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Seroprevalence of Neospora caninum infection in dairy and beef cattle in Paraguay
Veterinary Parasitology 110 (2002) 17–23
Seroprevalence of Neospora caninum infection in dairy and beef cattle in Paraguay Takeshi Osawa a,∗ , Jonathan Wastling b , Ladislao Acosta c , Carlos Ortellado c , Julio Ibarra c , Elisabeth A. Innes a a
b
Moredun Research Institute, International Research Centre, Pentland Science Park, Bush Loan, Penicuik EH26 OPZ, UK Institute of Biomedical and Life Sciences, Division of Infection and Immunity, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK c Laboratorio de Investigación y Diagnostico Veterinario, San Lorenzo, Paraguay, UK Received 2 July 2002; received in revised form 11 September 2002; accepted 11 September 2002
Abstract Seroprevalence of Neospora caninum in 879 beef and dairy cattle in different locations of Paraguay was determined by an ELISA. In the survey, 262 (29.8%) cattle were positive to N. caninum, and animals with anti-Neospora antibody titre were observed in all the locations tested in the country. Serum samples taken from a herd that exhibited persistent abortion had the highest percentage of animals being positive to the parasite (17/30, 56.7%). In the same herd, abortion was significantly more likely in animals with high anti-Neospora antibody titre. Immunoblot analysis demonstrated that the banding pattern from positive Paraguayan cattle was similar to that seen with the positive control sample. In conclusion, N. caninum infection is present among Paraguayan beef and dairy cattle, and it may be an important cause of bovine abortion in Paraguay. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Neospora caninum; ELISA; Paraguay; Seroprevalence
1. Introduction Neospora caninum infection is considered to be one of the major causes of abortions in cattle worldwide (Dubey and Lindsay, 1996). Bovine abortions due to Neospora infection has been reported to result in substantial economic loss to the cattle industry (Dubey, 1999). Thurmond and Hietala (1997) found that Neospora-seropositive dairy cows produced less ∗ Corresponding author. Present address: Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan. Tel.: +81-19-621-6278; fax: +81-19-621-6278. E-mail address: [email protected] (T. Osawa).
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 3 0 9 - 6
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T. Osawa et al. / Veterinary Parasitology 110 (2002) 17–23
milk and had a shortened production life, compared with seronegative herdmates. Barling et al. (2000) reported that Neospora-seropositive status was associated with significant reductions in postweaning weight gain and carcass weight in beef cattle. Cattle farming is one of the most important industries in South America, and the total number of cattle in this continent is nearly 300 million, more than the total number in Europe and North America (FAO, 1996). There have been several reports on N. caninum infection in cattle to date in South America (Campero et al., 1998; Gondim et al., 1999; Venturini et al., 1999; Corbellini et al., 2002). However, more work is required to understand the distribution of infection. In Paraguay, a country located in the middle of the continent, beef is the third largest export and accounts for 20% of the total export (FAO, 1996). No report exists so far on the incidence of neosporosis in Paraguay. We used an enzyme linked immunosorbent assay (ELISA) (Osawa et al., 1998) for a serological prevalence survey in this country, and conducted an immunoblot analysis in order to confirm the validity the ELISA. The objectives of this study were to examine whether N. caninum infection was present in Paraguay and to carry out a preliminary epidemiological study in selected herds in Paraguay. 2. Materials and methods 2.1. Area Paraguay is a landlocked country in South America with hot rainy summers, when temperatures reach over 35 ◦ C and mild winters with an average temperature of 18 ◦ C. Paraguay is located at latitude between 19 and 27◦ south and longitude between 55 and 62◦ west, and has 406,750 km2 (157,055 miles2 ) of land, a human population of 5.0 million, and a cattle population of 8.1 million (FAO, 1996). 