British Journal of Virology Short Communication Seroprevalence of Bluetongue and Associated Risk Factors in Costa Rican Sheep Flocks Rodolfo Villagra-Blanco1*, Gaby Dolz1, Danilo Montero-Caballero2, Juan José Romero-Zúñiga2 1Programa de Investigación en Medicina Poblacional, Escuela de Medicina Veterinaria, Universidad Nacional (UNA), P.O. Box 86-3000, Heredia, Costa Rica; 2 Cátedra de Salud de Hato y Control de la Producción, Escuela de Medicina Veterinaria, UNA, P.O. Box 86-3000, Heredia, Costa Rica. Abstract | Blood samples from a total of 359 sheep from 15 farms were analysed for the presence of antibodies against bluetongue virus (BTV) by commercial enzyme-linked immunosorbent assay (ELISA). Antibodies were detected in 290 sheep from fourteen different flocks, distributed in all analyzed regions (Central, Chorotega, Atlantic Huetar, North Huetar, and Central Pacific) determining regional seropositivity between 63.5% and 100.0%, as well as an overall prevalence of 80.8 %. The within flock seropositivity percentages ranged between 0% and 100.0%. Flocks with the highest seropositivity were found in low altitude regions close to the coast. Risk and protective factors determined in the present study were not in accordance with this insect borne disease. The results of this study indicate that BTV is endemic in sheep herds from Costa Rica, and animals seem not show clinical signs. We recommend carrying out further studies, to determine the presence of BTV in goats and wild ruminants, and to identify serotypes present in the country. Editor | Muhammad Munir, The Pirbright Institute, UK. Received | August 24, 2015; Accepted | October 28, 2015; Published | November 22, 2015 *Correspondence | Rodolfo Villagra-Blanco, Programa de Investigación en Medicina Poblacional, Escuela de Medicina Veterinaria, Universidad Nacional (UNA), P.O. Box 86-3000, Heredia, Costa Rica; E-mail: ravb870@gmail.com DOI | http://dx.doi.org/10.17582/journal.bjv/2015.2.5.74.79 Citation | Villagra-Blanco, R., G. Dolz, D. Montero-Caballero and J. J. Romero-Zúñiga. 2015. Seroprevalence of bluetongue and associated risk factors in Costa Rican sheep flocks. British Journal of Virology, 2(5): 74-79. Introduction Costa Rica. Various techniques have been used to de- tect antibodies against bluetongue virus (BTV), how- Bluetongue (BT) is a non-contagious viral disease ever only agar gel immunodiffusion test (AGIDT), affecting domestic and wild ruminants that is and enzyme-linked immunosorbent assay (ELISA) transmitted by insects, particularly biting midges of are accepted for international trade according the the Culicoides species (OIE, 2011). It is caused by a OIE Manual of Standards for Diagnostic Tests and double stranded RNA orbivirus of the family Reovir- Vaccines (Breard et al., 2004). idae, with more than 25 distinct serotypes distributed worldwide (Boden et al., 1971). The clinical signs of The incidence and geographical distribution of BTV the disease range from a mild febrile illness to ede- depend on seasonal conditions, the presence of vec- ma of lips and face, crusts on lips and muzzle, na- tors, and the availability of susceptible animals. The sal discharge, conjunctivitis and extensive erosions of midges prefer warm, moist conditions and are in their the oral mucosa, which can be mistaken for vesicu- greatest numbers and most active after rain periods lar stomatitis virus, an endemic occurring disease in (Animal Health Australia, 2008). More than 40 spe- our country (Rodríguez et al., 1996), and for foot and cies of Culicoides have been identified as vectors for mouth disease (Buxton & Frazer, 1977), not present in BTV in the country (Greiner et al., 1990; Tanya et October 2015 | Volume 2 | Issue 5 | Page 74 British Journal of Virology al., 1992), however Culicoides insignis was identified as the distribution of the agent was unknown, the same the primary vector, since it was detected in more than chance