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Seroprevalence of Neospora spp. among asymptomatic horses, aborted mares and horses demonstrating neurological signs in Israel
Veterinary Parasitology 148 (2007) 109–113 www.elsevier.com/locate/vetpar
Seroprevalence of Neospora spp. among asymptomatic horses, aborted mares and horses demonstrating neurological signs in Israel Eitan B. Kligler a, Varda Shkap b, Gad Baneth a, Zvia Mildenberg c, Amir Steinman a,* a
Koret School of Veterinary Medicine, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jeruslem, P.O. Box 12, Rehovot 76100, Israel b Division of Parasitology, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel c Equine Veterinary Medicine, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel Received 9 March 2007; received in revised form 1 May 2007; accepted 1 June 2007
Abstract Sera from 800 asymptomatic horses were examined for the presence of antibodies to Neospora caninum by immunofluorescence antibody test (IFAT). The presence of antibodies to N. caninum was also tested in sera from 52 mares that had aborted and 40 horses exhibiting neurological signs. A total of 95 (11.9%) of the 800 sera had antibodies for Neospora. Significantly higher seropositivity was obtained from horses that had neurological signs (21.2%) and from aborted mares (37.5%). There was significant linear-bylinear association between age and seropositivity. This is the first serologic survey for Neospora spp. antibodies performed on horses from the Middle East and the first to report significant difference in seropositivity between asymptomatic horses and horses exhibiting neurological signs. # 2007 Elsevier B.V. All rights reserved. Keywords: Neospora spp.; Horse; Seroprevalence: IFAT; Abortion; Neurological signs
1. Introduction Equine neosporosis is a protozoal disease caused by a cyst-forming coccidian of the phylum Apicomplexa. Two species, Neospora caninum and Neospora hughesi, have been identified as infecting the horse and were associated with neurological disease and fetal loss (Dubey and Porterfield, 1990; Pitel et al., 2003; Villalobos et al., 2006). Seroprevalences of Neospora spp. in asymptomatic horses were reported from North America (Dubey et al., * Corresponding author. Tel.: +972 3 968 8534; fax: +972 3 960 4079. E-mail address: [email protected] (A. Steinman). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.06.002
1999, 2003; Cheadle et al., 1999; Vardeleon et al., 2001), South America (Hoane et al., 2006; Villalobos et al., 2006), Europe (Pronost et al., 1999; Pitel et al., 2001; Ciaramella et al., 2004; Jakubek et al., 2006), New Zealand (Vardeleon et al., 2001) and South Korea (Gupta et al., 2002), and were found to vary considerably between the different geographic areas and between studies in the same region. Although exposure to Neospora spp. in horses seems to be common, clinical disease is mainly supported by case reports and seroprevalence studies in healthy versus horses with reproductive disorders. A suggestion on the possible role for Neospora spp. in equine reproductive diseases was based on differences between frequency of occurrence of serum antibodies against
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Neospora spp. in groups of aborting and healthy mares (McDole and Gay, 2002; Pitel et al., 2003; Villalobos et al., 2006). Neospora is suggested to have a potential role in neurologic disorders in horses (Vardeleon et al., 2001), therefore similar seroprevalence studies among healthy horses versus horses showing neurological symptoms might verify this claim. The aim of this study was to determine the seroprevalence of N. caninum among horses in Israel and to compare it with the seropositivity among mares with abortions and horses exhibiting neurological signs. 2. Materials and methods 2.1. Parasites and antigen preparation Tachyzoites of the N. caninum NC-1 isolate were grown in Vero cell monolayer cultures as described elsewhere (Shkap et al., 2002). Culture-derived tachyzoites were used for slide antigen preparation. The suspension of culture-grown parasites was semipurified through a Whatman’s CF-11 fibrous cellulose column. Tachyzoites with a final concentration of 3 103 ml 1 were dispersed on 12 spot glass slides. The air-dried antigen was fixed with cold acetone and stored at 70 8C pending use. 2.2. Serum samples Serum samples from 800 horses older than 1 year were collected in 48 farms in different geographical locations in Israel during 2002. In each farm, all horses older than 1 year of age were sampled. Serum samples from 52 aborted mares and 40 horses exhibiting neurological signs that were submitted to the diagnostic laboratory in Kimron Veterinary Institute between the years 2001 and 2004 were also examined. Sera were prepared and stored at 70 8C until use. 2.3. Antibody detection by the immunofluorescent antibody test (IFAT) Serum samples were tested at an initial screening dilution of 1:50 in phosphate buffered saline (PBS) (McDole and Gay, 2002). A control commercial antigen slide (VMRD, Pullman, Washington) was used as a ‘‘gold standard,’’ according to the manufacturer’s recommendations. Known positive and negative sera were included on each slide. All samples that showed fluorescence at the initial dilution were considered to be positive, and they were further diluted to endpoint titer. The highest dilution of serum exhibiting fluorescence of