2.2. Animals Blood samples were obtained between October 1996 and May 1997 from 879 animals (582 beef and 297 dairy animals) on 10 farms and 1 zone (Zone K) in Paraguay (Table 1). Zone K was composed of 28 small dairy farms from which 197 blood samples were collected (1–19 samples from each farm). Information on abortion history in these groups was not available, except Farm G that had recorded endemic abortion in the previous 3 years. The total number of cows in Farm G was 70, and samples were collected from 30 animals. Fourteen of the 30 cows had no history of abortion and the remaining 16 had aborted previously. One cow had aborted in 1994, three in 1995, eight in 1996 and four in 1997. Some of the aborting cows may have aborted more than once but no supporting data was available. Samples from non-aborting cows (n = 14) were randomly selected from the 54 cows that had no history of abortion. The herd was free from brucellosis and there was no evidence of other potential abortifacient agents such as infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVD), campylobacteriosis, leptospirosis, vibriosis or trichomoniasis. The age of the animals varied from less than 1 year to 10 years old, except two farms (Farm C and E) and Zone K where the age of the animals was not known. Venous blood
T. Osawa et al. / Veterinary Parasitology 110 (2002) 17–23
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Table 1 Bovine serum groups tested for the presence of antibodies to Neospora caninum in Paraguay Group
Region
Type of herd
Breed
Age (years)
Herd size
No. of animals tested
Herd A Herd B
Alto Paraguay Boquer´on
Beef Beef
3–11 0–9
1000a 61
444 61
Herd C Herd D Herd E Herd F Herd G Herd H Herd I Herd J Zone Kb
Boquer´on Presidente Hayes Paraguar´ı Central Central Central Concepci´on Concepci´on Cordillera, Paraguar´ı
Beef Beef Beef Dairy Dairy Dairy Dairy Dairy Dairy
Nelore Brahman, Charolais, St. Gertrudis nd Nelore, Brahman Aberdeen Angus Holstein Holstein, Brown Swiss Holstein Crossbred Holstein Crossbred
nd 4 nd 2.5 3–16 4–8 4–15 3–6 nd
nd nd nd nd 70 15 23 18 nd
48 10 19 25 30 8 22 15 197
Total
–
–
–
–
–
879
nd: no data. a Approximate figure. b K was composed of 28 small dairy farms.
was collected into evacuated blood collection tubes from the coccygeal vessels and serum was separated by centrifugation and stored at −20 ◦ C until assayed. 2.3. ELISA Between April and May 1997, serum samples were tested for the presence of IgG antibodies to N. caninum by an ELISA (Osawa et al., 1998) using water-soluble fraction of sonicated tachyzoites of NC-1 isolate (Dubey et al., 1988), as antigen. Control and sample sera were tested in duplicate. The optical density (OD) values of test samples were expressed as a percentage of a positive control (percent-positive OD, % OD) using the formula described by Buxton et al. (1988). The positive control serum was taken from a calf experimentally infected with N. caninum NC-1 tachyzoites (Osawa, 1997). The cut-off OD value, 0.4 for bovine sera was equivalent to 30% OD. Serum samples showing a percent positive OD equal to or greater than 30% were regarded as positive to indicate a detectable level of antibody titre and those showing equal to or greater than 60% OD were regarded as strongly positive to indicate a very high antibody level. 2.4. Immunoblot analysis Immunoblot analysis (Osawa et al., 1998) was performed on the serum samples taken in this study to detect appropriate Neospora-specific antigens. Proteins were separated by SDS-PAGE on a 12% discontinuous polyacrylamide gel under non-reducing conditions. The ELISA antigen was probed with six bovine serum samples, which were collected in different locations in Paraguay and were positive by the ELISA. The antigen also was probed with two control sera obtained from cattle in Scotland (one positive control serum
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T. Osawa et al. / Veterinary Parasitology 110 (2002) 17–23
in the ELISA and the other one was negative by the ELISA) as well as one bovine sample collected in Paraguay that was diagnosed as negative by the ELISA.
