additional file 2d). the t-cell subsets subjected to intracellular staining were stained according to previous studies [28, 29] , with few modifications. briefly, single-cell suspensions were treated with a mixture of 1 × cell stimulation cocktail (ebioscience, thermo fisher scientific, seoul, korea) and 1 × brefeldin a (ebioscience, thermo fisher scientific, seoul, korea) in rpmi growth media and incubated at 37 °c in a humidified chamber with 5% co 2 for 4-5 h. the cells were then stained with antibodies for various cell surface markers in cold facs buffer for 30 min at 4 °c, properly washed twice with cold facs buffer, and fixed with intracellular (ic) fixation buffer (ebioscience, thermo fisher scientific, seoul, korea) at 4 °c for 30 min. for intracellular staining, the cells were washed twice with permeabilization buffer (200 μl/well) and stained with cytokine-specific antibodies in cold permeabilization buffer at 4 °c for 30 min. subsequently, the cells were washed twice with permeabilization buffer. the gating strategy employed for obtaining various t-cell phenotypes after the gating of singlet lymphocytes is demonstrated in additional file 2e. a 100-μl suspension of the stained cell populations in facs buffer was run on an accuri c6 flow cytometer (bd accuri ™ c6 plus, bd biosciences, md, usa). bd accuri ™ c6 plus software version 1.0.23.1 (bd biosciences, md, usa) was used to analyse the data after setting compensation settings according to monocolour and isotype control stains. the data are presented as percentages of all the cell subsets. the cytokine levels in the sera and balf of the uninfected and infected pigs at 3, 10 and 28 dpc were measured using a porcine-specific procartaplex ™ multiplex immunoassay (thermofisher scientific, vienna-1030, austria) according to the manufacturer's instructions. magnetic microsphere technology based on porcine cytokine/chemokine antibody-immobilized magnetic beads was employed in the immunoassay for cytokine quantification [30] . the concentration of each cytokine was measured by running the samples on the luminex ® 200 ™ system (luminex corporation, austin, tx, usa). appropriate standards provided in the kit were utilized to determine the concentration of each cytokine. the machine was verified and calibrated using a luminex ® 100/200 ™ verification kit and a luminex ® 100/200 ™ calibration kit (luminex corporation, austin, tx, usa) prior to use. graphical presentations of the data were prepared using graphpad prism 7.00 (graphpad, san diego, ca, usa), and the data were statistically analysed using spss advanced statistics 17.0 software (spss, inc., chicago, il, usa). a nonparametric t-test (mann-whitney u test) was used to compare the viral loads in the lung tissues, the average daily weight gain (adwg), the phenotypes of various cell subsets and the cytokine responses between two groups. the normalized dead cd163 + cells were analysed by repeated anova (tukey post hoc test) to determine the overall difference, and pairwise comparisons were also performed at different days post-challenge. spearman rank correlation and linear regression were used to determine the associations between two parameters. differences were considered statistically significant if p < 0.05 and are indicated by asterisks and different letters over the bars. the highest viremia and lung viral loads in prrsv-ja142challenged pigs were detected between 3 and 10 dpc (figures 2a, b) . the mean peak virus titre in serum at 7 dpc was recorded as 10 4.58 tcid 50 /ml, whereas the mean peak live viral load in the lungs at 10 dpc, which was measured through the microtitration infectivity assay, was 10 3.34 tcid 50 /ml. the virus was gradually cleared by 35 dpc, at which point, the serum and lung viral loads had decreased to mean values of 10 0.76 and 10 0.08 tcid 50 / ml, respectively. as expected, the control group maintained an uninfected state throughout the experiment. the effect of viremia on body weight gain was observed in the infected pigs by calculating the adwg of the pigs ( figure 2c ). the adwg in the prrsv-ja142-challenged pigs was significantly lower than that in the control pigs at 10, 21 and 28 dpc. the adwg per control pig was recorded as 0.495 kg, whereas the adwg of the infected pigs was reduced to 0.316 kg. as expected, the