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Seroprevalence of Neospora, Toxoplasma gondii and Sarcocystis neurona antibodies in horses from Jeju island, South Korea
Veterinary Parasitology 106 (2002) 193–201
Seroprevalence of Neospora, Toxoplasma gondii and Sarcocystis neurona antibodies in horses from Jeju island, South Korea G.D. Gupta a , J. Lakritz a , Jae-Hoon Kim b , Dae-Yong Kim c , Jin-Kap Kim d , A.E. Marsh a,∗ a
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Connaway Hall, 1600 East Rollins Dr., Columbia, MO 65211, USA b National Veterinary Research and Quarantine Service, Anyang 430-824, South Korea c Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, South Korea d Korea Racing Association, Kwachon 427-711, South Korea Received 20 January 2002; received in revised form 19 March 2002; accepted 19 March 2002
Abstract Parasite-specific antibody responses to Neospora spp. and Toxoplasma gondii, antigens were detected using the indirect fluorescent antibody test (IFAT) and immunoblot analysis in a korean equine population located on Jeju island, South Korea (126◦ 12 E and 33◦ 34 N). For comparison, a naturally infected Neospora hughesi horse and an experimentally inoculated T. gondii equid (pony) were used. In addition, all samples were tested for antibodies to Sarcocystis neurona by immunoblot analysis. A total of 191 serum samples from clinically normal horses were evaluated. Only 2% (4 out of 191) and 2.6% (5 out of 191) of the samples had showed reactivity at 1:100 using the IFAT for Neospora spp. and T. gondii, respectively. For T. gondii, two samples matched the antigen banding pattern of the positive control by immunoblot analysis. No sample was positive for N. hughesi by immunoblot analysis in this study. Overall, there was a 1% seroprevalence for T. gondii antibodies in the horses tested based on immunoblot analysis. The seroprevalence for S. neurona and N. hughesi antibodies was 0%. We concluded that these horses are either not routinely exposed to these parasites or antibody titers are not sufficiently elevated to be detectable. It is most likely the former explanation since Jeju island equine farms are isolated from the main land, and the horses were all less than 3 years of age. This na¨ıve population of horses could be useful when evaluating S. neurona
∗ Corresponding author. Tel.: +1-573-884-2673; fax: +1-573-884-5414. E-mail address: [email protected] (A.E. Marsh).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 0 6 4 - X
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serodiagnostic tests or evaluating potential S. neurona vaccines since exposure risks to S. neurona and closely related parasites are negligible. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Equine protozoal myeloencephalitis (EPM); Sarcocystis neurona; Neospora; Toxoplasma gondii
1. Introduction Sarcocystis neurona, Neospora spp. and Toxoplasma gondii are related protozoan genera that can cause encephalomyelitis in livestock and laboratory animals. Equine protozoal myeloencephalitis (EPM) is caused by a protozoal infection of the central nervous system (CNS) (Rooney et al., 1970; Cusick et al., 1974; Madigan and Higgins, 1987; Marsh et al., 1996) and is the most commonly diagnosed neurologic disease of horses in North America (MacKay, 1997). Approximately 50% of horses in the US are seropositive to S. neurona (Bentz et al., 1997; Blythe et al., 1997; Saville et al., 1997). However, only a minority of all S. neurona seropositive horses actually develop clinical signs of neurological disease (Cohen and MacKay, 1997; MacKay, 1997). The diagnosis of EPM is usually based on results of a detailed clinical examination and cerebrospinal fluid (CSF) evaluation for S. neurona antibodies. Recently, there have been several reports of equine neosporosis associated with neurologic disease in horses (Lindsay et al., 1996; Marsh et al., 1996; Daft et al., 1997; Hamir et al., 1998; Cheadle et al., 1999; Pronost et al., 2000). Neospora hughesi can culminate in EPM just as S. neurona does. Limited information is currently available on the seroprevalence of Neospora antibodies in horses worldwide (Cheadle et al., 1999; Dubey et al., 1999a,b; Pitel et al., 2001). Although evidence that T. gondii causes neurologic disease in horses is not definite, there are reports of T. gondii infection in the eyes of aborted foals in UK (Turner and Savva, 1990, 1992) and in CNS of horses in Brazil (Macruz et al., 1975). Neospora caninum has been reported in Korean cattle (Bae et al., 2000) in similar rates as compared with reports from US (Jenkins et al., 2000). The purpose of this study was to evaluate normal horses from Jeju island, South Korea for the presence of antibodies that reacted with Neospora and Toxoplasma antigens by two methods, the indirect fluorescent antibody test (IFAT) and immunoblot analysis. These two methods were used to compare their usefulness in evaluating sera from horses that could potentially be exposed to these or closely related parasites such as Neospora spp. or T. gondii.