the whole Neospora organism was considered as the endpoint titer in the IFAT (Pare et al., 1995). 2.4. Data analysis The Chi-square test and Fisher’s exact test were applied to analyze the correlation between Neospora seroprevalence and various horse characteristics including: gender, age (1–2, 2–5, 5–10, over 10 years), breed (divided to six groups: 1, Arab, Arab cross, and local breed; 2, Quarter Horse, Quarter Horse cross; 3, Appaloosa, Mustang, Tennessee walking horse, and Andalusian; 4, Warmbloods; 5, Thoroughbred; 6, ponies), stable bedding (soil, sawdust, straw, shavings), type of fodder (buckwheat, hay, grazing), frequency of stall cleaning (once a day, every few days, every few weeks), living conditions (paddock or stall), geographical location (Coastal Plain, Galilee and the Golan Heights, Jerusalem and Mount Jeuda, Negev and Arava), proximity to other animals (no contact, contact with cattle, contact with dogs, contact with animals of other types). The two-sample t-test was used in order to compare the average age between horses that had anti-Neospora antibodies to those that were seronegative. A 95% confidence interval was calculated for the seroprevalence of anti-Neospora antibodies in each of the study groups. A p-value below 0.05 was considered statistically significant. The statistical analyses regarding geographical locations, age, gender, breed, stable bedding, type of fodder, frequency of cleaning stalls, living conditions, and proximity to other animals were done for the group of the 800 asymptomatic horses. 3. Results 3.1. Overall seropositivity and titer distribution A total of 95 (11.9%) of the 800 equine sera samples tested positive for antibodies against N. caninum. Significantly higher results were obtained from horses exhibiting neurological signs (21.2%; p = 0.049) and from aborted mares (37.5%; p < 0.001). The endpoint titers found in the group of 800 asymptomatic horses were mostly low. Of the sera tested, 65 (8.1%) had titers of 1:50, 10 (1.3%) had titer of 1:100, 9 (1.1%) had titer of 1:200, 6 (0.8%) had titer of 1:400, 2 (0.3%) had titer of 1:800, 2 (0.3%) had titer of 1:1600 and 1 (0.1%) had titer of 1:3200. The endpoint titers of the horses exhibiting neurological signs were 1:50 in 7 (13.5%) horses and 1:200 in 4
E.B. Kligler et al. / Veterinary Parasitology 148 (2007) 109–113
(7.7%) horses. Four (10%) of the aborted mares had an endpoint titer of 1:50, 7 (17.5%) had an endpoint titer of 1:100, 2 (5%) had an endpoint titer of 1:200 and 2 (5%) had an endpoint titer of 1:400. 3.2. Age The highest seropositivity rate (15.3%) was obtained in horses older than 10 years, while in the group of horses younger than 2 years the seropositivity was only 3.7%. About three times as many horses were found positive with the progression of age with a significant linear-by-linear association between age and seropositivity ( p = 0.043) (Table 1). 3.3. Gender The mares showed a seropositivity of 13.0% while the seropositivity for males was 10.9%. These values were not significantly different. 3.4. Breed Ponies exhibited the highest seropositive rate of 21.1% while Thoroughbreds showed the lowest positive reactivity of 5.4%. No significant differences in seropositivity were found among the different horse breed groups examined. 3.5. Parameters of environment and food The differences in seropositivity based on geographical location, stable bedding, frequency of stall cleaning, type of fodder, living conditions and proximity to other animals, were insignificant ( p = 0.157, 0.653, 0.693, 0.984, 0.063 and 0.636, respectively). Table 1 Seroprevalence of Neospora caninum among asymptomatic horses of different age groups by immunofluorescence antibody test (IFAT) demonstrating a significant linear-by-linear association between age and seropositivity ( p = 0.043) Age of horses (years)
Number of horses tested
Number of positive serum samples
Percent of positive sera
Up to 2 2–5 5–10 >10 Unknown age
27 184 271 307 11
1 21 24 47 2
3.7 11.4 8.9 15.3 18.2
Total
800
95
11.9
111