of infection on each farm was assumed, thus, 90.0% of the collections (Greiner et al., 1993; Sáenz all animals within each farm had an equal chance of and Greiner, 1994). being infected. A random selection of farms within regions was performed in order to get a representa- Prevalence of antibodies against BTV among sheep, tive sample population in each region and inside each goat and cattle had been reported in many tropical flock. The study was conducted in 15 Costa Rican and subtropical areas, considered endemic zones, in- sheep herds. According proportional allocation, the cluding Central America (Homan et al., 1985a,b; farms were distributed as follows: seven in the Central Mo et al., 1994; Mertens et al., 2009; Legisa et al., region (46.0%), two in the Chorotega region (13.5%), 2014). In Costa Rica, presence of BTV has been stud- two in the Central Pacific region (13.5%), two in the ied in cattle 30 years ago using AGIDT (Homan et North Huetar region (13.5%) and two in the Atlan- al., 1985a,b), and serotypes 1, 3 and 6 were identified tic Huetar region (13.5%). The Brunca region was (Thompson et al., 1992), indicating the importance of not analyzed, since it was not possible to find farms Central America as a possible source of BTV for the willing to participate in this study. However, less than rest of the continent (Mertens et al., 2009). However, 10.0% of animals were registered in this region. the serologic evidence of this agent in sheep flocks, an emerging industry in Costa Rica, had not been stud- Sample collection and survey ied to date. The aim of this study was to determine if Blood was collected between August 2012 and Feb- antibodies against BTV were present in Costa Rican ruary 2013. Tubes were transported using coolers sheep blood samples, using a commercial competitive for keeping a temperature between 4°C to 7°C. Af- ELISA, and to identify risk factors associated to this terwards in the laboratory the samples were centri- viral infection. fuged for 5 minutes at 10,000 g, sera was separated, and frozen at -20°C until processed by ELISA. A Materials and Methods questionnaire applied during a Maedi Visna research (Villagra-Blanco et al., 2015) was analyzed again to Studied population obtain information in order to determine risk factors Sheep flocks registered at the Costa Rican Associa- associated with BT disease such as housing, animal tion of Sheep Producers were sampled, most of them movement between herds, lamb husbandry, reproduc- were used commercially (87.0%), to produce tropical tive management and presence of compatible clinical hair breed lambs (100.0%), and these animals were signs of BT in each farm. maintained mainly in intensive systems (93.0%). The sampled sheep breeds were Dorper, Pellybuey, Kath- Enzyme-linked immunosorbent assay (ELISA) adin, Blackbelly, Texel, Suffolk, Santa Ines and their The IDScreen® Bluetongue Competition Multispecies crosses. ELISA (Montpellier, France) was used. This assay re- ported a sensitivity and specificity of 99.0% (Vanden- Sample size bussche et al., 2008). The methodology recommended The sample size was calculated with an estimat- by the manufacturer was used. ed population of 25,000 animals distributed in 138 sheep herds in Costa Rica (20.0% overall expected Statistical analysis prevalence, 95.0% confidence level and 5.0% expected Frequencies of the general characteristics and man- error), yielding a total of 244 samples to analyze; how- agement practices of the sheep flocks were calculated. ever, to enhance the power of the study, a total of 359 To assess the relationship between BTV and the man- sheep were sampled. Within each herd, the number agement practices, the odds ratio (OR) was calculat- of animals to be sampled was calculated to determine ed using a mixed effects logistic regression, being the presence or absence of antibodies against BTV, with sheep flock the random variable. The data was analyz- 