growth rate of the pigs was negatively affected by viremia following infection, and this negative correlation was significant (r = −0.3809; p ≤ 0.001) ( figure 2d ). moderate to severe interstitial pneumonia with alveolar wall thickening due to type 2 pneumocyte proliferation and inflammatory cell infiltration was detected in the infected pigs during the period of peak viremia. thus, the highest microscopic lung lesion score in the infected pigs was recorded at 10 dpc, and the scores of the lung lesions in these pigs decreased at later time points but remained at significantly higher levels compared with those in control pigs (figures 3a, b) . an elisa based on the nucleocapsid (n) protein was employed to measure the prrsv-specific antibody (igg) response in the infected and noninfected pigs ( figure 4a ). at 3 dpc, prrsv-ja142-specific igg were detected in the serum of the infected pigs. additionally, the sample-to-positive (sp) value gradually increased in the infected group from 7 to 35 dpc, whereas in the control group, the pigs did not produce any prrsv-specific igg at any time point. a low svn titre was observed in the challenged pigs at 28 and 35 dpc, when most of the virus was already cleared from the body ( figure 4b ). in general, the svn antibody responses predominantly appear at the later stages of infection, which is the phase at which most of the virus is cleared from the body, and might play a minor role in the clearance of virus. nk cells, which are a specialized subpopulation of lymphocytes, are the innate immune cells critically responsible for directly killing virus-infected cells, which ultimately leads to viral clearance in the host [31] . to observe the effect of prrsv infection on nk cells in pigs, the frequencies of two different nk cell subsets in the pbmc population were analysed. the percentages of nkp46 − nk cells (cd8 + nkp46 − in cd3 − ) in the pbmc populations of the uninfected and infected pigs were higher than those of nkp46 + nk cells (cd8 + nkp46 + in cd3 − ) (additional file 3a). moreover, compared with the control pigs, the infected pigs exhibited a significantly (p ≤ 0.05) increased frequency of nkp46 + nk cells at the early time point of 7 dpc, whereas the frequency of nkp46 − nk cells was slightly increased at 7 dpc and significantly increased at 14 dpc. the frequency of nkp46 + nk cells returned to normal values at 21 dpc, but the frequency of nkp46 − nk cells in the infected pigs remained at significantly higher values up to 28 dpc ( figures 5a, b) . the associations between nk cells in pbmc and serum viremia were evaluated post-infection ( figures 5c, d) . the nkp46 + nk cell population revealed a significant (r = 0.6621; p < 0.0001) positive correlation with viremia; however, no statistically significant correlation between the levels of nkp46 − nk cells and viremia was observed. the receptors of host cells determine the cell tropism of prrsv. among others, cd163, a cysteine-rich scavenger receptor (srcr), acts as the determinant receptor for prrsv entry and infection [32, 33] . the bal cells were subjected to cd163 staining, and the results show that although the virus did not affect the cd163 + cells until 3 dpc, the infected pigs exhibited a significant (p ≤ 0.05) reduction in the cd163 + cell percentage at 10 dpc, showing a gradual recovery with the decline in the viral loads at 35 dpc ( figure 6a ). the reduction in the cd163 + cell population was attributed to the death of cd163 + cells, which was confirmed by observing the viability of these cells through pi staining. the mean frequencies of dead cd163 + cells in the infected pigs, which were normalized to those in the uninfected pigs, was significantly (p ≤ 0.05) higher at 10 and 21 dpc (32.7% and 14.35%, respectively) ( figure 6b ). the class ii major histocompatibility complex (mhc-ii) is essential for the presentation of antigens to t cells and is constitutively expressed on macrophages and dc [34, 35] . the mhc-ii + cells were analysed to observe the changes in the dc/macrophage network in the lungs. the mhc-ii + cells, such as cd163 + cells, show significant (p ≤ 0.05) decreases at 10 and 21 dpc ( figure 6c) . interestingly, the percentage of am (cd172a + /cd163 high /mhc-ii + cells), which constituted the majority of mhc-ii + cells, decreased