4. Discussion Seroprevalence of Neospora spp. in horses was reported from many different areas of the world, however no reports are available from the Middle East. This study is the first to describe the presence of antibodies to Neospora spp. in horses in this region indicating that horses in Israel were exposed to Neospora spp. We cannot conclude which species infected the horses in this study, since N. caninum and N. hughesi cross-react serologically (Walsh et al., 2000). The role of Neospora spp. in equine abortions was first suggested by Dubey and Porterfield (1990) when Neospora tachyzoites were detected in tissues from an aborted equine fetus. However, compared to cattle where N. caninum was repeatedly demonstrated to be a major cause of abortion, evidence supporting the role of Neospora spp. in equine abortions is relatively scare (Villalobos et al., 2006). In this study, the prevalence of antibodies to Neospora spp. in aborted mares was considerably higher than in asymptomatic horses further supporting the role of Neospora spp. in equine abortions. Our findings are in agreement with previous studies in which the prevalence of serum antibodies against Neospora spp. in a population of horses with reproductive disorders were compared to asymptomatic horses and were significantly higher (Pitel et al., 2003; Villalobos et al., 2006). Other similar studies did not demonstrate a statistically significant difference between similar groups of horses (Pitel et al., 2001; McDole and Gay, 2002). N. caninum was first reported as a cause of neurological abnormalities in a dog more than two decades ago (Bjerka˚s et al., 1984). In recent years, the role of Neospora spp. in neurological disease of horses, such as equine protozoal myeloencephalitis (EPM) was demonstrated (Marsh et al., 1996; Daft et al., 1997; Hamir et al., 1998; Cheadle et al., 1999), however, neosporosis is currently not considered an important differential diagnosis in neurological diseases of horses. It was suggested by Cheadle et al. (1999) that Neospora infection in horses might be more prevalent than originally suspected and prediction of seropositivity in horses exhibiting central nervous system signs is difficult. In this study we showed for the first time, that the prevalence of antibodies to Neospora spp. in a group of horses exhibiting neurological signs was significantly higher compared to the prevalence in a group of asymptomatic horses. It is possible that over the past years Neospora infections was possibly misdiagnosed as EPM due to Sarcocystis neurona as
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was suggested previously (Pitel et al., 2001). This, however, is not likely to be the situation in our region, because of the absence of opossum (Didelphis virginiana), the definitive host of S. neurona in North America. Therefore, clinicians in areas where S. neurona and opossums are not present should be more aware of the possible role of Neospora in EPM, as suggested by Pitel et al. (2001). We have found direct association between the prevalence of antibodies to Neospora spp. and age. These results suggest that only a small proportion of horses were exposed to the parasite in the early stages of their life. These findings are in agreement with previous report from Italy (Ciaramella et al., 2004), but are in contrary to reports in cattle in which vertical transmission is the main route of infection (Hall et al., 2005). In light of this result, it appears that vertical transmission in horses might be less efficient than in cattle, which could be explained by differences in placentation as was suggested previously (Pitel et al., 2003). The association between age and seropositivity might explain some of the differences between studies. Difference in seroprevalence between groups of horses from various geographical locations was reported by Vardeleon et al. (2001). The prevalence of antibodies to N. hughesi was lower in race horses from Florida compared to horses from Missouri which were older (Vardeleon et al., 2001). Herd level risk factors such as the number of dogs on a farm, farm area, feeding pooled sources of colostrums and region were significantly associated with seropositivity to N. caninum in dairy farms in Brazil (Corbellini et al., 2006). In our study, other horse level risk factors including gender and breed, and various farm level risk factors such as stable bedding, frequency of stall cleaning and proximity to other animals were not significantly associated with seropositivity in this study. Horizontal transmission of Neospora spp. appears to be a major mode of transmission in horses, therefore it is important to determine which factors increase the probability of infection. Further studies are required in order to clarify the relationship between seropositivity and various horse and farm level risk factors, such as presence of dogs, which are a known source of post-natal transmission to cattle (Corbellini et al., 2006), which might help to decrease post-natal infection in horses. In conclusion, in this first study of Neospora in horses in Israel, seroprevalence was found to increase significantly with age. It was particularly high in horses with neurological disorders and in aborted mares.
