341 Veterinaria Italiana 2021, 57 (4), 341-345. doi: 10.12834/VetIt.2114.12867.1 Accepted: 27.05.2020 | Available on line: 31.12.2021 1Health and Biosecurity, CSIRO, Australia. 2Medical Entomology Lab, Institut Pasteur of French Guiana, 23 avenue Pasteur, 97300 Cayenne, French Guiana. 3Australian Animal Health Laboratory, CSIRO, Private Bag 24, Geelong, VIC 3220, Australia. 4Agricultural Research Council ‑ Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort 0110, South Africa. 5Department of Veterinary and Tropical Diseases, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa. 6District Veterinary Officer, Benalla, VIC, Australia. 7School of Biological Sciences, The University of Queensland, St Lucia, 4072, QLD, Australia. *Corresponding author at: Medical Entomology Lab, Institut Pasteur of French Guiana, 23 avenue Pasteur, 97300 Cayenne, French Guiana. E‑mail: jbduchemin@pasteur‑cayenne.fr. Jean-Bernard Duchemin1,2*, John R White3, Antonio Di Rubbo3, Shunin Shi3, Gert Johannes Venter4,5, Ian Holmes6 and Peter J. Walker7 Keywords Australia, Bluetongue virus, Culicoides, Oral susceptibility, Quantitative PCR, Vector competence. Summary Following the emerging bluetongue virus transmission in European temperate regions, we question the vector competence of the abundant Culicoides austropalpalis Lee and Reye in South-East temperate Australia. Field collected Culicoides midges were membrane fed with a bluetongue virus serotype 1 (BTV-1). The average feeding rate was 50%. After 13 days, survival rate was 25% and virus RNA presence was checked by quantitative PCR targeting viral genome segment 10. Virus RNA was found in 7.4% of individually tested females with relative viral RNA load values lower than freshly fed females, indicating that viral replication was low or null. A second qPCR targeting viral genome segment 1 confirmed the presence of virus RNA in only four out of 29 previously positive specimens. After 10 days culture on Culicoides cells, none of these four confimed positive samples did show subsequent cytopathogenic effect on Vero cells or BTV antigen detection by ELISA. As control for this virus activity detection, 12 days after microinjection of BTV-1, Culex annulirostris mosquitoes showed, after culture on Kc cells, cytopathogenic effect on Vero cells, with ELISA-confirmed infection. Despite its abundance in farm environment of the temperate Australian regions, the results of this study make C. austropalpalis of unlikely epidemiological importance in the transmission of BTV in Australia. Experimental bluetongue virus infection of Culicoides austropalpalis, collected from a farm environment in Victoria, Australia became endemic across part of northern Europe (Carpenter et al. 2009). These events suggested that the balance of factors that have allowed Australia to remain free of the disease may be relatively fragile and could be disrupted by the invasion of exotic vectors or more virulent viruses from Asia, changes in the distribution of endemic viruses or vectors, and/or adaptation of viruses to vectors with a more southerly distribution. The invasion of BTV and/or its vectors into southern sheep farming zones of Australia would have significant impacts both on animal health and the export trade. There are critical gaps in our understanding of the genetics, biology, Bluetongue (BT) is a debilitating arbovirus infection of sheep and sometimes cattle, transmitted by Culicoides biting midges (Diptera: Ceratopogonidae) with other domestic and wild ruminants serving as asymptomatic reservoirs, mostly. Multiple serotypes of bluetongue virus (BTV) are enzootic in northern and eastern Australia (Firth et  al. 2017). However, there has been no evidence to date of naturally occurring BT in Australian livestock. In 2006, BTV serotype 8 (BTV-8) emerged unexpectedly in northern Europe. Although the source of