3. Results Of the 879 Paraguayan cattle tested by the ELISA, 262 cattle (29.8%) were seropositive to N. caninum. In all 10 herds as well as Zone K, animals with anti-Neospora antibody titre were observed (Table 2). A herd that exhibited persistent abortion (Herd G) showed the highest percentage (17/30, 56.7%) of animals being seropositive to the parasite. To evaluate an association between anti-Neospora antibody titre and abortion, the percentage of seropositive animals in aborting cows in Herd G, which had persistent abortions, was compared with that in non-aborting cows in the same herd (Table 3). The percentage of animals with an anti-Neospora antibody titre of equal to or more than 30% OD in aborting cows was similar to that of non-aborting cows. However, the percentage of animals with an anti-Neospora antibody titre of equal to or more than 60% OD in aborting cows (i.e. ‘strongly’ positive) was significantly (P < 0.05; using Fisher’s exact probability test) higher than that in non-aborting cows (50.0% versus 14.3%). Table 2 Seroprevalence of Neospora caninum in beef and dairy herds in Paraguay Group
Sample size
No. of seropositive animals at 30% OD (% of sample size)
No. of seropositive animals at 60% OD (% of sample size)
Herd A Herd B Herd C Herd D Herd E Herd F Herd G Herd H Herd I Herd J Zone K
444 61 48 10 19 25 30 8 22 15 197
134 (30.2) 9 (14.8) 8 (16.7) 2 (20.0) 2 (10.5) 5 (20.0) 17 (56.7) 4 (50.0) 5 (22.7) 2 (13.3) 74 (35.6)
58 (13.1) 6 (9.8) 5 (10.4) 1 (10.0) 1 (5.3) 2 (8.0) 10 (33.3) 3 (37.5) 4 (18.2) 2 (13.3) 48 (24.4)
Total
879
262 (29.8)
140 (15.9)
OD: optical density. Table 3 Percentage of animals being positive (≥30% OD) and strongly positive (≥60% OD) to N. caninum in aborting and non-aborting cows in Herd G with a history of persistent abortion in Paraguay Animals
n
No. of positive (≥30% OD) (%)
No. of strongly positive (≥60% OD) (%)
Aborting cows Non-aborting cows
(16) (14)
10 (62.5) 7 (50.0)
8 (50.0∗ ) 2 (14.3∗ )
∗
A significant difference in strongly positive rate was observed between aborting cows and non-aborting cows by Fisher’s exact probability test (P < 0.05).
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Fig. 1. Neospora caninum (NC-1) tachyzoite immunoblots (14.4 g/lane) of sera (diluted 1:500) from Paraguayan cattle. Sera were diagnosed as positive (lanes 3–8) and negative (lane 9) by the ELISA. The serum samples were collected from a beef cow in Herd A (lane 3) and from dairy cows in Zone K (lanes 4 and 5), Herd G (lane 6), Herd H (lane 7), and Herd J (lanes 8 and 9). Lane 1 and 2: sera from Scottish cattle. Serum was taken from a calf experimentally infected with NC-1 tachyzoites and used as positive control in the ELISA (lane 1). The other serum was taken from a cow that had no history of abortion and diagnosed as negative by the ELISA (lane 2).
The result of the immunoblot analysis is shown in Fig. 1. All seropositive sera from Paraguayan cattle showed strong bands at about 19 and 36 kDa. The banding pattern of the immunoblots from positive Paraguayan cattle was similar to that seen with the positive control sera.
4. Discussion The seroprevalence for ten selected farms and one zone from seven different regions of Paraguay varied between 10.5 and 56.7%, and the overall estimated seroprevalence of N. caninum infection in the herds examined was 29.8%. The results strongly suggest that the parasite is widely spread among beef and dairy cattle in Paraguay. The figure of 29.8% is of a similar magnitude as that reported in dairy herds in the United States (Anderson et al., 1995) and Costa Rica (Romero et al., 2002). However, this should not be taken as the overall seroprevalence of N. caninum in Paraguay since sampling was not strictly random and did not adequately cover the entire country. In this study, strongly positive animals with anti-Neospora antibody titre of equal to or more than 60% OD were observed in all of the 10 herds and zone. Since the ELISA has been shown not to detect antibodies to Babesia spp. (Osawa et al., 1998), the high prevalence of babesiosis in this country (Payne and Osorio, 1990) should not have confounded the data here. Moreover, immunoblot analysis revealed that the sera from Paraguayan cattle that were diagnosed as positive in the ELISA showed a similar-pattern of response to positive control serum collected from a calf experimentally infected with N. caninum, NC-1 isolate tachyzoites. It has been reported that the antigens with apparent molecular weights of 19 and 36 kDa, which were revealed in this study, are likely to correspond to surface antigens
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T. Osawa et al. / Veterinary Parasitology 110 (2002) 17–23
of Neospora tachyzoites (Hemphill et al., 1997; Schares et al., 1999), and that the molecule of 36 kDa was present in Neospora, but was absent in Toxoplasma tachyzoites (Hemphill et al., 1997). Immunoblot analysis, therefore, confirmed the validity of the ELISA results, indicating that the seropositive animals diagnosed by ELISA had indeed been exposed to N. caninum. The percentage of animals that were ‘strongly positive’ to N. caninum in aborting cows in Herd G was significantly higher than that in non-aborting cows in the same herd. However, there was no significant difference between the two groups at an ELISA cut-off point of equal to or more than 30% OD. In this study ‘strongly positive’ was defined as an anti-Neospora antibody titre of equal to or more than 60% OD. Although this definition was arbitrary and the selected cut-off point of equal to or greater than 30% OD is likely to indicate the previous exposure of the animal to N. caninum, the higher level of antibody titre 60% OD may be more useful as an indicator of disease. A possible explanation for the fact that many non-aborting cows were seropositive relates to the pathogenesis of the disease and the host immune system. Cows with an anti-Neospora antibody titre of equal to or more than 30% OD but less than 60% OD may have been infected with the parasite, but the level of infection, i.e. number of N. caninum tachyzoites in the host tissue, may not have been high enough to cause clinical symptoms, i.e. abortion, or it could indicate exposure to infection sometime previously and the antibody response has since waned (Conrad et al., 1993; Sager et al., 2001). The finding, that there was an association between abortion and animals strongly positive to N. caninum, is similar to that obtained in a study examining a dairy herd in Scotland (Osawa, 1997), and it gives additional evidence that a high antibody titre may be a good indicator of possible clinical problems. Although no aborted material was examined directly for the presence of N. caninum, the high prevalence of anti-Neospora antibody in the serum samples collected from animals with an abortion history with no other identifiable cause suggests the possible involvement of this parasite in the abortion endemic.
