363 1Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Teramo, Italy. 2Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Foggia, Italy. 3Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Torino, Italy. 4Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici (NA), Italy. 5Institute of Animal Health, University of Las Palmas de Gran Canaria, Veterinary School, Arucas, Las Palmas, Spain. 6Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy. *Corresponding author at: Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Campo Boario, 64100 Teramo, Italy. Tel.: + 39 0861 332420, e-mail: g.difrancesco@izs.it Parole chiave Brucella ceti, Immunoistochimica, Neurobrucellosi, Stenella coeruleoalba. Riassunto I batteri del genere Brucella causano la brucellosi, una malattia infettiva comune all’uomo e ai mammiferi terrestri e acquatici. Dal 1994 diversi casi d‘infezione da Brucella sono stati segnalati nei mammiferi marini in tutto il mondo. I mammiferi marini infetti mostrano reperti lesivi analoghi a quelli osservati nei mammiferi terrestri con presenza di aborti, natimortalità, orchite e neurobrucellosi. Se da un lato i dati siero-epidemiologici suggeriscono che l’infezione da Brucella spp. è cosmopolita, la rilevazione mediante immunoistochimica di antigeni brucellari nei tessuti di animali infetti è spesso problematica. Obiettivo del presente studio è stato quello di valutare, mediante l’impiego di un anticorpo monoclonale nei confronti dell’antigene LPS di Brucella spp., l’immunoreattività del sistema nervoso centrale (SNC) in esemplari di stenella striata (Stenella coeruleoalba) B. ceti-infetti affetti da neurobrucellosi. L'anticorpo in questione si è dimostrato capace di riconoscere immunoistochimicamente le brucelle lisce sia nel SNC dei succitati animali B. ceti-infetti sia in più tessuti di ruminanti domestici Brucella spp.-infetti, essendo stato parimenti caratterizzato mediante ELISA e Western Blotting. In conclusione, i risultati di questo studio hanno rilevanza ai fini sia della diagnosi immunoistochimica sia della definizione patogenetica dell'infezione da B. ceti. Indagine immunoistochimica in esemplari di stenella striata (Stenella coeruleoalba) Brucella ceti-infetti con sintomi di neurobrucellosi Keywords Brucella ceti, Immunohistochemistry, Neurobrucellosis, Stenella coeruleoalba. Summary Bacteria of the genus Brucella cause brucellosis, an infectious disease common to humans as well as to terrestrial and aquatic mammals. Since 1994 several cases of Brucella spp. infection have been reported in marine mammals worldwide. While sero-epidemiological data suggest that Brucella spp. infection is widespread globally, detecting Brucella spp.- associated antigens by immunohistochemistry (IHC) in tissues from infected animals is often troublesome. The present study was aimed at investigating, by means of IHC based upon the utilization of an anti-Brucella LPS monoclonal antibody (MAb), the central nervous system (CNS) immunoreactivity shown by B. ceti-infected, neurobrucellosis-affected striped dolphins. The aforementioned MAb, previously characterized by means of ELISA and Western Blotting techniques, was able to immunohistochemically detect smooth brucellae both within the CNS from B. ceti-infected striped dolphins and within a range of tissues from Brucella spp.- infected domestic ruminants. In conclusion, the results of the present study are of relevance both from the B. ceti infection's diagnostic and pathogenetic standpoints. Gabriella Di Francesco1*, Antonio Petrini1, Anna Rita D'Angelo1, Ludovica Di Renzo1, Mirella Luciani1, Tiziana Di Febo1, Enzo Ruggieri1, Antonio Petrella2, Carla Grattarola3, Barbara Iulini3, Osvaldo Matteucci1, Giuseppe Lucifora4, Eva Sierra5, Antonio Fernández5, Roberto Giacominelli Stuffler6, Clotilde Angelucci6, Marina Baffoni6, Giovanni Di Guardo6 and Manuela Tittarelli1 Immunohistochemical investigations on Brucella ceti-infected, neurobrucellosis-affected striped dolphins (Stenella coeruleoalba) Veterinaria Italiana 2019, 55 (4), 363-367. doi: 10.12834/VetIt.1920.10224.2 Accepted: 30.10.2019 | Available on line: 31.12.2019 SHORT COMMUNICATION 364 Nick title Di Francesco et al. Veterinaria Italiana 2019, 55 (4), 363-367. doi: 10.12834/VetIt.1920.10224.2 subsequently enhanced, in recent years, through the inclusion of 9 cases of B. ceti infection in striped dolphins, 8 of which were found stranded between 2012 and 2019 along the Italian coastline, while the remaining