this virus is unclear, BTV-8 adapted to local Palaearctic Culicoides species, successfully over-wintered, and SHORT COMMUNICATION 342 Veterinaria Italiana 2021, 57 (4), 341-345. doi: 10.12834/VetIt.2114.12867.1 BTV-1 Culicoides austropalpalis competence Duchemin et al. Viral molecular detection by qPCR followed the technique of Veronesi and colleagues (Veronesi et  al. 2013). Briefly, samples were homogenized in 200  µl of Minimum Essential Medium for insect cell culture by bead beating at 6.0 m/s for 20 s with 10  x  1.0 mm beads. An aliquot of 100 µl was stored in -  80  °C for virus isolation. The remaining 100  µl was mixed with Magmax® nucleic acid extraction buffer for both DNA and RNA extractions. BTV-1 RNA presence and quantification was assessed by using a BTV real-time TaqMan PCR assay routinely run at AAHL and targets the NS3 gene expressed by virus genome segment 10 (Hofmann et al. 2008). Primers and probe were pre-mixed and stored in small aliquots at - 20 °C (Hofmann Fwd R10 189-207 5’-TGGAYAAAGCGATGTCAAA-3’, Hofmann Rev R10 285-266 5’-ACATCATCACGAAACGCTTC-3’, Hofmann Probe R10 245-264 5’-6FAM-ARGCTGCATTCGCAT CGTACGC-TAMRA-3’). Cycle threshold (Ct) values equal or higher than 40 were considered negative. Duplicates were required to yield the same results for a firm positive or negative conclusion. Specimens giving a positive/negative result for each duplicate were treated as inconclusive. In each qPCR run were included a strong and a weak positive controls with standard BTV RNAs, giving respective Ct  values around 24 and 32, and a negative control without viral RNA. To exclude any major effect of midge materials presence on molecular detection, a dilution curve was composed of four 10 x serial dilutions of tissue culture super natant (TCSN) virus mixed with individual unfed midges. Quality of RNA extraction was internally controlled by a separate TaqMan assay targeting 18S ribosomal RNA with specimens showing higher than 25 Ct values being discarded. The mean viral RNA relative index (invdiff ) was estimated with standardization after the eukayotic 18S RNA by the calculation of the multiplicative inverse of the difference between BTV and 18S Ct values [‘invdiff’ = 1 / (BTV Ct - 18S Ct)], (Figure 1). A subset of 31 positive RNAs, including freshly bloodfed specimens of midges and mosquitoes, were tested for another qPCR targeting BTV segment 1 (Shaw et  al. 2007) with another set of primers (BTVrsa 291-311For 5' -GCGTTCGAAGTTTACATCAAT- 3', BTVrsa 387-357Rev 5'-CAGTCATCTCTCTAGACACTCTATAATTACG- 3', Probe RSA-BTV 341-320 5' - CGGATCAAGTTCACTCCACGGT- 3'). Similar to the first qPCR, strong and weak BTV RNAs positive controls, as well as a negative one and 18S were used in this PCR run. The confirmed double-positive D 13 Culicoides specimens, with double-positive D 0 midges and mosquitoes as positive controls, were tested for presence of viable virus. More, egg homogenates previously injected with BTV-1 infected blood or PBS were included as supplementary, positive and negative controls, respectively. After ten day amplification culture on Kc/FLI C. variipennis cells (kindly provided by Istituto ecology and distribution of BTV and Culicoides in Australia, and the potential consequences of global warming. For a better understanding of the BTV transmission potential of the Culicoides species present in the southern parts of Australia, we have performed BTV experimental infection of Culicoides caught on a farm in the southern Australian state of Victoria. The collection and infection procedures are detailed in Venter and colleagues (Venter et al. 1998). Briefly, Onderstepoort UV traps were set up at a farm at Benalla, Victoria (145.9 E, 36.6 S, 170 m above sea level) for 33 trap-nights during March 2014. Three to four traps were set per night close to resting animals (sheep, horses) in paddocks. Insects were