Acknowledgements The authors would like to thank Dr. Faustino Benitez and his colleagues at the Faculty of Veterinary Sciences, Concepción, National University of Asunción, Paraguay, Dr. Nidia Ferreira and her colleagues at Laboratorio de Investigación y Diagnóstico Veterinario, San Lorenzo, Paraguay, and Dr. Yukari Koyama, Japan Overseas Cooperation Volunteers, Asunción, Paraguay, for their assistance in collecting the samples used in this study. This study was supported by Japan International Cooperation Agency (JICA), and in part by the Faculty of Veterinary Medicine, the University of Edinburgh (Birrell-Gray Travelling Scholarship). References Anderson, M.L., Palmer, C.W., Thurmond, M.C., Picanso, J.P., Blanchard, P.C., Breitmeyer, R.E., Layton, A.W., MacAllister, M., Daft, B., Kinde, H., Read, D.H., Dubey, J.P., Conrad, P.A., Barr, B.C., 1995. Evaluation of abortions in cattle attributable to neosporosis in selected dairy herds in California. J. Am. Vet. Med. Assoc. 207, 1206–1210.
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Barling, K.S., McNeill, J.W., Thompson, J.A., Paschal, J.C., McCollum III, F.T., Craig, T.M., Adams, L.G., 2000. Association of serologic status for Neospora caninum with postweaning weight gain and carcass measurements in beef calves. J. Am. Vet. Med. Assoc. 217, 1356–1360. Buxton, D., Blewett, D.A., Trees, A.J., McColgan, C., Finlayson, J., 1988. Further studies in the use of monensin in the control of experimental ovine toxoplasmosis. J. Comp. Pathol. 98, 225–236. Campero, C.M., Anderson, M.L., Conosciuto, G., Odriozola, H., Bretschneider, G., Poso, M.A., 1998. Neospora caninum-associated abortion in a dairy herd in Argentina. Vet. Rec. 143, 228–229. Corbellini, L.G., Driemeier, D., Cruz, C.F., Gondim, L.F., Wald, V., 2002. Neosporosis as a cause of abortion in dairy cattle in Rio Grande do Sul, southern Brazil. Vet. Parasitol. 103, 195–202. Conrad, P.A., Sverlow, K., Anderson, M., Rowe, J., BonDurant, R., Tuter, G., Breitmeyer, R., Palmer, C., Thurmond, M., Ardans, A., Dubey, J.P., Duhamel, G., Barr, B., 1993. Detection of serum antibody responses in cattle with natural or experimental Neospora infections. J. Vet. Diagn. Invest. 5, 572–578. Dubey, J.P., 1999. Recent advances in Neospora and neosporosis. Vet. Parasitol. 84, 349–367. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1–59. Dubey, J.P., Hattel, A.L., Lindsay, D.S., Topper, M.J., 1988. Neonatal Neospora caninum infection in dogs: isolation of the causative agent and experimental transmission. J. Am. Vet. Med. Assoc. 193, 1259–1263. FAO 1996. FAO Yearbook: Production, vol. 49, Food and Agriculture Organization of the United Nations, Rome. Gondim, L.F.P., Sartor, I.F., Hasegawa, M., Yamane, I., 1999. Seroprevalence of Neospora caninum in dairy cattle in Bahia. Brazil. Vet. Parasitol. 86, 71–75. Hemphill, A., Fuchs, N., Sonda, S., Gottstein, B., Hentrich, B., 1997. Identification and partial characterization of a 36 kDa surface protein on Neospora caninum tachyzoites. Parasitology 115, 371–380. Osawa, T., 1997. Development of an ELISA to detect antibodies to Neospora caninum in cattle, sheep, goats, and its use in epidemiological studies. Master of Philosophy Thesis, The University of Edinburgh. Osawa, T., Wastling, J., Maley, S., Buxton, D., Innes, E.A., 1998. A multiple antigen ELISA to detect Neospora-specific antibodies in bovine sera, bovine foetal fluids, ovine and caprine sera, ovine and caprine sera. Vet. Parasitol. 