Acknowledgements We thank Tali Bdolah-Abram for her assistance with statistical analysis. We also thank Dr. Igor Savitsky for his assistance in performing the serological assays. References Bjerka˚s, I., Mohn, S.F., Presthus, J., 1984. Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Z. Parasitenkd. 70, 271–274. Cheadle, M.A., Lindsay, D.S., Rowe, S., Dykstra, C.C., Williams, M.A., Spencer, J.A., Toivio-Kinnucan, M.A., Lenz, S.D., Newton, J.C., Rolsma, M.D., Blagburn, B.L., 1999. Prevalence of antibodies to Neospora sp. in horses from Alabama and characterisation of an isolate recovered from a naturally infected horse. Int. J. Parasitol. 29, 1537–1543. Ciaramella, P., Corona, M., Cortese, L., Piantedosi, D., Santoro, D., Di Loria, A., Rigato, R., 2004. Seroprevalence of Neospora spp. in asymptomatic horses in Italy. Vet. Parasitol. 123, 11–15. Corbellini, L.G., Smith, D.R., Pescador, C.A., Schmitz, M., Correa, A., Steffen, D.J., Driemeier, D., 2006. Herd-level risk factors for Neospora caninum seroprevalence in dairy farms in southern Brazil. Prev. Vet. Med. 74, 130–141. Daft, B.M., Barr, B.C., Collins, N., Sverlow, K., 1997. Neospora encephalomyelitis and polyradiculoneuritis in an aged mare with Cushing’s disease. Equine Vet. J. 29, 240–243. Dubey, J.P., Porterfield, M.L., 1990. Neospora caninum (Apicomplexa) in an aborted equine fetus. J. Parasitol. 76, 732–734. Dubey, J.P., Romand, S., Thulliez, P., Kwok, O.C., Shen, S.K., Gamble, H.R., 1999. Prevalence of antibodies to Neospora caninum in horses in North America. J. Parasitol. 85, 968– 969. Dubey, J.P., Mitchell, S.M., Morrow, J.K., Rhyan, J.C., Stewart, L.M., Granstrom, D.E., Romand, S., Thulliez, P., Saville, W.J., Lindsay, D.S., 2003. Prevalence of antibodies to Neospora caninum, Sarcocystis neurona, and Toxoplasma gondii in wild horses from central Wyoming. J. Parasitol. 89, 716–720. Gupta, G.D., Lakritz, J., Kim, J.H., Kim, D.Y., Kim, J.K., Marsh, A.E., 2002. Seroprevalence of Neospora, Toxoplasma gondii and Sarcocystis neurona antibodies in horses from Jeju island, South Korea. Vet. Parasitol. 106, 193–201. Hall, C.A., Reichel, M.P., Ellis, J.T., 2005. Neospora abortions in dairy cattle: diagnosis, mode of transmission and control. Vet. Parasitol. 128, 231–241. Hamir, A.N., Tornquist, S.J., Gerros, T.C., Topper, M.J., Dubey, J.P., 1998. Neospora caninum-associated equine protozoal myeloencephalitis. Vet. Parasitol. 79, 269–274. Hoane, J.S., Gennari, S.M., Dubey, J.P., Ribeiro, M.G., Borges, A.S., Yai, L.E., Aguiar, D.M., Cavalcante, G.T., Bonesi, G.L., Howe, D.K., 2006. Prevalence of Sarcocystis neurona and Neospora spp. infection in horses from Brazil based on presence of serum antibodies to parasite surface antigen. Vet. Parasitol. 136, 155–159. Jakubek, E.B., Lunden, A., Uggla, A., 2006. Seroprevalences of Toxoplasma gondii and Neospora sp. infections in Swedish horses. Vet. Parasitol. 138, 194–199. Marsh, A.E., Barr, B.C., Madigan, J., Lakritz, J., Nordhausen, R., Conrad, P.A., 1996. Neosporosis as a cause of equine protozoal myeloencephalitis. J. Am. Vet. Med. Assoc. 209, 1907–1913.