significantly at 10 dpc after infection (p ≤ 0.05) (figure 6d ). in contrast, the cd172a + /cd163 int /mhc-ii + cell (momɸ) and cd172a + /cd163 low /mhc-ii + cell (modc) frequencies were significantly (p ≤ 0.05) reduced in the infected pigs early during the infection process (3 dpc) ( figures 6e, f) . however, the cd172a + / cd163 int /mhc-ii + cell percentages in the infected pigs were significantly (p ≤ 0.05) increased at 10 and 21 dpc, and a higher (p ≤ 0.05) cd172a + /cd163 low /mhc-ii + cell frequency was also detected at 10, 21, 28 and 35 dpc in the infected pigs compared with the control pigs. cd172a + /cd163 − /mhc-ii + and cd172a − /cd163 − / mhc-ii + cells were not frequently detected among bal cells and accounted for less than 1% of the five subsets ( figures 6g, h) . at 10 dpc, a significantly higher (p ≤ 0.05) percentage of these cell populations was observed in the infected pigs, and this value decreased with the reduction in the viral load and the recovery of am. the associations between the lung viral loads and different subpopulations of macrophages and dc in bal were evaluated after pooling the results of infected pigs at each time point (figure 7) . during the infection, mhc-ii + cells and am displayed a significant negative correlation (p < 0.05) with lung viral loads; however, other subsets exhibited a significant (p < 0.05) positive correlation. during infection, prrsv-ja142 alters the dynamics of the respiratory dc/macrophage network by destroying am while increasing the populations of antigen-presenting cells (apc), which bridge the gap between innate and adaptive immune systems by presenting the foreign antigen to t cells. the peripheral immune response was measured by analysing the t-cell populations in the pbmc of uninfected and infected pigs at 7, 14, 21, 28 and 35 dpc (additional file 3a). the th1 (ifn-γ + in cd4 + cd8 − ) response in the infected pigs was significantly higher (p ≤ 0.05) than that in the control pigs at 21 dpc, and these responses continued to increase until 35 dpc. the cytotoxic t lymphocyte (ctl) (ifn-γ + in cd4 − cd8 + ) response in the infected pigs started to increase at 14 dpc and was significantly higher (p ≤ 0.05) compared with that in the control pigs at 28 dpc ( figure 8a ). at 21 dpc, the th17 (il17 + in cd4 + cd8 − ) cell response was significantly higher (p ≤ 0.05) in the infected pigs compared with the control pigs, and the response was further escalated at later time points. the il-17-producing cd4 − cd8 + cell population was significantly higher (p ≤ 0.05) in the infected pigs compared with uninfected pigs at 21 dpc. the delay in the induction of effector t cells was mainly perceived peripherally in blood. to observe the activation of the local adaptive immune responses by innate immune cells at the sites of replication and the persistence of prrsv, the t-cell phenotypes in the bln, bal and lung parenchyma of the euthanized control and infected pigs were analysed at 10, 21, 28 and 35 dpc. the percentages of various immune cells in the lungs, bal and bln of uninfected and prrsv-ja142infected pigs at different stages of infection are summarized in additional files 3b, c, and d. overall, various t-cell responses were significantly induced in the lungs as early as 10 dpc, and significant responses in bal and bln were first detected at 10 dpc and were maintained until 35 dpc. the th1 cell (ifn-γ + in cd4 + cd8 − ) frequency in all the local tissues was significantly (p ≤ 0.05) induced in the challenged pigs as early as 10 dpc, and the frequency in bal cells tended to be higher. the ctl (ifn-γ + in cd4 − cd8 + ) frequency was significantly (p ≤ 0.05) higher in the lungs of the infected pigs compared with that of the control pigs at 10 dpc, whereas the frequency in bln and bal cells was higher at the early time point and significantly higher at 21 dpc. the prrsv-ja142-infected pigs also displayed a higher induction (p ≤ 0.05) of th17 cells (il17 + in cd4 + cd8 − ) in the lymph nodes and lung tissues at 10 dpc, whereas a slight increase in these cells was observed in bal cells. the il-17-producing cd4 − cd8 + population was significantly (p ≤ 0.05) induced in the lungs and bal cells of the infected pigs at 10 dpc, whereas in bln, this cell population showed a slight increase at 10 dpc and a significant