2. Materials and methods 2.1. Parasite cultivation The N. hughesi isolate (NE1) and T. gondii (RH strain) were grown in HS-68 cells (CRL-1635, ATCC, Rockville, MD, USA) and ED cells (CCL-57, ATCC, Rockville, MD, USA), respectively, with protozoal culture medium consisting of either Dulbecco’s minimum essential or RPMI media supplemented with 5–10% fetal bovine serum, as previously described (Marsh et al., 1996). S. neurona (SN-UCD1) was cultivated as previously described (Marsh et al., 1996).
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2.2. Horses selected for testing A total of 191 (108 males and 83 females) sera were collected from horses on Jeju island in South Korea. They were all 1.5–2 years old thoroughbreds. These horses were born and raised on the island. They had never been transported off the island. Positive controls used in this study were a naturally infected N. hughesi horse (Marsh et al., 1996) and a naturally infected S. neurona (SN-UCD1) horse (Marsh et al., 1996). Pre- and post-immune sera from an experimentally inoculated equid (pony) with T. gondii (Dubey and Desmonts, 1987) was also used as a control. 2.3. Indirect fluorescent antibody test After approximately 70% of host cells had been infected with parasite clusters, the monolayers were removed with a cell scraper and the media (containing cells and parasites) was collected. This mixture was centrifuged at 1500 × g for 10 min. Supernatant was removed and the pellet resuspended in Ca2+ - and Mg2+ -free, phosphate buffered saline (PBS), centrifuged and washed twice. The final pellet was resuspended in 1 ml PBS. The parasite concentration was determined by counting on a hemocytometer. Stock solutions were diluted as necessary with PBS. For IFAT antigen preparation, aliquots of 5 l N. hughesi and T. gondii tachyzoites in PBS solution were dispensed to each 4 mm well of 8-well, heavy teflon coated antigen slides (Eerie Scientific, Portsmouth, NH). Tachyzoites concentration per well was 5 × 104 . Slides were air-dried at room temperature and fixed in cold acetone for 2 min. Slides were rinsed in PBS and then fixed in 2.5% formaldehyde for 15 min. The slides were rinsed in PBS three times for 5 min each and stored at −20 ◦ C after air-drying. Equine sera were tested for antibodies to the Neospara spp. and T. gondii by IFAT as previously described (Vardeleon et al., 2001). Positive samples were tested using two-fold dilutions starting at 1:50, and end titers were determined by lack of fluorescence. T. gondii and Neospora IFAT slides were used to test cross reactivity between these three parasites and to titer the positive control samples against homologues and heterologous antigens. Serial dilution of positive control sera of N. hughesi and T. gondii were tested to end-point titers by IFAT. 2.4. Antibody testing by immunoblotting Concentrated stock pellets of cultured S. neurona, T. gondii and N. hughesi parasites were used for immunoblot analysis (Laemmli, 1970; Gallagher and Smith, 1995; Vardeleon et al., 2001) with minor modification. Briefly, sample buffer (Invitrogen, Nu-PAGE LDS sample buffer, Carlsbad, CA, USA) was added and samples were heated at 70 ◦ C for 5 min. The non-reduced proteins were separated by electrophoresis in a Nu-PAGE MES, 4–12%, 1 mm gel. Prestained SDS-PAGE molecular weight markers (Invitrogen, Carlsbad, CA, USA) were included with each gel. The separated antigens were then transferred to nitrocellulose membranes (BioRad, Hercules, CA, USA). The nitrocellulose membrane was blocked in 5% non-fat dried milk in Tris buffered saline with tween; sera and membranes were used as previously described (Vardeleon et al., 2001). The T. gondii positive control sample was tested at 1:800 because reactive bands were not clear due to the strong reaction at lower
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Table 1 Results of horse samples by IFAT (at 1:100 and 1:200 serum dilution) and immunoblot analysis Serum samples
Serum dilutions Toxo-IFA
Toxo positive control Toxo negative control Neo positive control Neo negative control Horse (n = 183) Horse #12 Horse #25 Horse #30 Horse #50 Horse #117 Horse #135 Horse #138 Horse #147
Immunoblot reactivity Neo-IFA
1:100
1:200
1:100
1:200
Pos Neg Pos Neg Neg Pos Pos Neg Pos Pos Neg Pos Neg
Pos Neg Neg Neg ND Neg Pos ND Neg Pos ND Neg ND
Neg Neg Pos Neg Neg Neg Neg Pos Neg Pos Pos Neg Pos
Neg Neg Pos Neg ND Neg Neg Neg ND Pos Neg ND Neg
T. gondii
N. hughesi
Pos Neg Neg Neg ND Pos Pos ND Neg Neg ND Neg ND
Neg Neg Pos Neg ND ND ND Neg ND Neg Neg ND Neg
Neg: negative reaction; Pos: positive reaction; ND: not done; Toxo: T. gondii; Neo: N. hughesi.