95.0% confidence, assuming a sensitivity and speci- ed using SAS/STAT ver. 9.2 (SAS Institute Inc.). ficity of the ELISA of 99.0% (Vandenbussche et al., 2008; Niedbalski, 2011) using the formula described Results and Discussion by Cannon and Roe (1982). Since most of the sheep farms presented similar management conditions and Seropositive animals were detected in 14 (93.3%) October 2015 | Volume 2 | Issue 5 | Page 75 British Journal of Virology Table 1: Number and percentage of animals tested in15 sheep flocks and distribution of seropositive individuals ac- cording to flocks and regions Farm Region Total animals Animals Positive ani- Breed Flocks ana- Regional posi- in flock tested mals (%) lysed tivity 7 Central 80 25 25 (100.0) D,K 7 63.5 % 8 Central 103 25 11 (44.0) D,K 9 Central 136 26 16 (61.5) K,B,P 12 Central 100 25 21 (84.0) Om 13 Central 220 26 20 (76.9) Om 14 Central 300 28 8 (28.6) Om 15 Central 4 4 0 D,K Subtotal 943 159 101 (63.5) 5 Central Pacific 500 27 23 (85.2) Om 2 92.7% 10 Central Pacific 200 28 28 (100.0) Om Subtotal 700 55 51 (92.7) 2 Chorotega 115 25 25 (100.0) D,K,P 2 100.0% 3 Chorotega 140 26 26 (100.0) Om Subtotal 255 51 51 (100.0) 4 Atlantic Huetar 30 20 20 (100.0) K,P 2 100.0% 11 Atlantic Huetar 350 27 27 (100.0) D,K,S Subtotal 380 47 47 (100.0) 1 North Huetar 200 21 15 (71.4) D,K,P 2 85.1% 6 North Huetar 131 26 25 (96.2) D,K,T Subtotal 331 47 40 (85.1) TOTAL 2609 359 290 (80.8) 15 D: Dorper; K: Katahdin; P: Pelibuey; S: Suffolk; T: Texel; B: Blackbelly; Om: Other mixed breeds Table 2: Risk factors associated with BTV seropositivity Flocks with the highest seropositivity were found in in sheep flocks in Costa Rica low altitude regions close to the Atlantic and Pacif- Variable Animals OR CI (95 %) ic coast. Furthermore, only one small flock localized Positive Negative LL UL in a mountainous area (over 1,500 meters) with just Open flocks 212 147 2.54 1.49 4.35 four animals, all born inside this flock, was seronega- No quarantine areas 253 106 6.47 3.68 11.4 tive (Figure 1). According to Homan et al. (1985a, b) Costa Rica presented an inverse association between Partial stabling 333 26 1.10 1.06 1.14 antibody prevalence of cattle and altitude of the farm, OR: Odds Ratio; UL: Upper limit; LL: Lower limit; CI: Confi- observation that coincides with the results obtained dence Interval in our research. flocks; however, in the seronegative flock only four Two management practices were determined as risk animals were tested. From a total of 359 serum sam- factors for BTV seropositivity: buying animals from ples analysed, 290 sheep (80.8%) showed antibodies other farms without any sanitary control (59.1% of against BTV, the seropositivity in the regions ana- the participating farms, OR= 2.54; IC= 1.49 to 4.35), lysed ranged between 63.5% and 100.0%. Meanwhile, and the lack of quarantine areas or separated boxes the flock seropositivities determined ranged between for sick animals in each flock (70.47% of the stud- 0% and 100.0% (Table 1). This study was the first at- ied flocks, OR= 6.47; IC= 3.68 to 11.40). These risk tempt to detect BTV antibodies in sheep flocks in factors have been described in the literature as fac- Costa Rica, an emerging and fast growing industry in tors facilitating the infection of sheep and goat flocks the country. Previously, higher seroprevalences (from with different virus, bacteria and parasites (Vasileiou 15.0% to 75.0%) were reported in cattle by Homan et et al., 2015), including BTV (Mozaffari et al., 2014), al. (1985 a, b, 1990, 1992). especially if the animals are moved into high-humid October 2015 | Volume 2 | Issue 5 | Page 76 British Journal of Virology Figure 1: Location of the participating flocks with Bluetongue virus (BTV) seropositive sheep (black dots) and seron- egative animals (white dots) within the five analysed regions of Costa Rica endemic coast areas (Homan et al., 1990). Lack of ten by Culicoides, typically