individual was found beached ashore in Canary Islands (Spain) in 2004. The dolphin tissues were collected during post mortem examination, in tight agreement with the investigation protocols to be performed in the framework of the Italian National Stranding Network (INSN) for standard laboratory investigations on stranded cetacean specimens. Positive and negative controls were included in each IHC run, with the positive ones being represented by the lung, liver and placental tissues from ovine and bovine fetuses either naturally or experimentally infected by Brucella  spp. The brain from a Dolphin Morbillivirus-infected striped dolphin was additionally used as negative control tissue. Further negative controls were represented by tissue sections obtained from the 7 immunohistochemically positive, B.  ceti-infected striped dolphins under study, from which the primary anti-Brucella  Ab was omitted. More in detail, Brucella IHC was carried out using the MAb 4B5A against LPS Brucella diluted 1:10 to 1:100. Tissue sections were previously heat treated for antigen retrieval (at 121  °C for 8 minutes) in 0.01 M citrate buffer, pH 6.0. Immunoreactions were then visualized by means of a peroxidase technique (Envision Plus Kit, Dako at IZSAM and Vectastain elite ABC kit standard Vector at the Faculty of Veterinary Medicine, University of Teramo, Italy). The Brucella  spp. isolation and identification procedures were performed in accordance with the technique described in the OIE Manual of Diagnostic Tests and Vaccines (World Organisation for Animal Health 2017). With the only exception of the two individuals in wich B.  ceti infection was diagnosed only by means of biomolecular and IHC techniques (ID 1.5, Table I). All the tissue samples Brucella ceti was first isolated from an aborted bottlenose dolphin (Tursiops truncatus) fetus in 1994 (Ewalt et  al. 1994) and, since then, several cases of infection have been reported among free-ranging cetaceans worldwide (Guzman-Verri et al. 2012). The first case of B. ceti infection in the Mediterranean was recorded only in 2009 (Isidoro-Ayza et al. 2014), with the first case of B. ceti infection having been recorded along the Italian coastline in 2012 (Alba et  al. 2013). Besides the “classical” species, some recently discovered Brucella species have also demonstrated a zoonotic potential, as in the case of B. ceti (Whatmore et  al. 2008, De Massis et  al. 2019). Brucella  spp. infection in marine mammals is characterized by a pathogenicity similar to that of terrestrial mammals. In addition, a documented involvement of the central nervous system (CNS) in the striped dolphin (Stenella coeruleoalba), similarly to what described in the human species, has been reported (Guzman-Verri et  al. 2012), with neurobrucellosis having not been recorded in bovine, caprine, ovine, swine, or canine hosts. Nevertheless, this syndrome is a relatively common feature in non-treated human brucellosis-affected patients (Obiako et  al. 2010). Therefore, cetacean neurobrucellosis may serve as an interesting comparative neuropathology and neuropathogenesis model to understand how the bacterium is capable to cross the blood-brain barrier, thereby giving rise to host’s CNS invasion. While sero-epidemiological data suggest that Brucella  infection is widespread globally (Nymo et  al. 2011), detecting Brucella  spp.-associated antigens by immunohistochemistry (IHC) in tissues from naturally or experimentally infected animals is often troublesome The present study was aimed at investigating, by means of an anti-Brucella  LPS monoclonal antibody (MAb), the CNS immunoreactivity (IR) shown by B. ceti-infected, neurobrucellosis-affected striped dolphins, along with its comparative evaluation in a range of fetal tissues from B.  abortus- and B.  melitensis- infected ruminants. A MAb raised against Brucella  LPS was produced at Istituto Zooprofilattico Sperimentale dell’Abruzzo e Molise ‘G. Caporale’ (IZSAM) and characterized by Western blotting (WB) and indirect ELISA according to Di Febo and colleagues (Di Febo et  al. 2012) and Portanti and colleagues (Portanti et al. 2006), being subsequently characterized against B.  abortus RB51, B.  pinnipedialis and B.  ceti, which were not tested in the past experiments. Samples of lung, liver and placental tissues from 16 ovine fetuses originating from 15 ewes experimentally infected with B.  melitensis biotype 3, along with samples of lung, liver and placental tissues from 6 