dilutions. Reactive test samples were tested at 1:25, 1:50 and 1:100 dilution. Secondary antibody was evaluated at 1:500 and 1:1000 dilution. Sera from positive and negative controls were included on every blot.
3. Results 3.1. Detection of antibodies Serology results are summarized in Table 1. Of the 191 sera tested, four (2%) had reactivity at 1:100 for Neospora spp. by IFAT. When those samples were tested by immunoblot, 0% showed positive reactivity. Five (2.6%) sera reacted to T. gondii antigen by IFAT, but only two sera reacted by immunoblot using T. gondii antigen with a distinctive banding pattern corresponding to approximately 22 and 30 kDa antigens, which matched the positive control pattern (Fig. 1). The dilution of the secondary antibody did not clarify or change the immunoblot antigen pattern. All samples positive for T. gondii and Neospora by IFAT had titers ranging from 1:100 to 1:400. Only one sample was positive for both Neospora and T. gondii, having IFAT titers of 1:200 and 1:400, respectively, but negative for both by immunoblot. Testing the 191 sera samples by immunoblot analysis using S. neurona antigen exhibited 0% positive reactivity. When testing the specificity of reaction, only the confirmed N. hughesi positive serum reacted slightly to T. gondii antigen at 1:100 dilution by IFAT but there was no reaction at 1:200 dilution. On the N. hughesi IFAT slides, there was slight or negligible reactivity of T. gondii positive control at both 1:100 and 1:200 dilutions. S. neurona positive control did not react with either N. hughesi or T. gondii antigen by IFAT.
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Fig. 1. Immunoblot of T. gondii parasite antigens probed with sera from T. gondii experimentally inoculated pony (lane 1), horse #12 at 1:25 serum dilution with secondary antibody at 1:500 (lane 2) and 1:1000 dilution (lane 3), horse #25 at 1:50 serum dilution with secondary antibody at 1:500 (lane 4) and 1:1000 (lane 5), and horse #138 at 1:25 serum dilution with secondary antibody at 1:500 (lane 6) and 1:1000 (lane 7). Left side numbers indicate molecular weight in kDa.
4. Discussion This study evaluated clinically normal race horses from Jeju island, South Korea for the presence of N. hughesi, T. gondii and S. neurona antibodies. These horses were bred and raised for the horse racing industry. Jeju island is located off mainland South Korea. It is located 450 km south from the mainland. Although N. caninum and T. gondii have been reported in South Korea (Hur et al., 1998; Kim et al., 1997; Jeon and Yong, 2000), the prevalence or exposure to horses is unknown. The zero seroprevalence to S. neurona in horses from Jeju island was not surprising since the definitive host, D. virginiana is not present (Hall, 1981), but domestic dogs and cats are present. Cats serve as the definitive host for T. gondii (Frenkel et al., 1970). Dogs are definitive host of N. caninum (McAllister et al., 1998; Lindsay et al., 1999). The definitive host of N. hughesi is unknown. When the samples were tested by IFAT to N. hughesi, four (2%) tested positive at 1:100 but none were considered positive by immunoblot analysis. It may be possible that these horses are exposed to N. caninum or some other similar parasite having common surface epitopes that are also found on N. hughesi. Limited information is currently available on the seroprevalence of Neospora antibodies in horses (Cheadle et al., 1999; Dubey et al., 1999a,b; Cheadle et al., 2000; Vardeleon et al., 2001). At the present time, clinical equine neosporosis and the isolation of parasite has been reported only in USA, but bovine neosporosis principally in cattle has been reported in many continents (Bartels et al., 1999; Yamane et al., 1997; Schares et al., 1999). An important aspect of evaluating Neospora seroprevalence in the equine population is realizing the limitations of the testing methods. Screening by IFAT at 1:100 may result in false positives; therefore, the IFAT reactive samples were serially
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Fig. 2. Immunoblot of T. gondii parasite antigens probed with different serum dilutions at 1:1000 dilution of secondary antibody is as follows: Positive control at 1:800 serum dilution (1), negative control at 1:25 (lane 2) and 1:800 serum dilution (lane 3), horse #50 at 1:25 (lane 4) and 1:50 serum dilution (lane 5), and horse #117 at 1:25 (lane 6), 1:50 (lane 7) and 1:100 serum dilution (lane 8). Left side numbers indicate molecular weight in kDa.