collected alive and self-sorted by exiting through meshed funnel into small cardboard containers. They were provided with 10% sucrose solution on cotton pads and placed in iceboxes with cold packs before transport to the laboratory by car for 3 hours. They were acclimated for 2 to 6 days at 23 °C before experimental infections. Virus experiments were conducted in a biosafety level three (BSL-3) insectary at the Australian Animal Health Laboratory (AAHL). Supernatant of BTV serotype 1 (BTV-1) strain CS 156 (St George et  al. 1980) culture was diluted in heparinised cattle blood antibody-free for BTV to a final viral titre between 3  x  105 and 1  x  106 TCID 50 /ml. Sucrose ad libitum was stopped 24  h before infective challenge. Midges were offered an infective blood meal through one day-old chicken skin using a membrane feeding system (Hemotek®) in environmental cabinets at 15  °C, 50% relative humidity and darkness and allowed to feed for 60 min. After CO 2 anaesthesia and sorting on an entomological chill table, only blood-fed females were reserved in cardboard cups, with 10% sucrose and maintained at 23 °C in darkness for 13 day (D 13 ) extrinsic incubation for final testing. Unfed Culicoides were returned to containers for re-feeding on the following day. Controls for infection experiment were defined by infection of the non-vector Culex annulirostris colony mosquitoes challenged with the same BTV-1 strain either orally, as negative control, or by intra-thoracic microinjection of 69 nl of the undiluted virus batch corresponding to ~ 102 TCID 50 virus load. This micro-injection bypasses the midgut barrier and makes the mosquito able of viral infection in the hemolymph and as such, considered positive control. At D 13 , live midges and mosquitoes were anesthetised with CO 2 and sorted on the chill table, identified at the species level (Dyce et al. 2007) and stored at - 80 °C in individual tubes. The infection dynamics was assessed by comparison of presence and relative quantification of viral RNA at D 0 , in either per os or microinjection freshly challenged insects and at D 13 in midges and mosquitoes, after extrinsic incubation time. 343Veterinaria Italiana 2021, 57 (4), 341-345. doi: 10.12834/VetIt.2114.12867.1 Duchemin et al. BTV-1 Culicoides austropalpalis competence did not differ from per os orally challenged mosquito values (Kruskal-Wallis test, p = 0.22). The prevalence of positive specimens (Table I) at D 13 was 7.4% (31/416) for Culicoides, 0% for bloodfed mosquitoes (0/14) and 100% (9/9) for microinjected mosquitoes. Of 31 NS3 qPCR positive specimens, 29 (94%) were C.  austropalpalis, one C.  marksi and one undetermined. The positive RNA values for these two last species were not different from those for C.  austropalpalis. All the inconclusive samples were C.  austropalpalis. For Culicoides, D 13 average viral RNA relative values were 0.036 (SD  =  0.005), with positive values at 0.052, (SD  =  0.0049). Both were significantly (p  <  0.0001) lower than D 0 values. For microinjected mosquitoes, the D 13 average Ct values at 0.099 (SD  =  0.019) was higher than D 0 but not significantly (p  =  0.223). However, this D 13 value for micro-injected mosqutioes was significantly (p < 0.001) higher than the per-os infected mosquito at D 13 average value of 0.029 (SD = 0.002) (Figure 1). Of the 29 NS3 BTV-1 positive D 13 -infected Culicoides austropalpalis tested, only four were tested positive for RSA Segment 1 qPCR, six tested inconclusive and 19 were negative. As control, two D 0 infected Culicoides were positive. In total, approximately 1% of the challenged Culicoides females presented viral RNA from two different genes. None of these four specimens with viral RNA at D 13 showed CPE on Vero cells, after amplification attempt during ten days on Culicoides Kc cells. As control, CPE was demonstrated in 