E.B. Kligler et al. / Veterinary Parasitology 148 (2007) 109–113 McDole, M.G., Gay, J.M., 2002. Seroprevalence of antibodies against Neospora caninum in diagnostic equine serum samples and their possible association with fetal loss. Vet. Parasitol. 105, 257–260. Pare, J., Hietala, S.K., Thurmond, M.C., 1995. Interpretation of an indirect fluoresecent antibody test for diagnosis of Neospora sp Infection in cattle. J. Vet. Diagn. Invest. 7, 273–275. Pitel, P.H., Pronost, S., Romand, S., Thulliez, P., Fortier, G., Ballet, J.J., 2001. Prevalence of antibodies to Neospora caninum in horses in France. Equine Vet. J. 33, 205–207. Pitel, P.H., Romand, S., Pronost, S., Foucher, N., Gargala, G., Maillard, K., Thulliez, P., Collobert-Laugier, C., Tainturier, D., Fortier, G., Ballet, J.J., 2003. Investigation of Neospora sp. antibodies in aborted mares from Normandy, France. Vet. Parasitol. 118, 1–6. Pronost, S., Pitel, P.H., Romand, S., Thulliez, P., Collobert, C., Fortier, G., 1999. Neospora Caninum: first case in France in an aborted
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equine fetus, analysis and perspectives. Pract. Vet. Equine 31, 111–114. Shkap, V., Reske, A., Pipano, E., Fish, L., Baszler, T., 2002. Immunological relationship between Neospora caninum and Besnoitia besnoiti. Vet. Parasitol. 106, 35–43. Vardeleon, D., Marsh, A.E., Thorne, J.G., Loch, W., Young, R., Johnson, P.J., 2001. Prevalence of Neospora hughesi and Sarcocystis neurona antibodies in horses from various geographical locations. Vet. Parasitol. 95, 273–282. Villalobos, E.M., Ueno, T.E., de Souza, S.L., Cunha, E.M., do Carmo Custodio de Souza Hunold Lara, M., Gennari, S.M., Soares, R.M., 2006. Association between the presence of serum antibodies against Neospora spp. and fetal loss in equines. Vet. Parasitol. 142, 372–375. Walsh, C.P., Duncan, R.B., Zajac, A.M., Blagburn, B.L., Lindsay, D.S., 2000. Neospora hughesi: experimental infections in mice, gerbils and dogs. Vet. Parasitol. 92, 119–128.