diluted to end point titers. Immunoblot analysis was then used to compare reactivity of these samples to known positive controls. One sample, horse #117, was showing reactivity for N. hughesi and T. gondii by IFAT and negative by immunoblot. This horse may have been exposed or infected with some other related parasite of the apicomplexa family which cross-reacts on IFAT used herein, or the antibody titer has not been reached to the level (if exposed to either/both of them) that can show reactivity by immunoblot. It is most likely the former reason because horses #12 and 25 had lower IFAT titers than horse #117 for T. gondii but showed reactivity by immunoblot (Table 1). The IFAT results suggest some cross reactivity at the low dilutions of the Neospora spp. and T. gondii positive controls when tested against heterologous antigen (Table 1); further supporting the presence of similar surface epitopes on Neospora spp. and T. gondii (Howe et al., 1998). Five (2.6%) serum samples were positive for T. gondii by IFAT but only two, horses #12 and 25 showed characteristic banding pattern at approximately 22 and 30 kDa antigens (Fig. 1) which also corresponded to positive control where as the other three did not (Fig. 2). Surface proteins of protozoa have been the focus of extensive research for their potential as diagnostic antigens since they are the initial foreign proteins encountered by the host’s immune system. Harning et al. (1996) described the non-reduced 30 kDa protein (SAG1) as suitable for use in serodiagnostics for T. gondii by immunoblot. Velge-Roussel et al. (1994) showed the T. gondii B-cell epitopes are conformationally sensitive since T. gondii immune sera do not recognize the reduced SAG1 proteins. Similarly, a 22 kDa (SAG2) was described as a promising antigen for a serological diagnostic test of T. gondii since antibodies in sera from individuals of recently acquired infection (acute) reacted more strongly with SAG2 than sera from chronically infected individuals (Parmley et al., 1992). T. gondii positive control serum used in this experiment was collected after inoculation, so it may correspond to acute infection. Two samples, horses #12 and 25 (Fig. 1, lanes 2–5) showed reaction to the approximately 22 kDa protein but not as strong as the positive control. It may be possible that these horses were infected for a longer duration than the positive control. Although T. gondii can infect horses as revealed by serological surveys and isolation
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of viable T. gondii parasites from equine tissues (Dubey and Beattie, 1988), definitive evidence that it causes clinical disease such as EPM is unknown. The final deposition of T. gondii infected horses that are removed from racing or breeding careers is a concern if the carcass is used as an unprocessed (inadequate freezing or cooking) food source. This study would suggest that horses on Jeju island are not routinely and frequently exposed to oocysts of T. gondii or N. hughesi. It would be useful to compare the results of this study with the seroprevalence of horses exported from Jeju island and maintained for a period of time on mainland South Korea. The national average seroprevalence of Neospora antibodies in cattle raised on mainland South Korea is approximately 21%; however, it is only approximately 4% in cattle raised on Jeju island (Yong, unpublished data). Finally, this population of isolated horses could be used to evaluate duration of immune response to S. neurona vaccine since these thoroughbred horses do not have environmental exposure but are under standard racing exercise and training stress.
Acknowledgements The University of Missouri College of Veterinary Medicine and Brain Korea 21 Project supported this project. We are thankful to Dr. J.P. Dubey for T. gondii positive control. We also thank Aislinn Halaney and Kitty Rodrigues for technical assistance.