100% of D 0 -infected Culicoides [n  =  4, average Optical Density (DO) 2.178 +/- 0.102] and 100% of mosquitoes infected at starting stage of infection (n =  9, average OD 2.229 +/- 0.57). We performed experimental infections on midges caught at a Victorian farm of mixed cattle and sheep, and challenged more than 3,000 Culicoides by membrane feeding of BTV-1 infected blood meal. We obtained a feeding rate of approximately 50%, comparable to experiments conducted in Europe by Carpenter and colleagues (Carpenter et al. 2008) on field collected midges. Our 25% survival rate was lower than the 39% obtained by Carpenter and colleagues (Carpenter et al. 2008) but the incubation period was longer: 13 days instead of 7-10 days and a membrane-feeding instead of pad-feeding technique was used. The viral RNA detection method Zooprofilattico Sperimentale dell’Abruzzo e del Molise, IZSAM, Italy) (Wechsler et  al. 1989), samples were tested for cytopathogenic effect (CPE) on Vero cells, and BTV antigen detection by ELISA technique following Stanilawek and colleagues (Stanilawek et al. 1996) and Hawkes and colleagues (Hawkes et al. 2000), using Nunc Maxisorp ELISA immuno-plates (Thermo Fisher ScientificTM) and insect cell supernatants. The controls of the cell culture phase were used. Statistics and graphics were obtained by using R (R Core Team 2017) and ggplot2 package (Wickham 2016). The rate of blood-feeding for Culicoides was 49.8% (n  =  3293). The rate for mosquitoes was 90.6% (n = 53). The immediate (D 0 ) mortality rate was 15% for Culicoides. The percentage survival for Culicoides at D 13 was 25.2% (n  =  1641), 58.3% for bloodfed mosquitoes (n  =  24), and 37.5% for microinjected mosquitoes (n = 24). Among the 416 Culicoides females tested by qPCR, 94.5% were Culicoides austropalpalis Lee and Reye, 1.7% Culicoides marksi Lee and Reye, 1.2% Culicoides victoriae Macfie, 0.7% Culicoides bundyensis Lee and Reye, 0.7% Culicoides ornatus grp and 1.2% were not determined. The mean Ct  values for TaqMan PCR assay of virus and midge serial dilution (from TCID 50 101 to 104) were 26.8, 23.7, 20.1, and 16.6, respectively. The invdiff (Figure  1) for freshly bloodfed Culicoides (D 0 ) were 0.071 (SD = 0.007) and 0.065 for mosquitoes (SD = 0.006), and do not differ significantly (Kruskal-Wallis test p  =  0.27). The D 0 value for microinjected mosquitoes was 0.074 and Table I. Prevalence of positive, negative and inconclusive Ct values for NS3 BTV-1 at RT-PCR D 13 . Positive (%) Negative (%) Inconclusive (%) Culicoides blood fed (n = 416) 31 (7.4) 338 (81.3) 47 (11.3) Mosquitoes blood fed (n = 14) 0 (0) 12 (85.7) 2 (14.3) Mosquitoes microinjected (n = 9) 9 (100) 0 (0) 0 (0) C ul ic oi de s p er o s M o sq u it o p er o s 0.12 0.10 0.08 0.06 0.04 M o sq u it o m ic ro -i n je ct ed C ul ic oi de s p er o s M o sq u it o p er o s M o sq u it o m ic ro -i n je ct ed D 0 D 12-13 In ve rs e o f d i� (B T V C t – 18 S C t) Figure 1. Boxplots of normalized BTV RNA index standardized to the 18S ribsoomal RNA [Y-axis: invdiff = 1/(BTV Ct – 18S Ct)]. On the left side, red boxplots with values at day 0 for, from the left to the right: per os infected Culicoides, per os infected mosquitoes, and microinjection infected msoquitoes. On the right side, with same order, blue boxplots for day 13 values. 