as adults, and controlling sanitary control and lack of quarantine areas are not exposure to these mosquitoes in Costa Rica might considered risk factors for BTV (Bosnić et al., 2015), actually lead to clinical disease if infection is delayed the only risk factor would be exposure to Culicoides (Holbrook, 1996). (Sáenz and Greiner, 1994), which was not analyzed in the present study. Finally, no clinical signs of disease were observed in the analyzed sheep, findings that are in accordance On the other hand, all seronegative individuals with Mo et al. (1994). Sheep in endemic areas are (19.2%) belonged to flocks with partial stabling, in- naturally resistant to BT, and clinical disease is only dicating this management practice as a protective fac- observed when non-native ruminants, particular- tor for BTV infection (OR= 1.10; IC= 1.06 to 1.14) ly European breeds, are introduced into these areas (Table 2), since stabled animals are likely exposed to (OIE, 2011). fewer Culicoides (Meiswinkel et al., 2000). However, BT disease occurs, when seronegative animals are bit- Measures for preventing and controlling the disease October 2015 | Volume 2 | Issue 5 | Page 77 British Journal of Virology in endemic areas are based mainly on sentinel mon- ra, Australia. itoring programs, in combination with surveillance • Greiner, E.C., Alexander, F.C., Roach, J., St. John, programs of insect vectors (OIE, 2011). V.S., King, T.H., Taylor, W.P. and E.P. Gibbs. 1990. Bluetongue epidemiology in the Caribbean The positive results obtained in this study confirmed region: Serological and entomological evidence the presence of antibodies against BTV in Costa Ri- from a pilot study in Barbados. Med. Vet. Ento- can sheep flocks. Risk and protective factors deter- mol. 4: 289-295. mined in the present study were not in accordance • Greiner, E.C., Mo, C.L., Homan, E.J., Gonzalez, with this insect borne disease, probably due to the J., Oviedo, M.T., Thompson, L.H. and E.P. Gibbs. type of study (cross-sectional). We recommend car- 1993. Epidemiology of bluetongue in Central rying out further studies, to determine the presence America and the Caribbean: Initial entomolog- of BTV in goats and wild ruminants, and to identify ical findings. Regional Bluetongue Team. Med. serotypes present in the country. Vet. Entomol. 7:309-315. • Holbrook, F.R. 1996. Biting midges and the agents Acknowledgements they transmit. In: Beaty, B.J. and W.C. Marquardt (Eds): The Biology of Disease Vectors. University Thanks to all farmers that participated in this research. Press of Colorado, USA. pp. 110-116. We wish to thank Roberto Leiva and Jose Segura for • Homan, E. J., Lorbacher de Ruiz, H., Dona- their help. to, A.P., Taylor, W.P. and T. M. Yuill. 1985a. A preliminary survey of the epidemiology of blue- Conflict of interest tongue in Costa Rica and Northern Colombia. J. Hyg., Camb. 94: 357-363. There is no conflict of interest in this study. • Homan, E.J., Lorbacher, H., Donato, A., Taylor, W. and T. M. Yuill. 1985b. Bluetongue virus infec- References tion in Costa Rican and Colombian cattle. Prog Clin Biol Res 178, 559–561. • Animal Health Australia. 2008. Ausvetplan: Dis- • Homan, E.J., Mo, C.L., Thompson, L.H., Bar- ease Strategy: Bluetongue (Version 3.0) Aus- reto, C.H., Oviedo, M.T., Gibbs, E.P.J. and E.C. tralian Veterinary Emergency Plan (AUSVET- Greiner. 1990. Epidemiologic study of bluetongue PLAN), Edition 3, Primary Industries Ministerial viruses in Central America and the Caribbean: Council, Canberra, ACT. 1986–1988, Regional Bluetongue Team. Am J Vet • Breard, E., Hamblin, C., Hammoumi, S., Sailleau, Res 51 (7), 1089–1094. C., Dauphin, G. and S. Zientara. 2004. The epi- • Homan, E.J., Gibbs, E.P.J., Walker, J.S., Walton, demiology and diagnosis of bluetongue with par- T.E., Yuill, T.M., Gonzalez, J., Barreto, C.H. and ticular reference to Corsica. Res Vet Sci 77: 1-8. E.C. Greiner. Interamerican Bluetongue Team, • Boden, E.C., Shope, R.E. and F.A. Murphy. 