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Seroprevalence of Neospora, Toxoplasma gondii and Sarcocystis neurona antibodies in horses from Jeju island, South Korea G.D. Gupta a , J. Lakritz a , Jae-Hoon Kim b , Dae-Yong Kim c , Jin-Kap Kim d , A.E. Marsh a,∗ a
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Connaway Hall, 1600 East Rollins Dr., Columbia, MO 65211, USA b National Veterinary Research and Quarantine Service, Anyang 430-824, South Korea c Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, South Korea d Korea Racing Association, Kwachon 427-711, South Korea Received 20 January 2002; received in revised form 19 March 2002; accepted 19 March 2002
Abstract Parasite-specific antibody responses to Neospora spp. and Toxoplasma gondii, antigens were detected using the indirect fluorescent antibody test (IFAT) and immunoblot analysis in a korean equine population located on Jeju island, South Korea (126◦ 12 E and 33◦ 34 N). For comparison, a naturally infected Neospora hughesi horse and an experimentally inoculated T. gondii equid (pony) were used. In addition, all samples were tested for antibodies to Sarcocystis neurona by immunoblot analysis. A total of 191 serum samples from clinically normal horses were evaluated. Only 2% (4 out of 191) and 2.6% (5 out of 191) of the samples had showed reactivity at 1:100 using the IFAT for Neospora spp. and T. gondii, respectively. For T. gondii, two samples matched the antigen banding pattern of the positive control by immunoblot analysis. No sample was positive for N. hughesi by immunoblot analysis in this study. Overall, there was a 1% seroprevalence for T. gondii antibodies in the horses tested based on immunoblot analysis. The seroprevalence for S. neurona and N. hughesi antibodies was 0%. We concluded that these horses are either not routinely exposed to these parasites or antibody titers are not sufficiently elevated to be detectable. It is most likely the former explanation since Jeju island equine farms are isolated from the main land, and the horses were all less than 3 years of age. This na¨ıve population of horses could be useful when evaluating S. neurona
∗ Corresponding author. Tel.: +1-573-884-2673; fax: +1-573-884-5414. E-mail address: [email protected] (A.E. Marsh).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 0 6 4 - X
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serodiagnostic tests or evaluating potential S. neurona vaccines since exposure risks to S. neurona and closely related parasites are negligible. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Equine protozoal myeloencephalitis (EPM); Sarcocystis neurona; Neospora; Toxoplasma gondii
1. Introduction Sarcocystis neurona, Neospora spp. and Toxoplasma gondii are related protozoan genera that can cause encephalomyelitis in livestock and laboratory animals. Equine protozoal myeloencephalitis (EPM) is caused by a protozoal infection of the central nervous system (CNS) (Rooney et al., 1970; Cusick et al., 1974; Madigan and Higgins, 1987; Marsh et al., 1996) and is the most commonly diagnosed neurologic disease of horses in North America (MacKay, 1997). Approximately 50% of horses in the US are seropositive to S. neurona (Bentz et al., 1997; Blythe et al., 1997; Saville et al., 1997). However, only a minority of all S. neurona seropositive horses actually develop clinical signs of neurological disease (Cohen and MacKay, 1997; MacKay, 1997). The diagnosis of EPM is usually based on results of a detailed clinical examination and cerebrospinal fluid (CSF) evaluation for S. neurona antibodies. Recently, there have been several reports of equine neosporosis associated with neurologic disease in horses (Lindsay et al., 1996; Marsh et al., 1996; Daft et al., 1997; Hamir et al., 1998; Cheadle et al., 1999; Pronost et al., 2000). Neospora hughesi can culminate in EPM just as S. neurona does. Limited information is currently available on the seroprevalence of Neospora antibodies in horses worldwide (Cheadle et al., 1999; Dubey et al., 1999a,b; Pitel et al., 2001). Although evidence that T. gondii causes neurologic disease in horses is not definite, there are reports of T. gondii infection in the eyes of aborted foals in UK (Turner and Savva, 1990, 1992) and in CNS of horses in Brazil (Macruz et al., 1975). Neospora caninum has been reported in Korean cattle (Bae et al., 2000) in similar rates as compared with reports from US (Jenkins et al., 2000). The purpose of this study was to evaluate normal horses from Jeju island, South Korea for the presence of antibodies that reacted with Neospora and Toxoplasma antigens by two methods, the indirect fluorescent antibody test (IFAT) and immunoblot analysis. These two methods were used to compare their usefulness in evaluating sera from horses that could potentially be exposed to these or closely related parasites such as Neospora spp. or T. gondii.