79, 19–34. Payne, R.C., Osorio, O., 1990. Tick-borne diseases of cattle in Paraguay. Part I. Seroepidemiological studies on anaplasmosis and babesiosis. Trop. Anim. Health Prod. 22, 53–60. Romero, J.J., Perez, E., Dolz, G., Frankena, K., 2002. Factors associated with Neospora caninum serostatus in cattle of 20 specialised Costa Rican dairy herds. Prev. Vet. Med. 53, 263–273. Sager, H., Fischer, I., Furrer, K., Strasser, M., Waldvogel, A., Boerlin, P., Audige, L., Gottstein, B., 2001. A Swiss case-control study to assess Neospora caninum-associated bovine abortions by PCR, histopathology and serology. Vet. Parasitol. 102, 1–15. Schares, G., Dubremetz, J.F., Dubey, J.P., Barwald, A., Loyens, A., Conraths, F.J., 1999. Neospora caninum: identification of 19-, 38-, and 40-kDa surface antigens and a 33-kDa dense granule antigen using monoclonal antibodies. Exp. Parasitol. 92, 109–119. Thurmond, M.C., Hietala, S.K., 1997. Effect of Neospora caninum infection on milk production in first-lactation dairy cows. J. Am. Vet. Med. Assoc. 210, 672–674. Venturini, M.C., Venturini, L., Bacigalupe, D., Machuca, M., Echaide, I., Basso, W., Unzaga, J.M., Di Lorenzo, C., Guglielmore, A., Jenkins, M.C., Dubey, J.P., 1999. Neospora caninum infections in bovine foetuses and dairy cows with abortions in Argentina. Int. J. Parasitol. 29, 1705–1708.
Seroprevalence of Neospora caninum infection in dairy and beef cattle in Paraguay Takeshi Osawa a,∗ , Jonathan Wastling b , Ladislao Acosta c , Carlos Ortellado c , Julio Ibarra c , Elisabeth A. Innes a a
b
Moredun Research Institute, International Research Centre, Pentland Science Park, Bush Loan, Penicuik EH26 OPZ, UK Institute of Biomedical and Life Sciences, Division of Infection and Immunity, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK c Laboratorio de Investigación y Diagnostico Veterinario, San Lorenzo, Paraguay, UK Received 2 July 2002; received in revised form 11 September 2002; accepted 11 September 2002
Abstract Seroprevalence of Neospora caninum in 879 beef and dairy cattle in different locations of Paraguay was determined by an ELISA. In the survey, 262 (29.8%) cattle were positive to N. caninum, and animals with anti-Neospora antibody titre were observed in all the locations tested in the country. Serum samples taken from a herd that exhibited persistent abortion had the highest percentage of animals being positive to the parasite (17/30, 56.7%). In the same herd, abortion was significantly more likely in animals with high anti-Neospora antibody titre. Immunoblot analysis demonstrated that the banding pattern from positive Paraguayan cattle was similar to that seen with the positive control sample. In conclusion, N. caninum infection is present among Paraguayan beef and dairy cattle, and it may be an important cause of bovine abortion in Paraguay. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Neospora caninum; ELISA; Paraguay; Seroprevalence
1. Introduction Neospora caninum infection is considered to be one of the major causes of abortions in cattle worldwide (Dubey and Lindsay, 1996). Bovine abortions due to Neospora infection has been reported to result in substantial economic loss to the cattle industry (Dubey, 1999). Thurmond and Hietala (1997) found that Neospora-seropositive dairy cows produced less ∗ Corresponding author. Present address: Department of Veterinary Medicine, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan. Tel.: +81-19-621-6278; fax: +81-19-621-6278. E-mail address: [email protected] (T. Osawa).
