Abstract: Neospora spp. are an obligate intracellular protozoan parasite and are recognized as a major cause of abortion in farm animals. Epidemiological and field studies concerning Neospora spp are very much dependent on serology. The objective of the present study was to determine the seroprevalence of Neospora spp. in Arabian horses used for sporting and showing purposes in Riyadh city, Saudi Arabia. In total, 163 serum samples from physically healthy horses were analyzed for anti-Neospora antibodies using the Neospora Modified Agglutination Test (N-MAT). Antibodies to Neospora spp were 39 (23.9%) of the 163 horses with 1:40 serum dilution, where 1:80 serum dilution was applied as significant cut off dilution and the serum positivity was reduced to 28 (17.1%) of the 163 horses. The differences in age and horse sex were not statistically significant (p<0.05). However, this study is the first seroprevalence study to determine Neospora spp antibodies by a Neospora Modified Agglutination Test (N-MAT) in horses from arid areas in Saudi Arabia.
INTRODUCTION
Neospora spp. is protozoan parasite and have been identified in a wide range of animal species in particular farm animals such as cattle, sheep, goats, dogs and horses farm, being associated with neonatal mortality and abortion in these animals (Dubey et al., 2003, 2007). In horses, Neospora caninum or Neospora hughesi can be cause neurological diseases called Equine Protozoal Myeloencephalitis (EPM) (Marsh et al., 1998; Vardeleon et al., 2001). The clinical signs have been recognized for more than three decades (Beech and Dodd, 1974) and include ataxia, abnormal gait, difficulty ingesting, paralysis of muscles of the eyes, face or mouth and loss of sensation along the horse’s face, neck and body (MacKay, 1997). Antibody to N. caninum and N, hughesi in horse populations were reported in many parts of the world such as in North America, with prevalence (2-31%) (Cheadle et al., 1999; Dubey et al., 1999, 2003), 2.5-15% in South America (Hoane et al., 2006), 1-28% in Europe (Pitel et al., 2001; Ciaramella et al., 2004; Jakubek et al., 2006; Bartova et al., 2010), 2% in New Zealand (Vardeleon et al., 2001) and South Korea (Gupta et al., 2002).
In Saudi Arabia, the No. of horses according to estimates of the Saudi Ministry of Agriculture in 2011 were more than 45,000 horse and more than 300 horse were imported per year from USA and some European countries under restricted import regulations (Ministry of Agriculture in Saudi Arabia, 2011). Although, there is lack of information about the prevalence of Neospora spp infection in horses in Saudi Arabia. Therefore, this study was carried out to determine the seroprevalence of such infection in Arabian horses from Riyadh city Saudi Arabia using Neospora Modified Agglutination Test (N-MAT).
MATERIALS AND METHODS
Blood collection and preparation: From September 2012 to March 2013, blood samples were collected from 163 physically healthy adult horses for sporting purposes from Riyadh city, Saudi Arabia. Blood samples (5-10 mL) were collected from the jugular vein of each horse in vacuum tubes without anticoagulant. The blood samples were transported to the laboratory of Parasitology, Department of Biological Sciences, Faculty of Science and Humanities, Shaqra University. After clotting, samples were centrifuged at 3000 rpm for 10-15 min, serum was decanted and stored at 20°C until assayed for the antibodies to Neospora spp.
Serological test for Neospora spp: Sera obtained was stored at 20°C and subsequently thawed at 35°C immediately before testing. For detection of antibodies to Neospora spp., sera were initially screened at 1:40 and 1:80 dilutions using Neospora Agglutination Test (NAT), as described by Packham et al. (1998). Sera were double diluted from 1:10-:80 with phosphate-buffered saline containing 0.2 M 2-mercaptoethanol and 50 μL of each dilution was put in a well of 96 U-bottom microtiter plates. Then 50 μL of 3.5x107 mL-1 suspension of tachyzoites of the NC-1 strain of N. caninum resuspended in alkaline buffer (7.02 g of NaCl, 3.09 g of H3BO3, 24 mL of 1N NaOH, 4 g of horse serum [HS] albumin [fraction V], 50 mg of eosin Y, dH2O to 1 L, 0.1% sodium azide as a preservative; pH 8.7) were added to each serum dilution of samples as well as positive and negative controls. The wells were then mixed thoroughly by pipetting them up and down several times, covered and incubated overnight at 37°C with 5% CO2. A cut-off titer of 1:80 was considered as significant for the presence of antibodies according to Packham et al. (1998) and Pitel et al. (2001). Reactions were considered positive when the tachyzoites were spread on the entire bottom of the well of the micro titer plate and those showing button formation were considered negative.