2. Materials and methods 2.1. Parasite cultivation The N. hughesi isolate (NE1) and T. gondii (RH strain) were grown in HS-68 cells (CRL-1635, ATCC, Rockville, MD, USA) and ED cells (CCL-57, ATCC, Rockville, MD, USA), respectively, with protozoal culture medium consisting of either Dulbecco’s minimum essential or RPMI media supplemented with 5–10% fetal bovine serum, as previously described (Marsh et al., 1996). S. neurona (SN-UCD1) was cultivated as previously described (Marsh et al., 1996).
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2.2. Horses selected for testing A total of 191 (108 males and 83 females) sera were collected from horses on Jeju island in South Korea. They were all 1.5–2 years old thoroughbreds. These horses were born and raised on the island. They had never been transported off the island. Positive controls used in this study were a naturally infected N. hughesi horse (Marsh et al., 1996) and a naturally infected S. neurona (SN-UCD1) horse (Marsh et al., 1996). Pre- and post-immune sera from an experimentally inoculated equid (pony) with T. gondii (Dubey and Desmonts, 1987) was also used as a control. 2.3. Indirect fluorescent antibody test After approximately 70% of host cells had been infected with parasite clusters, the monolayers were removed with a cell scraper and the media (containing cells and parasites) was collected. This mixture was centrifuged at 1500 × g for 10 min. Supernatant was removed and the pellet resuspended in Ca2+ - and Mg2+ -free, phosphate buffered saline (PBS), centrifuged and washed twice. The final pellet was resuspended in 1 ml PBS. The parasite concentration was determined by counting on a hemocytometer. Stock solutions were diluted as necessary with PBS. For IFAT antigen preparation, aliquots of 5 l N. hughesi and T. gondii tachyzoites in PBS solution were dispensed to each 4 mm well of 8-well, heavy teflon coated antigen slides (Eerie Scientific, Portsmouth, NH). Tachyzoites concentration per well was 5 × 104 . Slides were air-dried at room temperature and fixed in cold acetone for 2 min. Slides were rinsed in PBS and then fixed in 2.5% formaldehyde for 15 min. The slides were rinsed in PBS three times for 5 min each and stored at −20 ◦ C after air-drying. Equine sera were tested for antibodies to the Neospara spp. and T. gondii by IFAT as previously described (Vardeleon et al., 2001). Positive samples were tested using two-fold dilutions starting at 1:50, and end titers were determined by lack of fluorescence. T. gondii and Neospora IFAT slides were used to test cross reactivity between these three parasites and to titer the positive control samples against homologues and heterologous antigens. Serial dilution of positive control sera of N. hughesi and T. gondii were tested to end-point titers by IFAT. 2.4. Antibody testing by immunoblotting Concentrated stock pellets of cultured S. neurona, T. gondii and N. hughesi parasites were used for immunoblot analysis (Laemmli, 1970; Gallagher and Smith, 1995; Vardeleon et al., 2001) with minor modification. Briefly, sample buffer (Invitrogen, Nu-PAGE LDS sample buffer, Carlsbad, CA, USA) was added and samples were heated at 70 ◦ C for 5 min. The non-reduced proteins were separated by electrophoresis in a Nu-PAGE MES, 4–12%, 1 mm gel. Prestained SDS-PAGE molecular weight markers (Invitrogen, Carlsbad, CA, USA) were included with each gel. The separated antigens were then transferred to nitrocellulose membranes (BioRad, Hercules, CA, USA). The nitrocellulose membrane was blocked in 5% non-fat dried milk in Tris buffered saline with tween; sera and membranes were used as previously described (Vardeleon et al., 2001). The T. gondii positive control sample was tested at 1:800 because reactive bands were not clear due to the strong reaction at lower
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Table 1 Results of horse samples by IFAT (at 1:100 and 1:200 serum dilution) and immunoblot analysis Serum samples
Serum dilutions Toxo-IFA
Toxo positive control Toxo negative control Neo positive control Neo negative control Horse (n = 183) Horse #12 Horse #25 Horse #30 Horse #50 Horse #117 Horse #135 Horse #138 Horse #147
Immunoblot reactivity Neo-IFA
1:100
1:200
1:100
1:200
Pos Neg Pos Neg Neg Pos Pos Neg Pos Pos Neg Pos Neg
Pos Neg Neg Neg ND Neg Pos ND Neg Pos ND Neg ND
Neg Neg Pos Neg Neg Neg Neg Pos Neg Pos Pos Neg Pos
Neg Neg Pos Neg ND Neg Neg Neg ND Pos Neg ND Neg
T. gondii
N. hughesi
Pos Neg Neg Neg ND Pos Pos ND Neg Neg ND Neg ND
Neg Neg Pos Neg ND ND ND Neg ND Neg Neg ND Neg
Neg: negative reaction; Pos: positive reaction; ND: not done; Toxo: T. gondii; Neo: N. hughesi.