additional aborted fetuses carried by sheep belonging to Brucella-free flocks, were preliminarly investigated, 20 years ago, against Brucella  spp. The study was Table I. Results of Brucella ceti. IHC in the CNS from infected striped dolphins (Stenella coeruleoalba). ID IHC(IZSAM) IHC (UniTe) Bovine fetal lung tissue (positive control) 1_430 ES 2004 Positive Positive Positive 2_3479 IT 2012 Negative Negative Positive 3_4555 IT 2012 Positive Positive Positive 4_5566IT 2014 Negative Negative Positive 5_16769IT 2017 Positive Positive Positive 6_346IT 2017 Positive Positive Positive 7_202IT 2018 Positive Positive Positive 8_47465IT 2018 Positive Positive Positive 9_2785IT2019 Positive Positive Positive 365 Di Francesco et al. Nick title Veterinaria Italiana 2019, 55 (4), 363-367. doi: 10.12834/VetIt.1920.10224.2 the different neuro-topographical concentrations of B.  ceti organisms, not coincident with that of the microscopic field under study. An additional factor to be considered refers to the experimental conditions used in a portion of this work, that are counterparted by the ‘field conditions’ under which post mortem examinations are routinely carried out on stranded cetacean specimens, including also the adverse effects exerted by post mortem autolysis. Based upon the herein presented results, Brucella spp. IHC should be regarded as a laboratory procedure which is useful not only when analyzing were routinely processed for histopathology and Brucella  immunohistochemistry (IHC), whose reliability and reproducibility were also evaluated by means of an ‘inter-laboratory comparison’, which involved two independent Pathologists (based at IZSAM and at Faculty of Veterinary Medicine, University of Teramo, Italy, respectively) (Table I). The results of the characterization of 4B5A MAb are shown in Table II (i-ELISA) and Figure 1 (WB). Both i-ELISA and WB confirmed that the aforementioned MAb reacted with Brucella smooth strains, with the typical LPS-ladder pattern exhibited in WB analysis. Conversely, the same MAb did not react with rough Brucella  strains (Table I). The results of IHC investigations are reported in Tables II and III. More in detail, Brucella  spp.-associated antigens were detected in pulmonary necrotic cell debris as well as in the cytoplasm of both alveolar macrophages and neutrophils from the B. abortus-infected bovine fetuses (Figure 2A) as well as in liver cells from the B.  melitensis-infected ovine fetuses under study (Figure  2B). Within the CNS from B.  ceti-infected dolphins, macrophage-like cells were seen harbouring more or less consistent loads of microbial antigen (Figure 2, C and D). Neither background staining nor artifacts or positive IR were observed in negative control tissues. The results obtained in the present study clearly showed a strong IR against Brucella  LPS in tissues from all the Brucella  spp.-infected, herein investigated bovine, ovine and striped dolphins (7 out 9 individuals) (Figure 2, C and D). In this respect, the negative IHC results observed in the CNS from 2 B. ceti-infected dolphins may be due either to the low sensitivity of Brucella spp. IHC when low bacterial concentrations are present in infected tissues, or to Table II. Indirect ELISA: cross-reactivities of MAb 4B5A anti-Brucella LPS. Bacterial strain MAb 4B5A* Brucella melitensis biovar 2 Positive Brucella melitensis biovar 1 16M Positive Brucella melitensis biovar 1 Rev.1 Positive Brucella abortus strain S19 Positive Brucella abortus strain S99 Positive Brucella abortus strain S99 (LPS) Positive Brucella abortus biovar 2 Positive Brucella abortus biovar 3 Positive Brucella abortus biovar 6 Positive Brucella suis biovar 1 Positive Brucella ovis Negative Brucella abortus RB51 Negative Brucella ceti Positive Brucella pinnipedialis Positive Table III. Brucella melitensis detection by immunohistochemistry in infected animals. Ovine tissues Tested IHC POS Bovine fetal lung tissue (positive control) Fetal lung 26 16 OK Controls 6 0 OK Fetal liver 26 16 OK Controls 6 0 OK Total 64 32 Figure 1. Western blotting of Mab 4B5A vs Brucella abortus S99 (Lane 1), Brucella melitensis 16M (Lane 2), Brucella suis biotype 1 (Lane 3), Brucella ceti (Lane 4), Brucella pinnipedialis (Lane 5). 