344 Veterinaria Italiana 2021, 57 (4), 341-345. doi: 10.12834/VetIt.2114.12867.1 BTV-1 Culicoides austropalpalis competence Duchemin et al. readily extrapolate our results to other serotypes and especially exotic BTVs. Culicoides austropalpalis is reported to be particularly abundant in eastern Australia (Kettle and Elson 1975), in south east Queensland (Wild 1984) and in Victoria (present work). However, its vector capacity for livestock pathogens is potentially compromised by its predominantly ornithophilic behaviour (Kat et  al. 1978). Similarly, African bird feeder Culicoides, being found BTV positive in experimental infections (Paweska et  al. 2002) and isolations (Nevill et  al. 1992), have been considered of low epidemiological importance. However, feeding behaviour is rarely fully restrictive and C. austropalpalis has been found to also feed on cattle at a relatively high rate of 46% (Van der Saag et  al. 2016), and also on marsupials (Kay et al. 1978). Detection of viral RNA of Wallal virus (an orbivirus responsible for kangaroo blindness) in C.  austropalpalis (Hooper et  al. 1999) suggests it may be a vector but BTV has never been isolated from pools of C.  austropalpalis (Standfast et  al. 1984). Therefore, despite its abundance in the farm environment in Australian temperate regions, our data and its mainly ornithophilic diet indicate that C. austropalpalis is of low, if not null, epidemiological importance for BTV transmission. developed by Veronesi and colleagues (Veronesi et  al. 2013) allowed individual testing of midges with up-scaling to more than 400  midges. The dilution curve for positive controls gave consistent Ct values. Seven percent of the midges tested gave positive results for the presence of viral RNA 13 days after infective feeding. However, the invdiff were siginificantly lower than at D 0 , indicating that viral replication was low or null. The Culex annulirostris mosquitoes used as D 0 control gave not significantly different viral RNA values, despite the probable blood meal volume difference between the two insects. However, and as expected with mosquitoes and BTV, results became negative with time after oral challenge. However, following microinjection and the midgut barrier bypass, increased viral RNA indices, indicative of viral replication, justified the mosquito model for positive control, as with other insect models (Shaw et al. 2012). Differences in vector competence according to virus serotypes or strains have been described (Bellis et  al. 1994). Our experiments were conducted to determine for vector competence for BTV-1, the most common serotype (Firth et al. 2017) circulating in the adjacent more northerly state of New South Wales and one of the most probable to invade southern grazing regions. However, we cannot 345Veterinaria Italiana 2021, 57 (4), 341-345. doi: 10.12834/VetIt.2114.12867.1 Duchemin et al. BTV-1 Culicoides austropalpalis competence Bellis G.A., Gibson D.S., Polkinghorne I.G., Johnson S.J. & Flanagan M. 1994. Infection of Culicoides brevitarsis and C. wadai (Diptera: Ceratopogonidae) with four Australian serotypes of bluetongue virus. J Med Entomol, 31 (3), 382-387. Carpenter S., McArthur C., Selby R., Ward R., Nolan D.V., Luntz A.J.M., Dallas J.F., Tripet F. & Mellor P.S. 2008. Experimental infection studies of UK Culicoides species midges with bluetongue virus serotypes 8 and 9. Vet Rec, 163 (20), 589-592. Carpenter S., Wilson A. & Mellor P.S. 2009. Culicoides and the emergence of bluetongue virus in northern Europe. Trends Microbiol, 17 (4), 172-178. Dyce A.L., Bellis G.A. & Muller M.J. 2007. Pictorial atlas of Australasian Culicoides wings (Diptera: Ceratopogonidae). Australian Biological Resources Study, Canberra. Firth C., Blasdell K.R., Amos-Ritchie R., Sendow I., Agnihotri K., Boyle D.B., Daniels P., Kirkland P.D. & Walker P.J. 2017. Genomic analysis of bluetongue virus episystems in Australia and Indonesia. Vet Res, 48 (1), 82. Hawkes R.A., Kirkland P.D., Sanders D.A., Zhang F., Li Z., Davis R.J., Zhang N. 2000. Laboratory and field studies of an antigen capture ELISA for bluetongue virus. J Virol Methods, 85 (1-2), 137-149. Hofmann M., Griot C., Chaignat V., Perler L. & Thür B. 2008. Blauzungenkrankheit erreicht die Schweiz. Schweizer Archiv Für Tierheilkunde, 150 (2), 49-56. Hooper P.T., Lunt R.A., Gould A.R., Hyatt A.D., Russell G.M., Kattenbelt J.A., Blacksell S.D., Reddacliff