dilutions. Reactive test samples were tested at 1:25, 1:50 and 1:100 dilution. Secondary antibody was evaluated at 1:500 and 1:1000 dilution. Sera from positive and negative controls were included on every blot.
3. Results 3.1. Detection of antibodies Serology results are summarized in Table 1. Of the 191 sera tested, four (2%) had reactivity at 1:100 for Neospora spp. by IFAT. When those samples were tested by immunoblot, 0% showed positive reactivity. Five (2.6%) sera reacted to T. gondii antigen by IFAT, but only two sera reacted by immunoblot using T. gondii antigen with a distinctive banding pattern corresponding to approximately 22 and 30 kDa antigens, which matched the positive control pattern (Fig. 1). The dilution of the secondary antibody did not clarify or change the immunoblot antigen pattern. All samples positive for T. gondii and Neospora by IFAT had titers ranging from 1:100 to 1:400. Only one sample was positive for both Neospora and T. gondii, having IFAT titers of 1:200 and 1:400, respectively, but negative for both by immunoblot. Testing the 191 sera samples by immunoblot analysis using S. neurona antigen exhibited 0% positive reactivity. When testing the specificity of reaction, only the confirmed N. hughesi positive serum reacted slightly to T. gondii antigen at 1:100 dilution by IFAT but there was no reaction at 1:200 dilution. On the N. hughesi IFAT slides, there was slight or negligible reactivity of T. gondii positive control at both 1:100 and 1:200 dilutions. S. neurona positive control did not react with either N. hughesi or T. gondii antigen by IFAT.
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Fig. 1. Immunoblot of T. gondii parasite antigens probed with sera from T. gondii experimentally inoculated pony (lane 1), horse #12 at 1:25 serum dilution with secondary antibody at 1:500 (lane 2) and 1:1000 dilution (lane 3), horse #25 at 1:50 serum dilution with secondary antibody at 1:500 (lane 4) and 1:1000 (lane 5), and horse #138 at 1:25 serum dilution with secondary antibody at 1:500 (lane 6) and 1:1000 (lane 7). Left side numbers indicate molecular weight in kDa.
4. Discussion This study evaluated clinically normal race horses from Jeju island, South Korea for the presence of N. hughesi, T. gondii and S. neurona antibodies. These horses were bred and raised for the horse racing industry. Jeju island is located off mainland South Korea. It is located 450 km south from the mainland. Although N. caninum and T. gondii have been reported in South Korea (Hur et al., 1998; Kim et al., 1997; Jeon and Yong, 2000), the prevalence or exposure to horses is unknown. The zero seroprevalence to S. neurona in horses from Jeju island was not surprising since the definitive host, D. virginiana is not present (Hall, 1981), but domestic dogs and cats are present. Cats serve as the definitive host for T. gondii (Frenkel et al., 1970). Dogs are definitive host of N. caninum (McAllister et al., 1998; Lindsay et al., 1999). The definitive host of N. hughesi is unknown. When the samples were tested by IFAT to N. hughesi, four (2%) tested positive at 1:100 but none were considered positive by immunoblot analysis. It may be possible that these horses are exposed to N. caninum or some other similar parasite having common surface epitopes that are also found on N. hughesi. Limited information is currently available on the seroprevalence of Neospora antibodies in horses (Cheadle et al., 1999; Dubey et al., 1999a,b; Cheadle et al., 2000; Vardeleon et al., 2001). At the present time, clinical equine neosporosis and the isolation of parasite has been reported only in USA, but bovine neosporosis principally in cattle has been reported in many continents (Bartels et al., 1999; Yamane et al., 1997; Schares et al., 1999). An important aspect of evaluating Neospora seroprevalence in the equine population is realizing the limitations of the testing methods. Screening by IFAT at 1:100 may result in false positives; therefore, the IFAT reactive samples were serially
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Fig. 2. Immunoblot of T. gondii parasite antigens probed with different serum dilutions at 1:1000 dilution of secondary antibody is as follows: Positive control at 1:800 serum dilution (1), negative control at 1:25 (lane 2) and 1:800 serum dilution (lane 3), horse #50 at 1:25 (lane 4) and 1:50 serum dilution (lane 5), and horse #117 at 1:25 (lane 6), 1:50 (lane 7) and 1:100 serum dilution (lane 8). Left side numbers indicate molecular weight in kDa.