Seroprevalence of Neospora spp. among asymptomatic horses, aborted mares and horses demonstrating neurological signs in Israel Eitan B. Kligler a, Varda Shkap b, Gad Baneth a, Zvia Mildenberg c, Amir Steinman a,* a
Koret School of Veterinary Medicine, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jeruslem, P.O. Box 12, Rehovot 76100, Israel b Division of Parasitology, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel c Equine Veterinary Medicine, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel Received 9 March 2007; received in revised form 1 May 2007; accepted 1 June 2007
Abstract Sera from 800 asymptomatic horses were examined for the presence of antibodies to Neospora caninum by immunofluorescence antibody test (IFAT). The presence of antibodies to N. caninum was also tested in sera from 52 mares that had aborted and 40 horses exhibiting neurological signs. A total of 95 (11.9%) of the 800 sera had antibodies for Neospora. Significantly higher seropositivity was obtained from horses that had neurological signs (21.2%) and from aborted mares (37.5%). There was significant linear-bylinear association between age and seropositivity. This is the first serologic survey for Neospora spp. antibodies performed on horses from the Middle East and the first to report significant difference in seropositivity between asymptomatic horses and horses exhibiting neurological signs. # 2007 Elsevier B.V. All rights reserved. Keywords: Neospora spp.; Horse; Seroprevalence: IFAT; Abortion; Neurological signs
1. Introduction Equine neosporosis is a protozoal disease caused by a cyst-forming coccidian of the phylum Apicomplexa. Two species, Neospora caninum and Neospora hughesi, have been identified as infecting the horse and were associated with neurological disease and fetal loss (Dubey and Porterfield, 1990; Pitel et al., 2003; Villalobos et al., 2006). Seroprevalences of Neospora spp. in asymptomatic horses were reported from North America (Dubey et al., * Corresponding author. Tel.: +972 3 968 8534; fax: +972 3 960 4079. E-mail address: [email protected] (A. Steinman). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.06.002
1999, 2003; Cheadle et al., 1999; Vardeleon et al., 2001), South America (Hoane et al., 2006; Villalobos et al., 2006), Europe (Pronost et al., 1999; Pitel et al., 2001; Ciaramella et al., 2004; Jakubek et al., 2006), New Zealand (Vardeleon et al., 2001) and South Korea (Gupta et al., 2002), and were found to vary considerably between the different geographic areas and between studies in the same region. Although exposure to Neospora spp. in horses seems to be common, clinical disease is mainly supported by case reports and seroprevalence studies in healthy versus horses with reproductive disorders. A suggestion on the possible role for Neospora spp. in equine reproductive diseases was based on differences between frequency of occurrence of serum antibodies against
110
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Neospora spp. in groups of aborting and healthy mares (McDole and Gay, 2002; Pitel et al., 2003; Villalobos et al., 2006). Neospora is suggested to have a potential role in neurologic disorders in horses (Vardeleon et al., 2001), therefore similar seroprevalence studies among healthy horses versus horses showing neurological symptoms might verify this claim. The aim of this study was to determine the seroprevalence of N. caninum among horses in Israel and to compare it with the seropositivity among mares with abortions and horses exhibiting neurological signs. 2. Materials and methods 2.1. Parasites and antigen preparation Tachyzoites of the N. caninum NC-1 isolate were grown in Vero cell monolayer cultures as described elsewhere (Shkap et al., 2002). Culture-derived tachyzoites were used for slide antigen preparation. The suspension of culture-grown parasites was semipurified through a Whatman’s CF-11 fibrous cellulose column. Tachyzoites with a final concentration of 3 103 ml 1 were dispersed on 12 spot glass slides. The air-dried antigen was fixed with cold acetone and stored at 70 8C pending use. 2.2. Serum samples Serum samples from 800 horses older than 1 year were collected in 48 farms in different geographical locations in Israel during 2002. In each farm, all horses older than 1 year of age were sampled. Serum samples from 52 aborted mares and 40 horses exhibiting neurological signs that were submitted to the diagnostic laboratory in Kimron Veterinary Institute between the years 2001 and 2004 were also examined. Sera were prepared and stored at 70 8C until use. 2.3. Antibody detection by the immunofluorescent antibody test (IFAT) Serum samples were tested at an initial screening dilution of 1:50 in phosphate buffered saline (PBS) (McDole and Gay, 2002). A control commercial antigen slide (VMRD, Pullman, Washington) was used as a ‘‘gold standard,’’ according to the manufacturer’s recommendations. Known positive and negative sera were included on each slide. All samples that showed fluorescence at the initial dilution were considered to be positive, and they were further diluted to endpoint titer. The highest dilution of serum exhibiting fluorescence of