L.A., Kirkland P.D., Davis R.J., Durham P.J., Bishop A.L. & Waddington J. 1999. Epidemic of blindness in kangaroos - Evidence of a viral aetiology. Austr Vet J, 77 (8), 529-536. Kay B.H., Boreham P.F.L., Dyce A.L. & Standfast H.A. 1978. Blood feeding of biting midges (Diptera: Ceratopogonidae) ast Kowanyama, Cape York Peninsula, North Queensland. Austr J Entomol, 17 (2),  145-149. Kettle D.S. & Elson M.M. 1975. Variation in larvae and adults of Culicoides austropalpalis Lee and Reye in S.E. Queensland. J Natural History, 9 (3), 321-336. Nevill E., Erasmus B.J., Venter G.J., Walton T. & Osburn B. 1992. A six-year survey of viruses associated with Culicoides biting midges throughout South Africa (Diptera: Ceratopogonidae) In Bluetongue, African horse sickness and related orbiviruses. Proc. 2nd International Symposium (T.E. Walton & B.I. Osburn, eds). CRC Press, Boca Raton, Florida, 314 319. Paweska J.T., Venter G.J. & Mellor P.S. 2002. Vector competence of South African Culicoides species for bluetongue virus serotype 1 (BTV-1) with special reference to the effect of temperature on the rate of virus replication in C. imicola and C. bolitinos. Med Vet Entomol, 16 (1), 10-21. R Core Team. 2017. R: A language and environment References for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https:// www.R-project.org/. Shaw A.E., Monaghan P., Alpar H.O., Anthony S., Darpel K.E., Batten C.A., Guercio A., Alimena G., Vitale M., Bankowska K., Carpenter S., Jones H., Oura C.A.L., King D.P., Elliott H., Mellor P.S. & Mertens P.P.C. 2007. Development and initial evaluation of a real-time RT-PCR assay to detect bluetongue virus genome segment 1. J Virol Methods, 145 (2), 115-126. Shaw A.E., Veronesi E., Maurin G., Ftaich N., Guiguen F., Rixon F., Ratinier M., Mertens P., Carpenter S., Palmarini M., Terzian C. & Arnaud F. 2012. Drosophila melanogaster as a model organism for Bluetongue virus replication and tropism. J Virol, 86 (17), 9015-9024. St George T.D., Cybinski D.H., Della-Porta A.J., McPhee D.A., Wark M.C. & Bainbridge M.H. 1980. The isolation of two bluetongue viruses from healthy cattle in Australia. Austr Vet J, 56 (11), 562-563. Standfast H.A., Dyce A.L., St George T.D., Muller M.J., Doherty R.L., Carley J.G. & Filippich C. 1984. Isolation of arboviruses from insects collected at Beatrice Hill, Northern Territory of Australia, 1974-1976. Austr J Biol Sci, 37 (5-6), 351-366. Stanislawek W.L., Lunt R.A,. Blacksell S.D., Newberry K.M., Hooper P.T., White J.R. 1996. Detection by ELISA of bluetongue antigen directly in the blood of experimentally infected sheep. Vet Microbiol, 52 (1-2), 1-12. Van Der Saag M.R., Gu X., Ward M.P. & Kirkland P.D. 2016. Development and evaluation of real-time PCR assays for bloodmeal identification in Culicoides midges. Med Vet Entomol, 30 (2), 155-165. Venter G.J., Paweska J.T., Van Dijk A.A., Mellor P.S. & Tabachnick W.J. 1998. Vector competence of Culicoides bolitinos and C. imicola for South African bluetongue virus serotypes 1, 3 and 4. Med Vet Entomol, 12 (4), 378-385. Veronesi E., Antony F., Gubbins S., Golding N., Blackwell A., Mertens P.P., Brownlie J., Darpel K.E., Mellor P.S. & Carpenter S. 2013. Measurement of the infection and dissemination of Bluetongue virus in Culicoides biting midges using a semi-quantitative RT-PCR assay and isolation of infectious virus. PLoS ONE, 8 (8), e70800. Wechsler S.T., McHolland L.E. & Tabachnick W.J. 1989. Cell lines from Culicoides variipennis (Diptera: Ceratopogonidae) support replication of Bluetongue virus. J Invertebrate Pathol, 54 (3), 385-393 Wickham H. 2016. ggplot2: Elegant graphics for data analysis. Springer-Verlag, New York. https://ggplot2. tidyverse.org. Wild C. 1984. The influence of meteorological factors on capture of Culicoides in flight (Diptera: Ceratopogonidae). The University of Queensland, School of Biological Sciences.