diluted to end point titers. Immunoblot analysis was then used to compare reactivity of these samples to known positive controls. One sample, horse #117, was showing reactivity for N. hughesi and T. gondii by IFAT and negative by immunoblot. This horse may have been exposed or infected with some other related parasite of the apicomplexa family which cross-reacts on IFAT used herein, or the antibody titer has not been reached to the level (if exposed to either/both of them) that can show reactivity by immunoblot. It is most likely the former reason because horses #12 and 25 had lower IFAT titers than horse #117 for T. gondii but showed reactivity by immunoblot (Table 1). The IFAT results suggest some cross reactivity at the low dilutions of the Neospora spp. and T. gondii positive controls when tested against heterologous antigen (Table 1); further supporting the presence of similar surface epitopes on Neospora spp. and T. gondii (Howe et al., 1998). Five (2.6%) serum samples were positive for T. gondii by IFAT but only two, horses #12 and 25 showed characteristic banding pattern at approximately 22 and 30 kDa antigens (Fig. 1) which also corresponded to positive control where as the other three did not (Fig. 2). Surface proteins of protozoa have been the focus of extensive research for their potential as diagnostic antigens since they are the initial foreign proteins encountered by the host’s immune system. Harning et al. (1996) described the non-reduced 30 kDa protein (SAG1) as suitable for use in serodiagnostics for T. gondii by immunoblot. Velge-Roussel et al. (1994) showed the T. gondii B-cell epitopes are conformationally sensitive since T. gondii immune sera do not recognize the reduced SAG1 proteins. Similarly, a 22 kDa (SAG2) was described as a promising antigen for a serological diagnostic test of T. gondii since antibodies in sera from individuals of recently acquired infection (acute) reacted more strongly with SAG2 than sera from chronically infected individuals (Parmley et al., 1992). T. gondii positive control serum used in this experiment was collected after inoculation, so it may correspond to acute infection. Two samples, horses #12 and 25 (Fig. 1, lanes 2–5) showed reaction to the approximately 22 kDa protein but not as strong as the positive control. It may be possible that these horses were infected for a longer duration than the positive control. Although T. gondii can infect horses as revealed by serological surveys and isolation
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of viable T. gondii parasites from equine tissues (Dubey and Beattie, 1988), definitive evidence that it causes clinical disease such as EPM is unknown. The final deposition of T. gondii infected horses that are removed from racing or breeding careers is a concern if the carcass is used as an unprocessed (inadequate freezing or cooking) food source. This study would suggest that horses on Jeju island are not routinely and frequently exposed to oocysts of T. gondii or N. hughesi. It would be useful to compare the results of this study with the seroprevalence of horses exported from Jeju island and maintained for a period of time on mainland South Korea. The national average seroprevalence of Neospora antibodies in cattle raised on mainland South Korea is approximately 21%; however, it is only approximately 4% in cattle raised on Jeju island (Yong, unpublished data). Finally, this population of isolated horses could be used to evaluate duration of immune response to S. neurona vaccine since these thoroughbred horses do not have environmental exposure but are under standard racing exercise and training stress.
Acknowledgements The University of Missouri College of Veterinary Medicine and Brain Korea 21 Project supported this project. We are thankful to Dr. J.P. Dubey for T. gondii positive control. We also thank Aislinn Halaney and Kitty Rodrigues for technical assistance.
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