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 3 0 9 - 6
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milk and had a shortened production life, compared with seronegative herdmates. Barling et al. (2000) reported that Neospora-seropositive status was associated with significant reductions in postweaning weight gain and carcass weight in beef cattle. Cattle farming is one of the most important industries in South America, and the total number of cattle in this continent is nearly 300 million, more than the total number in Europe and North America (FAO, 1996). There have been several reports on N. caninum infection in cattle to date in South America (Campero et al., 1998; Gondim et al., 1999; Venturini et al., 1999; Corbellini et al., 2002). However, more work is required to understand the distribution of infection. In Paraguay, a country located in the middle of the continent, beef is the third largest export and accounts for 20% of the total export (FAO, 1996). No report exists so far on the incidence of neosporosis in Paraguay. We used an enzyme linked immunosorbent assay (ELISA) (Osawa et al., 1998) for a serological prevalence survey in this country, and conducted an immunoblot analysis in order to confirm the validity the ELISA. The objectives of this study were to examine whether N. caninum infection was present in Paraguay and to carry out a preliminary epidemiological study in selected herds in Paraguay. 2. Materials and methods 2.1. Area Paraguay is a landlocked country in South America with hot rainy summers, when temperatures reach over 35 ◦ C and mild winters with an average temperature of 18 ◦ C. Paraguay is located at latitude between 19 and 27◦ south and longitude between 55 and 62◦ west, and has 406,750 km2 (157,055 miles2 ) of land, a human population of 5.0 million, and a cattle population of 8.1 million (FAO, 1996). 2.2. Animals Blood samples were obtained between October 1996 and May 1997 from 879 animals (582 beef and 297 dairy animals) on 10 farms and 1 zone (Zone K) in Paraguay (Table 1). Zone K was composed of 28 small dairy farms from which 197 blood samples were collected (1–19 samples from each farm). Information on abortion history in these groups was not available, except Farm G that had recorded endemic abortion in the previous 3 years. The total number of cows in Farm G was 70, and samples were collected from 30 animals. Fourteen of the 30 cows had no history of abortion and the remaining 16 had aborted previously. One cow had aborted in 1994, three in 1995, eight in 1996 and four in 1997. Some of the aborting cows may have aborted more than once but no supporting data was available. Samples from non-aborting cows (n = 14) were randomly selected from the 54 cows that had no history of abortion. The herd was free from brucellosis and there was no evidence of other potential abortifacient agents such as infectious bovine rhinotracheitis (IBR), bovine viral diarrhea (BVD), campylobacteriosis, leptospirosis, vibriosis or trichomoniasis. The age of the animals varied from less than 1 year to 10 years old, except two farms (Farm C and E) and Zone K where the age of the animals was not known. Venous blood
T. Osawa et al. / Veterinary Parasitology 110 (2002) 17–23
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Table 1 Bovine serum groups tested for the presence of antibodies to Neospora caninum in Paraguay Group
Region
Type of herd
Breed
Age (years)
Herd size
No. of animals tested
Herd A Herd B
Alto Paraguay Boquer´on
Beef Beef
3–11 0–9
1000a 61
444 61
Herd C Herd D Herd E Herd F Herd G Herd H Herd I Herd J Zone Kb
Boquer´on Presidente Hayes Paraguar´ı Central Central Central Concepci´on Concepci´on Cordillera, Paraguar´ı
Beef Beef Beef Dairy Dairy Dairy Dairy Dairy Dairy
Nelore Brahman, Charolais, St. Gertrudis nd Nelore, Brahman Aberdeen Angus Holstein Holstein, Brown Swiss Holstein Crossbred Holstein Crossbred
nd 4 nd 2.5 3–16 4–8 4–15 3–6 nd
nd nd nd nd 70 15 23 18 nd
48 10 19 25 30 8 22 15 197
Total
–
–
–
–
–
879
nd: no data. a Approximate figure. b K was composed of 28 small dairy farms.