increase (p ≤ 0.05) at 21 dpc (figures 8b-d) . therefore, compared with the weak and delayed peripheral responses, early and effective cellular immune responses were triggered in local tissues by prrsv-ja142 infection. tregs are well known for their immunosuppressive activities, and previous studies have demonstrated that prrsv infection induces treg responses that might be responsible for ineffective adaptive immune responses [36] [37] [38] . unlike previous studies, no upregulation of tregs (cd25 + foxp3 + in cd4 + cd8 − ) was detected in pbmc isolated from prrsv-infected pigs. moreover, tregs were significantly (p ≤ 0.05) reduced in the bal throughout the course of infection, but no such decline was observed in the lungs (figure 9 ). however, the treg frequencies in bln of the infected pigs initially exhibited a significant decline at 10 dpc but then increased significantly at 21 dpc. the levels of seven different innate and adaptive cytokine/chemokine proteins in the sera and balf at 3, 10 and 28 dpc were compared between the uninfected and infected pigs ( figure 10) . the results reveal that interferon-α (ifn-α) was significantly (p ≤ 0.05) induced in the sera and balf of the infected pigs at 3 dpc. however, at peak viremia (10 dpc), the ifn-α level was reduced peripherally in the sera of the infected pigs, whereas the cytokine level was elevated locally in the balf. nevertheless, significant increases in the level of this cytokine (p ≤ 0.05) were maintained both locally and systemically in the infected pigs compared with the uninfected pigs. furthermore, proinflammatory cytokines/chemokines, such as tumour necrosis factor-α (tnf-α), interleukin-1β (il-1β), il-6, il-8 and il-12, show a similar pattern of early induction at 3 dpc in the sera of the infected pigs followed by decreases as viremia increased. in the sera, significant (p ≤ 0.05) changes in il-1β were only observed in the infected pigs at 3 and 28 dpc. however, significant (p ≤ 0.05) induction of il-1β and il-6 locally in the balf was observed in the infected pigs at 3 dpc. in addition, elevations in all the proinflammatory cytokine/chemokine levels were observed in the infected pigs as the viral load increased, and significant increases in the levels of il-1β, il-6, il-8 and il-12 were observed in the infected pigs compared with the uninfected pigs at 10 dpc. moreover, il-10, an anti-inflammatory cytokine, was significantly induced (p ≤ 0.05) at 3 and 10 dpc in the balf of the infected pigs. overall, the clearance of the virus from the pigs also coincided with the increased secretion of anti-viral and proinflammatory figure 10 local and systemic levels of cytokines/chemokines in the pigs. the concentrations of cytokines/chemokines in the a sera and b balf of pigs belonging to both groups at 3, 10, and 28 dpc were measured using a multiplex luminex-based cytokine immunoassay. the bars in the graphs represent the mean ± sem of the cytokine levels, and the asterisks (*) indicate a statistically significant difference between the averages found for the uninfected (nc) pigs and those obtained for the infected pigs at each time point (* indicates p ≤ 0.05 and ** indicates p ≤ 0.01). cytokines locally in the balf of prrsv-infected pigs, although no considerable shifts were detected in the serum. despite exhaustive research, the understanding of the protective immune responses against prrsv in pigs remains limited. previous studies have mainly focused on certain aspects of immune responses and/or have used a narrow time window to study the immune responses during prrsv infection; therefore, the available data offer a limited picture of the host defence system. to the best of our knowledge, the present study constitutes the first investigation of various aspects of immune responses, such as local vs. systemic and innate vs adaptive, during the course of prrsv infection to obtain a broader picture of the host defence against prrsv. here, we demonstrate the critical role of local immune responses in the clearance of prrsv from pigs due to the early induction of dc/macrophage subsets, the activation of protective t cells in local lymphoid and lung tissues, and the stimulation of proinflammatory cytokines locally in the lungs. in pigs that were intramuscularly inoculated with the biologically characterized