the whole Neospora organism was considered as the endpoint titer in the IFAT (Pare et al., 1995). 2.4. Data analysis The Chi-square test and Fisher’s exact test were applied to analyze the correlation between Neospora seroprevalence and various horse characteristics including: gender, age (1–2, 2–5, 5–10, over 10 years), breed (divided to six groups: 1, Arab, Arab cross, and local breed; 2, Quarter Horse, Quarter Horse cross; 3, Appaloosa, Mustang, Tennessee walking horse, and Andalusian; 4, Warmbloods; 5, Thoroughbred; 6, ponies), stable bedding (soil, sawdust, straw, shavings), type of fodder (buckwheat, hay, grazing), frequency of stall cleaning (once a day, every few days, every few weeks), living conditions (paddock or stall), geographical location (Coastal Plain, Galilee and the Golan Heights, Jerusalem and Mount Jeuda, Negev and Arava), proximity to other animals (no contact, contact with cattle, contact with dogs, contact with animals of other types). The two-sample t-test was used in order to compare the average age between horses that had anti-Neospora antibodies to those that were seronegative. A 95% confidence interval was calculated for the seroprevalence of anti-Neospora antibodies in each of the study groups. A p-value below 0.05 was considered statistically significant. The statistical analyses regarding geographical locations, age, gender, breed, stable bedding, type of fodder, frequency of cleaning stalls, living conditions, and proximity to other animals were done for the group of the 800 asymptomatic horses. 3. Results 3.1. Overall seropositivity and titer distribution A total of 95 (11.9%) of the 800 equine sera samples tested positive for antibodies against N. caninum. Significantly higher results were obtained from horses exhibiting neurological signs (21.2%; p = 0.049) and from aborted mares (37.5%; p < 0.001). The endpoint titers found in the group of 800 asymptomatic horses were mostly low. Of the sera tested, 65 (8.1%) had titers of 1:50, 10 (1.3%) had titer of 1:100, 9 (1.1%) had titer of 1:200, 6 (0.8%) had titer of 1:400, 2 (0.3%) had titer of 1:800, 2 (0.3%) had titer of 1:1600 and 1 (0.1%) had titer of 1:3200. The endpoint titers of the horses exhibiting neurological signs were 1:50 in 7 (13.5%) horses and 1:200 in 4
E.B. Kligler et al. / Veterinary Parasitology 148 (2007) 109–113
(7.7%) horses. Four (10%) of the aborted mares had an endpoint titer of 1:50, 7 (17.5%) had an endpoint titer of 1:100, 2 (5%) had an endpoint titer of 1:200 and 2 (5%) had an endpoint titer of 1:400. 3.2. Age The highest seropositivity rate (15.3%) was obtained in horses older than 10 years, while in the group of horses younger than 2 years the seropositivity was only 3.7%. About three times as many horses were found positive with the progression of age with a significant linear-by-linear association between age and seropositivity ( p = 0.043) (Table 1). 3.3. Gender The mares showed a seropositivity of 13.0% while the seropositivity for males was 10.9%. These values were not significantly different. 3.4. Breed Ponies exhibited the highest seropositive rate of 21.1% while Thoroughbreds showed the lowest positive reactivity of 5.4%. No significant differences in seropositivity were found among the different horse breed groups examined. 3.5. Parameters of environment and food The differences in seropositivity based on geographical location, stable bedding, frequency of stall cleaning, type of fodder, living conditions and proximity to other animals, were insignificant ( p = 0.157, 0.653, 0.693, 0.984, 0.063 and 0.636, respectively). Table 1 Seroprevalence of Neospora caninum among asymptomatic horses of different age groups by immunofluorescence antibody test (IFAT) demonstrating a significant linear-by-linear association between age and seropositivity ( p = 0.043) Age of horses (years)
Number of horses tested
Number of positive serum samples
Percent of positive sera
Up to 2 2–5 5–10 >10 Unknown age
27 184 271 307 11
1 21 24 47 2
3.7 11.4 8.9 15.3 18.2
Total
800
95
11.9
111