was collected into evacuated blood collection tubes from the coccygeal vessels and serum was separated by centrifugation and stored at −20 ◦ C until assayed. 2.3. ELISA Between April and May 1997, serum samples were tested for the presence of IgG antibodies to N. caninum by an ELISA (Osawa et al., 1998) using water-soluble fraction of sonicated tachyzoites of NC-1 isolate (Dubey et al., 1988), as antigen. Control and sample sera were tested in duplicate. The optical density (OD) values of test samples were expressed as a percentage of a positive control (percent-positive OD, % OD) using the formula described by Buxton et al. (1988). The positive control serum was taken from a calf experimentally infected with N. caninum NC-1 tachyzoites (Osawa, 1997). The cut-off OD value, 0.4 for bovine sera was equivalent to 30% OD. Serum samples showing a percent positive OD equal to or greater than 30% were regarded as positive to indicate a detectable level of antibody titre and those showing equal to or greater than 60% OD were regarded as strongly positive to indicate a very high antibody level. 2.4. Immunoblot analysis Immunoblot analysis (Osawa et al., 1998) was performed on the serum samples taken in this study to detect appropriate Neospora-specific antigens. Proteins were separated by SDS-PAGE on a 12% discontinuous polyacrylamide gel under non-reducing conditions. The ELISA antigen was probed with six bovine serum samples, which were collected in different locations in Paraguay and were positive by the ELISA. The antigen also was probed with two control sera obtained from cattle in Scotland (one positive control serum
20
T. Osawa et al. / Veterinary Parasitology 110 (2002) 17–23
in the ELISA and the other one was negative by the ELISA) as well as one bovine sample collected in Paraguay that was diagnosed as negative by the ELISA.
3. Results Of the 879 Paraguayan cattle tested by the ELISA, 262 cattle (29.8%) were seropositive to N. caninum. In all 10 herds as well as Zone K, animals with anti-Neospora antibody titre were observed (Table 2). A herd that exhibited persistent abortion (Herd G) showed the highest percentage (17/30, 56.7%) of animals being seropositive to the parasite. To evaluate an association between anti-Neospora antibody titre and abortion, the percentage of seropositive animals in aborting cows in Herd G, which had persistent abortions, was compared with that in non-aborting cows in the same herd (Table 3). The percentage of animals with an anti-Neospora antibody titre of equal to or more than 30% OD in aborting cows was similar to that of non-aborting cows. However, the percentage of animals with an anti-Neospora antibody titre of equal to or more than 60% OD in aborting cows (i.e. ‘strongly’ positive) was significantly (P < 0.05; using Fisher’s exact probability test) higher than that in non-aborting cows (50.0% versus 14.3%). Table 2 Seroprevalence of Neospora caninum in beef and dairy herds in Paraguay Group
Sample size
No. of seropositive animals at 30% OD (% of sample size)
No. of seropositive animals at 60% OD (% of sample size)
Herd A Herd B Herd C Herd D Herd E Herd F Herd G Herd H Herd I Herd J Zone K
444 61 48 10 19 25 30 8 22 15 197
134 (30.2) 9 (14.8) 8 (16.7) 2 (20.0) 2 (10.5) 5 (20.0) 17 (56.7) 4 (50.0) 5 (22.7) 2 (13.3) 74 (35.6)
58 (13.1) 6 (9.8) 5 (10.4) 1 (10.0) 1 (5.3) 2 (8.0) 10 (33.3) 3 (37.5) 4 (18.2) 2 (13.3) 48 (24.4)
Total
879
262 (29.8)
140 (15.9)
OD: optical density. Table 3 Percentage of animals being positive (≥30% OD) and strongly positive (≥60% OD) to N. caninum in aborting and non-aborting cows in Herd G with a history of persistent abortion in Paraguay Animals
n
No. of positive (≥30% OD) (%)
No. of strongly positive (≥60% OD) (%)
Aborting cows Non-aborting cows
(16) (14)
10 (62.5) 7 (50.0)
8 (50.0∗ ) 2 (14.3∗ )
∗
A significant difference in strongly positive rate was observed between aborting cows and non-aborting cows by Fisher’s exact probability test (P < 0.05).
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Fig. 1. Neospora caninum (NC-1) tachyzoite immunoblots (14.4 g/lane) of sera (diluted 1:500) from Paraguayan cattle. Sera were diagnosed as positive (lanes 3–8) and negative (lane 9) by the ELISA. The serum samples were collected from a beef cow in Herd A (lane 3) and from dairy cows in Zone K (lanes 4 and 5), Herd G (lane 6), Herd H (lane 7), and Herd J (lanes 8 and 9). Lane 1 and 2: sera from Scottish cattle. Serum was taken from a calf experimentally infected with NC-1 tachyzoites and used as positive control in the ELISA (lane 1). The other serum was taken from a cow that had no history of abortion and diagnosed as negative by the ELISA (lane 2).
The result of the immunoblot analysis is shown in Fig. 1. All seropositive sera from Paraguayan cattle showed strong bands at about 19 and 36 kDa. The banding pattern of the immunoblots from positive Paraguayan cattle was similar to that seen with the positive control sera.