Age estimation of horses: In this study, horses were divided in 4 age groups ( = 2, 3-5, 6-8 and = 9 years) based on their dentition. In younger horses (0<2years), it was possible to make a good estimate from the number of baby and adult teeth that have erupted or emerged from the gum. Further, the baby teeth are smaller and whiter with a rounded gum line. In horses from 3-5 years old, there are two adult incisors in wear in the centre of the jaw flanked by baby teeth (Richardson et al., 1995). The dental star appears in horses sequentially in more lateral teeth at 6, 7 and 8 years old (Pasca et al., 2006). The length of the groove of Galwayne was the indicator which was used to estimate age greater than 9 years, (Muylle et al., 1998; Pasca et al., 2006). In the present study, it was difficult to estimate the age of older horses (<12) because the cups had disappeared from all the lower incisors (Richardson et al., 1994; Muylle et al.,1998). The age groupings used in this study reduced any errors that variable dentition imposed on the judgment of age.
| Table 1: | Prevalence of serum antibodies to Neospora spp in arabian horses from Riyadh city, Saudi Arabia |
| Table 2: | Distribution of NAT titres according to the horse gender |
Statistical analysis: Data analyses were performed using the SPSS software (Statistical Package for Social Sciences, version 16.0, [2008] for Windows, Sydney, Australia). The Chi square test was applied to compare the rates of seropositivity between the age and the sex of the horses. Statistical significance in this study was defined as p<0.05.
RESULTS
Antibodies to Neospora spp were found in 39 (23.9%) of the 163 horses of all ages (Table 1). Also, 39 out of 163 (23.9%) horses, reacted positively for Neospora spp antibodies with 1:40 serum dilution, where 1:80 serum dilution was applied as significant cut off dilution and the serum positivity was reduced to 28 (17.1%) of the 163 horses. Prevalences in mares were similar to those in males, the mares showed a seropositivity of 21( 21.8%) while the seropositivity for males was 18 (26.8%). These values indicated that there is not any association between the presence of antibodies to Neospora spp. and the sex of the horses (Table 2). The differences in age and horse sex were not statistically significant (p<0.05).
DISCUSSION
This is the first Neospora spp seroprevalence study on horses in Saudi Arabia using; Neospora modified agglutination test (N-MAT); however, these results can only be compared with those obtained in other countries in the same geographic area. In Turkey and Iran, it was found that 24 and 28% of horses had Neospora antibodies in IFAT and cELISA tests (Bartova et al., 2010; Yagoob, 2012), respectively. In other countries such as the Czech Republic and Italy, it was found that 9.3 28% of horses had Neospora spp. antibodies by Indirect Fluorescent Antibody Test (IFAT) Kilbas et al. (2008); Ciaramella et al. (2004), respectively. In South Korea, 2% of horses reacted positively for Neospora spp Gupta et al. (2002). In the United States, antibodies to N. caninum were found to be (8-12%) horses in IFAT and 21-31% in agglutination (Cheadle et al., 1999; McDole and Gay, 2002 and Dubey et al., 2003). The variation of the results may be due to the sample sizes of the different studies and also to the different methodologies used.
In the previous studies, there is a lack of sensitivity and specificity of the serological methods used to detect Neospora spp For example, Packham et al. (1998) compared the sensitivity and specificity of ELISA with modified direct agglutination test (N-MAT) and (IFAT) to detect antibodies against N. caninum in cattle sera. The N-MAT had superior sensitivity (100) and specificity (97%) compared with ELISA (74 and 94%, respectively) and had a higher sensitivity but lower specificity than the IFAT (98 and 99%, respectively). Hoane et al. (2005) were used recombinant antigen (rNhSAG1) to detect N. caninum and N. hughesi antibodies in horse serum. In their work, the ELISA results showed the highest sensitivity and specificity at 94.4 and 95.0%, respectively. These results suggested that rNhSAG1 recombinant antigens can be used as gold promise for serodiagnosis of N. caninum and N. hughesi infection in horse serum.
While the Neospora spp. can cause reproductive and neurological diseases in horses (Daft et al., 1997; Hamir et al., 1998; Pitel et al., 2003; Hoane et al., 2006) but the horses in the current study were physically normal and there was no history of abortion or neurological diseases. Also, the sample size was a bit small to draw any clinical conclusion compared with previous studies.
CONCLUSION
The results of the present study confirm the presence of Neospora spp. antibodies in Arabian horses from Saudi Arabia. The impact of neosporosis on horse farms and production, due to clinical neosporosis and potential risk factors of its transmission to other animals in Saudi Arabia, needs further investigations.
ACKNOWLEDGMENTS
Authors are grateful to the Deanship of Scientific Research, Shaqra University. Also, we would like to thank all horse owners for their consent to collect blood samples and the Staff Members of the Department of Biological Sciences, Faculty of Science, Shaqra University for their kind technical support during this investigation.