1971. 1992. Central American and Caribbean region- Physicochemical and morphological relationships al bluetongue epidemiology study, antecedents of some arthropod-borne viruses to bluetongue and geo-graphic review. In: Bluetongue, African virus-a new taxonomic group. Physicochemical Horsesicknes and Related Orbiviruses, Proced- and serological studies. J Gen Virol 3:261–271. ings of the Second International Symposium. • Bosnić, S., Beck, R., Listeš, E., Lojkić, I., Savini, • Legisa, D. M., Gonzalez, F.N. and M. J. Dus San- G. and B. Roić. 2015. Bluetongue virus in Oryx tos. 2014. Bluetongue virus in South America, antelope (Oryx leucoryx) during the quarantine Central America and the Caribbean. Virus Re- period in 2010 in Croatia. Vet Ital. 51: 139-143. search 182: 87–94. • Buxton, A. and G. Frazer. 1977. Reoviruses (and • Meiswinkel, R., Baylis, M. and K. Labuschagne. other diplorna viruses). In Animal microbiology, 2000. Stabling and protection of horses from Cu- Vol. 2. Blackwell Scientific Publications, Oxford, licoides bolitinos (Diptera: Ceratopogonidae), a re- 629-632. cently identified vector of African horse sickness. • Cannon, R.M. and R.T. Roe. 1982. Livestock dis- Bull Entomol Res. 90:509-15. ease survey: a field manual for veterinarians. Aus- • Mertens, P., Baylis, M. and P.S. Mellor. 2009. tralian Government Publishing Service, Camber- Bluetongue, vol 1, 1st Elsevier, London UK, pp. October 2015 | Volume 2 | Issue 5 | Page 78 British Journal of Virology 483. • Tanya, V.N., Griener, E.C. and E.P. Gibbs. 1992. • Mo, C.L., Thompson, L.H., Homan, E.J., Oviedo, Evaluation of Culicoides insignis (Diptera: Cera- M.T., Greiner, E.C., Gonzalez, J. and M.R. Sae- topogonidae) as a vector of bluetongue virus. Vet. nz. 1994. Bluetongue virus isolations from vectors Microbiol 32: 1-14. and ruminants in Central America and the Carib- • Thompson, L.H., Mo, C.L., Oviedo, M.T. and E.J. bean. Interamerican Bluetongue Team. Am J Vet Homan: Interamerican Bluetongue Team, 1992. Res 55 (2), 211–215. Prevalence and incidence of Bluetongue Viruses • Mozaffari, A.A., Khalili, M. and S. Sabahi. 2014. in the Caribbean Basin: serologic and virologic High seroprevalence of bluetongue virus antibod- findings. Bluetongue, African Horsesickness and ies in goats in southeast Iran. Asian Pac J Trop related Orbiviruses. Biomed 4: 275-278. • Vandenbussche F., Vanbinst, T., Verheyden, B., • Niedbalski, W. 2011. Evaluation of commercial Van Dessel, W., Demeestere, L., Houdart, P., ELISA kits for the detection of antibodies against Bertels, G., Praet, N., Berkvens, D., Mintiens, K., bluetongue virus. Pol J Vet Sci 14(4): 615-619 Goris, N and K. De Clercq. 2008. Evaluation of • OIE (World Organization for Animal Health). antibody-ELISA and real-time RT-PCR for the 2011. OIE Terrestrial Animal Health Code, diagnosis and profiling of bluetongue virus sero- (http://www.oie.int/eng/nomes/mcode/en_chap- type 8 during the epidemic in Belgium in 2006. itre_2.1.1.htm). Vet Microbiol. 129:15-27. • Rodríguez, L.L., Fitch, W.M. and S.T. Nichol. • Vasileiou, N.G.C., Fthenakis, G.C and E. Pap- 1996. Ecological factors rather than temporal fac- adopoulos. 2015. Dissemination of parasites by tors dominate the evolution of vesicular stomatitis animal movements in small ruminant farms. Vet virus. Proc. Natl. Acad. Sci. USA 93(23): 13030- Parasitol. May 8. pii: S0304-4017(15)00223-X. 13035. • Villagra-Blanco, R., Dolz, G., Solórzano-Mo- • Sáenz, M.R. and E.C. Greiner. 1994. Culicoides rales, A., Alfaro-Alarcón, A., Montero-Caballe- aspirated from cattle in  Costa Rica, Honduras, ro, D. and J.J. Romero-Zúñiga. 2015. Presence of Panama and Puerto Rico, and their role as poten- Maedi-Visna in Costa Rican sheep flocks Small tial vectors of bluetongue viruses. Regional Blue- Ruminant Res 124: 132–136. tongue Team. Med Vet Entomol. 8(1):15-9. October 2015 | Volume 2 | Issue 5 | Page 79