4. Discussion The seroprevalence for ten selected farms and one zone from seven different regions of Paraguay varied between 10.5 and 56.7%, and the overall estimated seroprevalence of N. caninum infection in the herds examined was 29.8%. The results strongly suggest that the parasite is widely spread among beef and dairy cattle in Paraguay. The figure of 29.8% is of a similar magnitude as that reported in dairy herds in the United States (Anderson et al., 1995) and Costa Rica (Romero et al., 2002). However, this should not be taken as the overall seroprevalence of N. caninum in Paraguay since sampling was not strictly random and did not adequately cover the entire country. In this study, strongly positive animals with anti-Neospora antibody titre of equal to or more than 60% OD were observed in all of the 10 herds and zone. Since the ELISA has been shown not to detect antibodies to Babesia spp. (Osawa et al., 1998), the high prevalence of babesiosis in this country (Payne and Osorio, 1990) should not have confounded the data here. Moreover, immunoblot analysis revealed that the sera from Paraguayan cattle that were diagnosed as positive in the ELISA showed a similar-pattern of response to positive control serum collected from a calf experimentally infected with N. caninum, NC-1 isolate tachyzoites. It has been reported that the antigens with apparent molecular weights of 19 and 36 kDa, which were revealed in this study, are likely to correspond to surface antigens
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T. Osawa et al. / Veterinary Parasitology 110 (2002) 17–23
of Neospora tachyzoites (Hemphill et al., 1997; Schares et al., 1999), and that the molecule of 36 kDa was present in Neospora, but was absent in Toxoplasma tachyzoites (Hemphill et al., 1997). Immunoblot analysis, therefore, confirmed the validity of the ELISA results, indicating that the seropositive animals diagnosed by ELISA had indeed been exposed to N. caninum. The percentage of animals that were ‘strongly positive’ to N. caninum in aborting cows in Herd G was significantly higher than that in non-aborting cows in the same herd. However, there was no significant difference between the two groups at an ELISA cut-off point of equal to or more than 30% OD. In this study ‘strongly positive’ was defined as an anti-Neospora antibody titre of equal to or more than 60% OD. Although this definition was arbitrary and the selected cut-off point of equal to or greater than 30% OD is likely to indicate the previous exposure of the animal to N. caninum, the higher level of antibody titre 60% OD may be more useful as an indicator of disease. A possible explanation for the fact that many non-aborting cows were seropositive relates to the pathogenesis of the disease and the host immune system. Cows with an anti-Neospora antibody titre of equal to or more than 30% OD but less than 60% OD may have been infected with the parasite, but the level of infection, i.e. number of N. caninum tachyzoites in the host tissue, may not have been high enough to cause clinical symptoms, i.e. abortion, or it could indicate exposure to infection sometime previously and the antibody response has since waned (Conrad et al., 1993; Sager et al., 2001). The finding, that there was an association between abortion and animals strongly positive to N. caninum, is similar to that obtained in a study examining a dairy herd in Scotland (Osawa, 1997), and it gives additional evidence that a high antibody titre may be a good indicator of possible clinical problems. Although no aborted material was examined directly for the presence of N. caninum, the high prevalence of anti-Neospora antibody in the serum samples collected from animals with an abortion history with no other identifiable cause suggests the possible involvement of this parasite in the abortion endemic.
Acknowledgements The authors would like to thank Dr. Faustino Benitez and his colleagues at the Faculty of Veterinary Sciences, Concepción, National University of Asunción, Paraguay, Dr. Nidia Ferreira and her colleagues at Laboratorio de Investigación y Diagnóstico Veterinario, San Lorenzo, Paraguay, and Dr. Yukari Koyama, Japan Overseas Cooperation Volunteers, Asunción, Paraguay, for their assistance in collecting the samples used in this study. This study was supported by Japan International Cooperation Agency (JICA), and in part by the Faculty of Veterinary Medicine, the University of Edinburgh (Birrell-Gray Travelling Scholarship). References Anderson, M.L., Palmer, C.W., Thurmond, M.C., Picanso, J.P., Blanchard, P.C., Breitmeyer, R.E., Layton, A.W., MacAllister, M., Daft, B., Kinde, H., Read, D.H., Dubey, J.P., Conrad, P.A., Barr, B.C., 1995. Evaluation of abortions in cattle attributable to neosporosis in selected dairy herds in California. J. Am. Vet. Med. Assoc. 207, 1206–1210.
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