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Association of antibodies against Neospora caninum in mares with reproductive problems and presence of seropositive dogs as a risk factor R.A. Abreu a , R.R. Weiss a,∗ , V. Thomaz-Soccol a , R. Locatelli-Dittrich b , L.M. Laskoski b , M.A.F. Bertol a , M.O. Koch a , S.M. Alban a , K.T. Green c a Programa de Pós-Graduac¸ão em Engenharia de Bioprocessos e Biotecnologia, Universidade Federal do Paraná, Rua Cel. Francisco H. dos Santos, 100, CEP 81531-990 Curitiba, Paraná, Brazil b Programa de Pós-Graduac¸ão em Ciências Veterinárias, Universidade Federal do Paraná, Curitiba, Paraná, Brazil c Médica veterinária autônoma, Brazil

a r t i c l e

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Article history: Received 14 January 2014 Received in revised form 14 March 2014 Accepted 18 March 2014

Keywords: Neospora caninum Toxoplasma gondii Serology Mares Reproductive problems Risk factors

a b s t r a c t Sera from 112 mares from 5 horse-breeding farms was examined for the presence of antibodies to Neospora caninum and Toxoplasma gondii by an indirect fluorescent antibody test (IFAT), as well as from dogs and cattle present on these properties for the presence of antibodies to N. caninum. Among the 112 mares, 35 had a history of reproductive problems in the last breeding season and 77 had no reproductive problems. The rates of seroprevalence of N. caninum in mares with and without a history of reproductive problems were 25.71% and 6.49% and from T. gondii 2.85% and 1.29%, respectively. In dogs and cattle, the rates of seroprevalence of N. caninum were 10.52% and 15.55%, respectively. A positive correlation was found between the presence of antibodies against N. caninum (p = 0.010) in mares and the occurrence of reproductive problems using the Fisher’s exact test. Significantly higher seroprevalence for N. caninum in mares was observed on the farm that had seropositive dogs (p = 0.018). Cattle on this farm were also seropositive. No significant differences in seropositivity were found on farms where dogs were seronegative, or absent. This result suggests, for the first time, the presence of seropositive dogs as a risk factor for N. caninum in mares and the necessity for further investigation of the epidemiology of this parasite in horse-breeding farms with reproductive problems and the presence of cattle and dogs. This is the first report on the occurrence of antibodies against N. caninum in horses from the state of Santa Catarina, Brazil. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Equine neosporosis is caused by the parasites Neospora caninum and Neospora hughesi, which are obligate intracellular protozoans. The definitive hosts of N. caninum are

∗ Corresponding author. Tel.: +55 41 3350 5613; fax: +55 41 3350 5623. E-mail address: [email protected] (R.R. Weiss).

dogs, coyotes and wolves and intermediate hosts include diverse animal species such as: cattle, sheep, dogs and horses (Dubey and Schares, 2011). Infection by N. caninum can occur by horizontal and vertical transmission (Dubey et al., 2007). The definitive host of N. hughesi is still unknown (Hoane et al., 2006). Abortion and neonatal diseases are associated with N. caninum infection in horses (Villalobos et al., 2006; Kligler et al., 2007) whereas N. hughesi infection is associated

http://dx.doi.org/10.1016/j.vetpar.2014.03.022 0304-4017/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Abreu, R.A., et al., Association of antibodies against Neospora caninum in mares with reproductive problems and presence of seropositive dogs as a risk factor. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.03.022

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with neurological diseases, principally equine protozoal myeloencephalitis (EPM) (Finno et al., 2007; Kligler et al., 2007). In Brazil, the seroprevalence of Neospora spp. ranges from 2.5 to 47% in healthy horses (Hoane et al., 2006; Locatelli-Dittrich et al., 2006). The presence of dogs on farms plays an important epidemiological role in the infection of cattle and other intermediate hosts, and as such, are a potential risk factor for the occurrence of N. caninum in these species (Basso et al., 2001). Canine infection can occur by the ingestion of carcasses, fetuses and the placental remains of contaminated bovine and other intermediate hosts (Gondim et al., 2002). Toxoplasma gondii is another protozoan that is being investigated in equines, which is one of the domestic species most resistant to infection by this parasite. Infection in horses is generally inapparent, being characterized by the maintenance of titer and the presence of tissue cysts (Langoni et al., 2007). In general, the seroprevalence is lower than in other livestock species (Dubey and Jones, 2008). Previous studies in Brazil have indicated that seropositivity for T. gondii in horses ranges from 2.7 to 5.9% (Locatelli-Dittrich et al., 2006; Coiro et al., 2012). This study aimed to correlate the seroprevalence of N. caninum and T. gondii in mares with the occurrence of reproductive problems and to investigate the presence of dogs and cattle with the seroprevalence of N. caninum, identifying if these animals represent a risk factor for equine neosporosis in the states of Paraná and Santa Catarina in southern Brazil.