1 2 3 4 5kDa 260 160 110 80 60 50 40 30 20 15 10 366 Nick title Di Francesco et al. Veterinaria Italiana 2019, 55 (4), 363-367. doi: 10.12834/VetIt.1920.10224.2 health viewpoint, considering the documented zoonotic potential of Brucella microorganisms. Moreover, MAb 4B5A anti Brucella LPS could represent a diagnostic and research laboratory reagent, whose use may be highly recommended also for the IHC diagnosis and pathogenetic characterization of B. ceti and B. pinnipedialis infections in cetaceans and in pinnipeds, respectively. Acknowledgements This work was carried out within the framework of an ad hoc Research Project on Brucella ceti infection, funded by the Italian Ministry of Health and headed/ coordinated by Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ (Grant code IZS AM 03/16 RC). ovine and bovine infected tissues, but also in the case of B.  ceti-infected, neurobrucellosis-affected striped dolphin CNS tissue specimens (in which macrophage-like cells were seen harbouring more or less consistent loads of bacterial antigen), providing a method capable of achieving a direct and reliable IHC diagnosis of Brucella infection. Furthermore, and not less important, the consistent background and the non-specific reactions observed when using an anti-Brucella  -polyclonal Ab were not seen when MAb 4B5A was used. The additional knowledge provided by this study on the detection of Brucella infection in cetaceans may be helpful not only from a diagnostic standpoint but also for increasing our awareness on the (neuro)pathogenesis of Brucella infection in aquatic mammals and, not less important, also from a public Figure 2. Brucella spp.-associated antigen positive immunohistochemical labeling in bovine fetal lung (A), in ovine fetal liver (B) as well as in CNS (cervical spinal cord) tissues (C, D) from neurobrucellosis-affected, B. ceti-infected striped dolphins. Brucella spp. IHC with MAb 4B5A, Mayer’s hematoxylin counterstain, different magnifications. A B C D 367 Di Francesco et al. Nick title Veterinaria Italiana 2019, 55 (4), 363-367. doi: 10.12834/VetIt.1920.10224.2 Alba P., Terracciano G., Franco A., Lorenzetti S., Cocumelli C., Fichi G., Eleni C., Zygmunt M.S., Cloeckaert A. & Battisti A. 2013. The presence of Brucella ceti ST26 in a striped dolphin (Stenella coeruleoalba) with meningoencephalitis from the Mediterranean Sea. Vet Microbiol ,164, 158-163. Baucheron S., Grayon M., Zygmunt M.S. & Cloeckaert A. 2002. Lipopolysaccharide heterogeneity in Brucella strains isolated from marine mammals. Res Microbiol, 153, 277-280. De Massis F., Zilli K., Di Donato G., Nuvoloni R., Pelini S., Sacchini L., D’Alterio N. & Di Giannatale E. 2019. Distribution of Brucella field strains isolated from livestock, wildlife populations, and humans in Italy from 2007 to 2015. PLoS One, https://doi.org/10.1371/ journal.pone.0213689. Di Febo T., Luciani M., Portanti O., Bonfini B., Lelli R. & Tittarelli M. 2012. Development and evaluation of diagnostic tests for the serological diagnosis of brucellosis in swine. Vet It, 48, 145-156. Ewalt D.R., Payeur J.B., Martin B.M., Cummins D.R. & Miller W.G. 1994. Characteristics of a Brucella species from a bottlenose dolphin (Tursiops truncatus). J Vet Diagn Invest, 6 (4), 448-452. Groussaud P., Shankster S.J., Koylass M.S. & Whatmore A.M. 2007. Molecular typing divides marine mammal strains References of Brucella into at least three groups with distinct host preferences. J Med Microbiol, 56, 1512-1518. Guzmán-Verri C., González-Barrientos R., Hernández-Mora G., Morales J.A., Baquero-Calvo E., Chaves-Olarte E. & Moreno E. 2012. Brucella ceti and brucellosis in cetaceans. Front Cell Infect Microbiol, 2, 3. pmid:22919595. Isidoro-Ayza M., Ruiz-Villalobos N., Pérez L., Guzmán-Verri C., Muñoz P.M., Alegre F., Barberán M., Chacón-Díaz C., Olarte E.C., Barrientos R.G., Moreno E., Blasco J.M. & Domingo M. 2014. Brucella ceti infection in dolphins from the Western Mediterranean sea. BMC Vet Res, 10, 206. Nymo I.H., Tryland M. & Godfroid J. 2011. A review of Brucella infection in marine mammals, with special emphasis on Brucella pinnipedialis in the hooded seal (Cystophora cristata). Vet Res, 42, 93. Portanti O., Tittarelli M., Di Febo T., Luciani M., Mercante M.T., Conte A. & Lelli R. 2006. Development and validation of a competitive ELISA kit for the serological diagnosis of ovine, caprine and bovine Brucellosis. J Vet Med B, 53, 494-498. World Organisation for Animal Health (OIE) 2019. Brucella abortus, B. melitensis and B. suis (infection with B. abortus, B. melitensis and B. suis). In Manual of diagnostic tests and vaccines for terrestrial animals, Part 3, Chapter 3.1.4. (version adopted in May 2016), OIE Paris, 355-398.