4. Discussion Seroprevalence of Neospora spp. in horses was reported from many different areas of the world, however no reports are available from the Middle East. This study is the first to describe the presence of antibodies to Neospora spp. in horses in this region indicating that horses in Israel were exposed to Neospora spp. We cannot conclude which species infected the horses in this study, since N. caninum and N. hughesi cross-react serologically (Walsh et al., 2000). The role of Neospora spp. in equine abortions was first suggested by Dubey and Porterfield (1990) when Neospora tachyzoites were detected in tissues from an aborted equine fetus. However, compared to cattle where N. caninum was repeatedly demonstrated to be a major cause of abortion, evidence supporting the role of Neospora spp. in equine abortions is relatively scare (Villalobos et al., 2006). In this study, the prevalence of antibodies to Neospora spp. in aborted mares was considerably higher than in asymptomatic horses further supporting the role of Neospora spp. in equine abortions. Our findings are in agreement with previous studies in which the prevalence of serum antibodies against Neospora spp. in a population of horses with reproductive disorders were compared to asymptomatic horses and were significantly higher (Pitel et al., 2003; Villalobos et al., 2006). Other similar studies did not demonstrate a statistically significant difference between similar groups of horses (Pitel et al., 2001; McDole and Gay, 2002). N. caninum was first reported as a cause of neurological abnormalities in a dog more than two decades ago (Bjerka˚s et al., 1984). In recent years, the role of Neospora spp. in neurological disease of horses, such as equine protozoal myeloencephalitis (EPM) was demonstrated (Marsh et al., 1996; Daft et al., 1997; Hamir et al., 1998; Cheadle et al., 1999), however, neosporosis is currently not considered an important differential diagnosis in neurological diseases of horses. It was suggested by Cheadle et al. (1999) that Neospora infection in horses might be more prevalent than originally suspected and prediction of seropositivity in horses exhibiting central nervous system signs is difficult. In this study we showed for the first time, that the prevalence of antibodies to Neospora spp. in a group of horses exhibiting neurological signs was significantly higher compared to the prevalence in a group of asymptomatic horses. It is possible that over the past years Neospora infections was possibly misdiagnosed as EPM due to Sarcocystis neurona as
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was suggested previously (Pitel et al., 2001). This, however, is not likely to be the situation in our region, because of the absence of opossum (Didelphis virginiana), the definitive host of S. neurona in North America. Therefore, clinicians in areas where S. neurona and opossums are not present should be more aware of the possible role of Neospora in EPM, as suggested by Pitel et al. (2001). We have found direct association between the prevalence of antibodies to Neospora spp. and age. These results suggest that only a small proportion of horses were exposed to the parasite in the early stages of their life. These findings are in agreement with previous report from Italy (Ciaramella et al., 2004), but are in contrary to reports in cattle in which vertical transmission is the main route of infection (Hall et al., 2005). In light of this result, it appears that vertical transmission in horses might be less efficient than in cattle, which could be explained by differences in placentation as was suggested previously (Pitel et al., 2003). The association between age and seropositivity might explain some of the differences between studies. Difference in seroprevalence between groups of horses from various geographical locations was reported by Vardeleon et al. (2001). The prevalence of antibodies to N. hughesi was lower in race horses from Florida compared to horses from Missouri which were older (Vardeleon et al., 2001). Herd level risk factors such as the number of dogs on a farm, farm area, feeding pooled sources of colostrums and region were significantly associated with seropositivity to N. caninum in dairy farms in Brazil (Corbellini et al., 2006). In our study, other horse level risk factors including gender and breed, and various farm level risk factors such as stable bedding, frequency of stall cleaning and proximity to other animals were not significantly associated with seropositivity in this study. Horizontal transmission of Neospora spp. appears to be a major mode of transmission in horses, therefore it is important to determine which factors increase the probability of infection. Further studies are required in order to clarify the relationship between seropositivity and various horse and farm level risk factors, such as presence of dogs, which are a known source of post-natal transmission to cattle (Corbellini et al., 2006), which might help to decrease post-natal infection in horses. In conclusion, in this first study of Neospora in horses in Israel, seroprevalence was found to increase significantly with age. It was particularly high in horses with neurological disorders and in aborted mares.
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