2. Materials and methods

embryonic absorption and 4 mares presented histories of stillbirths. Blood samples from 19 dogs of different breeds and ages and of both sexes from 4 properties were collected for serological analysis. Of the 5 farms, only 1 had horses, dogs, and cattle. On this farm, 22 blood samples were collected from mares, of which 10 had a history of reproductive problems and 12 were without a history of reproductive problems; there were 7 blood samples from dogs and 45 blood samples from cattle (only samples from mature female bovines were collected). The cattle and dogs did not present clinical signs of neosporosis. 2.2. Serology Sera were separated and stored at −20◦ C until used. The indirect fluorescent antibody test (IFAT) was utilized to test for circulating IgG antibodies specific for antigens from N. caninum and T. gondii, NC-1 and RH strains respectively. Specific conjugates were used for each species: anti-horse IgG (Sigma–Aldrich® , St Louis, MO, USA), anti-dog IgG (Sigma–Aldrich® , St Louis, MO, USA), and anti-bovine IgG (Sigma–Aldrich® , St Louis, MO, USA) antibodies. For dog and horse serum samples the initial screening dilution was 1:50 in accordance with Locatelli-Dittrich et al. (2006, 2008), respectively, in phosphate buffered saline (PBS), and the positives were diluted to an endpoint titer. In cattle, the cut-off dilution was 1:100 (LocatelliDittrich et al., 2001). Only samples that presented complete peripheral tachyzoite fluorescence were considered positive. Previously known positive and negative sera for N. caninum in horses, dogs and cattle were included on each slide according to the species analyzed as well as horses for T. gondii.

2.1. Animals 2.3. Statistical analysis Five horse-breeding farms in the states of Paraná and Santa Catarina in southern Brazil were chosen to estimate the occurrence of N. caninum and T. gondii antibodies. Blood was obtained from the jugular vein of 112 horses in August 2011. They were all adult females from the following breeds: Crioula, Mangalarga Marchador and Thoroughbred. All mares were in good physical condition and were subjected to routine veterinary examinations. The mares analyzed from each farm were for the last 3 years on the same farm. Among the 112 mares, 35 had a history of reproductive problems in the last breeding season, of which 29 had one or more abortions registered, principally between the 7th and 9th month of gestation, 2 mares presented

The statistical analysis of the association between the seroprevalence for N. caninum and T. gondii and the occurrence of reproductive problems, as well as, the association between the seroprevalence for N. caninum in mares and the presence of dogs and cattle was performed by Fisher’s exact test, considering p < 0.05. 3. Results The overall frequencies of occurrence of antibodies against N. caninum in mares, dogs, and cattle were 12.5% (14/112), 10.52% (2/19), and 15.55% (7/45), respectively

Table 1 Seroprevalence (%) of Neospora caninum in mares with and without history of reproductive problems, and in dogs and cattle analyzed by farm. Farms

Mares (IFAT ≥ 1:50)

Dogs (IFAT ≥ 1:50)

Cattle (IFAT ≥ 1:100)

16.66 (2/12) 1.85 (1/54) 28.57 (2/7) 0.0 (0/1) 0.0 (0/3)

28.57 (2/7) 0.0 (0/8) – 0.0 (0/2) 0.0 (0/2)

15.55 (7/45) – – – –

6.49 (5/77)

10.52 (2/19)

15.55 (7/45)

With reproductive problems

Without reproductive problems

1 2 3 4 5

50.0 (5/10) 16.66 (1/6) 0.0 (0/2) 22.22 (2/9) 12.0 (1/8)

Total

25.71 (9/35)

Please cite this article in press as: Abreu, R.A., et al., Association of antibodies against Neospora caninum in mares with reproductive problems and presence of seropositive dogs as a risk factor. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.03.022

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R.A. Abreu et al. / Veterinary Parasitology xxx (2014) xxx–xxx Table 2 Absolute numbers and percentage of seropositive mares to Neospora caninum and Toxoplasma gondii according to the presence or absence of reproductive problems. Mares

Neospora caninum (%)

Toxoplasma gondii (%)

With reproductive problems, N = 35 Without reproductive problems, N = 77

25.71a

2.85a

6.49b

1.31a

Total, N = 112

12.50

1.78

Values with different superscripts in the same column indicate significant statistical difference according to Fisher’s exact test (p < 0.05).