prrsv-ja142 strain [20] , the viral titre reached its peak value in the serum and lungs between 3 and 10 dpc. in addition, moderate to severe interstitial pneumonia and a significant decrease in the adwg, which are characteristics of prrsv infection [39] , were detected in the challenged pigs by 10 dpc. mild histopathological lesions (with a lesion score lower than 1) characterized by alveolar wall thickening due to type 2 pneumocyte proliferation were also observed in some of the uninfected healthy pigs that were free of other respiratory pathogens. these observations were likely obtained due to the stress induced by environmental factors, such as housing conditions, weaning, individual space, and ambient temperature variations, as previously reported [40, 41] . similar to previous reports [7] , the prrsv-ja142 strain induced delayed (≥ 28 dpc) and weak (≤ 8) svn titres in infected pigs. an svn antibody titre of 32 offers sterilizing immunity, whereas a titre greater than 8 is considered protective against prrsv infection [42] . the anti-prrsv antibody response induced by prrsv-ja142 in the infected pigs crossed the threshold of 0.4 (s/p ratio) between 3 and 10 dpc, and this response was in accordance with the response produced by other prrsv strains [5] , but the role of these antibodies in protection against prrsv infection is unknown [12] . nk cells, an important component of the innate host defence system, play a critical role in the resolution of viral infections [31] . in general, the potential roles of nk cells in relation to prrsv immunity are poorly understood [43, 44] . in the current study, two defined nk cell subpopulations were distinguished in pbmc based on an approach similar to one previously described [25] . similar to previous observations, the nkp46 + nk cell frequency in pbmc collected from both control and infected pigs was lower than that of nkp46 − cells. nkp46 + and the nkp46 − nk cells execute analogous cytolytic activities but produce different levels of ifnγ, with nkp46 + nk cells producing higher amounts of ifn-γ. moreover, nkp46 can be expressed in nkp46 − nk cells after stimulation with interleukins (il)-2, il-12 and il-18 [25] . in the current study, early increases in the frequencies of nkp46 + and nkp46 − nk cells in prrsv-ja142-challenged pigs were clearly detected. previous studies have shown similar increases in the cd3 − cd4 − cd8 + cell population after infection with different strains of prrsv [7, 45] . induced proliferation of nk cells has also been perceived after influenza infection, suggesting that this subset of lymphocytes is capable of antigen-specific clonal expansion [46] . prrsv, however, significantly suppresses nk cell-mediated cytotoxicity to evade the host immune response [45] , but these previous studies did not consider the subsets of nk cells. the specific action of different subsets of nk cells on prrsv has not yet been explored, and further studies are needed to explain the role of nk cells in the containment of the virus during infection. the main targets of prrsv are cells belonging to the monocyte and macrophage lineages, particularly am. however, dc are also reportedly vulnerable to prrsv infection [47] . the dc/macrophage network, which senses the foreign antigen and initiates the immune response, constitutes one of the main components of the respiratory immune system [10] . it is plausible to expect that the viral infection of these cells alters this network and thus affects downstream immune responses. therefore, dissecting how these immune cells respond to prrsv infection is important to better understand the nature of prrs. until now, limited studies have investigated the alterations in the respiratory dc/macrophage network in pigs during disease progression [8, 10] , and the association of the alteration in this immune network with the t-cell response has not been reported. here, we attempted to explore the dynamics of this important host defence mechanism in relation to prrsv infection. during infection, prrsv replicates in the cd163 + cells in the lungs and induces apoptosis at the early stages of infection [33] . in the current study, a flow cytometric analysis of bal cells revealed that prrsv-ja142 decreased the cd163 + cell population in the infected pigs at the time when peak vir