(Table 1). All the properties presented at least 1 seropositive mare for N. caninum, where the prevalence varies from 3.33 to 31.81%. Significantly higher seroprevalence for N. caninum in mares was observed on the farm that had seropositive dogs (p = 0.018). Cattle on this farm were also seropositive. No significant differences in seropositivity were found on farms where dogs were seronegative, or absent. Among the mares with a history of reproductive problems, 25.71% (9/35) were seropositive for N. caninum. Of the mares without a history of reproductive problems, 6.49% (5/77) were seropositive to the parasite (Table 2). None of the mares had IFAT titer greater than 1:50. The results showed an association between the seropositivity to N. caninum and the occurrence of reproductive problems (p = 0.010), and abortion was the only reproductive problem found in the positive mares. The rates of seroprevalence of T. gondii found in mares with and without a history of reproductive problems were 2.85% (1/35) and 1.31% (1/77), respectively. There was no association between the occurrence of reproductive problems and seroprevalence of the parasite (p = 0.306). 4. Discussion Seroprevalence of Neospora spp. in horses has been reported in many different countries, while the association between seropositivity to the parasite and reproductive problems found in this study were similar in the USA, France, Brazil and Israel (McDole and Gay, 2002; Pitel et al., 2003; Villalobos et al., 2006; Kligler et al., 2007). This is the first known case reported of the presence of dogs and cattle being associated with the seropositivity to N. caninum in horses. The overall seroprevalence for N. caninum antibodies in mares with reproductive problems (25.71%) in this study was higher than in the study conducted in São Paulo, Brazil by Villalobos et al. (2006) that had a seroprevalence of 15.1% using IFAT (1:50). This difference might be due to geographic differences or the presence of the parasite in the region. Locatelli-Dittrich et al. (2006) reported the highest seroprevalence of this parasite in mares (47%), alerting to the possibility of exposure of the horses to the parasite in the state of Paraná, Brazil. The endpoint titers in mares to N. caninum were low (1:50), including in mares with reproductive problems. In Israel, Kligler et al. (2007) reported a seroprevalence of

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37.5% in aborted mares, 4 (10%) and 7 (17.5%) of these mares had endpoint titers of 1:50 and 1:100, respectively. McDole and Gay (2002) reported that if the antibody titer to N. caninum is associated with abortion in mares, the risk is lower than in cattle. Antibodies against T. gondii were detected in only 2 mares (1.78%). Antibodies to T. gondii have been reported in horses from many countries; however, seroprevalence studies also suggest that there is a low prevalence of T. gondii in equine populations (Dubey et al., 1999). In Brazil, Locatelli-Dittrich et al. (2006) found only 1 mare seropositive to T. gondii (2.7%) in the state of Paraná also utilizing IFAT (1:50), whereas Coiro et al. (2012) found 42 (5.9%) horses seropositive to this parasite in the state of São Paulo utilizing the same test but the titer considered positive was 1:16. The comparison of results among different studies becomes difficult due to the type of serologic methods employed and the titer considered positive. Furthermore, the selection of the samples and the differences among the populations tested can influence the result. In this study, dogs and cattle seropositive to N. caninum were from the same farm. In other studies, the occurrence of N. caninum in these species varied in accordance with the region of study, type and cut-off dilution of the serologic methods utilized, along with the history of the farm, the animals examined, and alimentary habits of the dogs. In the state of Paraná, Brazil, Locatelli-Dittrich et al. (2008) did not find significant differences in seroprevalence of N. caninum in cattle from farms with or without seropositive dogs whereas Guimarães Junior et al. (2004) associated the presence of dogs with the seroprevalence of this parasite in cattle. The presence of seropositive dogs (10.52%) in farm 1 could indicate the exposure of these animals to the parasite, considering the presence of seropositive cattle and mares on this farm but did not signify the necessarily elimination of oocysts (Basso et al., 2001). Additionally, at the farms 2, 4, and 5 with seronegative dogs, the possibility of contamination by oocysts can not be excluded. In experimental infections with tissue from infected cattle, dogs are not seroconverted, confirming that the absence of antibodies does not mean that the dog is free from infection. Once, serologically negative dogs may have eliminated or may continue to eliminate oocysts of N. caninum (McAllister et al., 1998; Schares et al., 2005). However, little is known about the frequency of elimination of oocysts by dogs and the survival of these oocysts in the environment (Basso et al., 2001). Other factors that could be involved in the seroprevalence of N. caninum in mares from the farms without seropositive dogs is the possibility of dogs of neighborhood farms contaminating the paddocks and also the presence of wild animals on these farms. In Brazil, antibodies against N. caninum were found in opossums (Yai et al., 2003), capybaras (Truppel et al., 2010), and wild canids (Mattos et al., 2008). Additional studies need to be conducted to demonstrate the role of these animals in the cycle of Neospora spp. The highest seroprevalence of N. caninum in mares, including those with a history of reproductive failure (50%), was observed on the farm that had seropositive dogs. Cattle on this farm were also seropositive. There was a statistically

Please cite this article in press as: Abreu, R.A., et al., Association of antibodies against Neospora caninum in mares with reproductive problems and presence of seropositive dogs as a risk factor. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.03.022

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significant difference (p = 0.018) between the seroprevalence of N. caninum in mares and the presence of dogs and cattle. On the farms that had only dogs, no statistically significant difference was observed (p = 0.351). This study demonstrates the importance that dogs and cattle can have on the epidemiology of N. caninum on horsebreeding farms. The presence of seropositive cattle and dogs may mean that this farm has a higher pressure of the parasite cycle and more easily can infect the mares. Additional studies need to be conducted to evaluate the risk of N. caninum infection in horses that live with dogs and cattle on the same farm, and a definitive diagnosis of dogs as a source of infection should be carried out by analyzing oocysts on the feces. Diagnosis of this protozoan in aborted fetuses is necessary by isolation of the parasite in cell culture or PCR to confirm conclusively as a cause of abortions in mares. 5. Conclusion N. caninum is present in horses in the southern states of Paraná and Santa Catarina. A positive association was found between the seroprevalence of this parasite and the occurrence of reproductive problems. The presence of seropositive dogs associated as a risk factor for the seroprevalence of N. caninum in mares reveals the necessity for further investigation of the epidemiology of this parasite in horse-breeding farms with reproductive problems and the presence of cattle and dogs. Seroprevalence for T. gondii suggest that there is a low prevalence of this parasite in equine populations. References Basso, W., Venturini, L., Venturini, M.C., Moore, P., Rambeau, M., Unzaga, J.M., Campero, C., Bacigalupe, D., Dubey, J.P., 2001. Prevalence of Neospora caninum infection in dogs from beef cattle farms, dairy farms, and from urban areas of Argentina. J. Parasitol. 87, 906–907. Coiro, C.J., Langoni, H., Silva, R.C., 2012. Epidemiological aspects in the Leptospira spp. and Toxoplasma gondii infection in horses from Botucatu, São Paulo, Brazil. J. Equine Vet. Sci. 32, 620–623. Dubey, J.P., Kerber, C.E., Granstrom, D.E., 1999. Serologic prevalence of Sarcocystis neurona, Toxoplasma gondii and Neospora caninum in horses in Brazil. J. Am. Vet. Med. Assoc. 215, 970–972. Dubey, J.P., Schares, G., Ortega-Mora, L.M., 2007. Epidemiology and control of neosporosis and Neospora caninum. Clin. Microbiol. Rev. 20, 323–367. Dubey, J.P., Jones, J.L., 2008. Toxoplasma gondii infection in humans and animals in the United States. Int. J. Parasitol. 38, 1257–1278. Dubey, J.P., Schares, G., 2011. Neosporosis in animals – the last five years. Vet. Parasitol. 180, 90–108. Finno, C.J., Aleman, M., Pusterla, N., 2007. Equine protozoal myeloencephalitis associated with neosporosis in 3 horses. J. Vet. Intern. Med. 21, 1405–1408. Gondim, L.F.P., Gao, L., McAllister, M.M., 2002. Improved production of Neospora caninum oocysts, cyclical oral transmission between

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Please cite this article in press as: Abreu, R.A., et al., Association of antibodies against Neospora caninum in mares with reproductive problems and presence of seropositive dogs as a risk factor. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.03.022

Association of antibodies against Neospora caninum in mares with reproductive problems and presence of seropositive dogs as a risk factor.

Sera from 112 mares from 5 horse-breeding farms was examined for the presence of antibodies to Neospora caninum and Toxoplasma gondii by an indirect f...
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