Veterinary Parasitology 110 (2002) 17–23

Seroprevalence ofNeospora caninum infectionin dairy and beef cattle in Paraguay

Takeshi Osawaa,∗, Jonathan Wastlingb, Ladislao Acostac,Carlos Ortelladoc, Julio Ibarrac, Elisabeth A. Innesaa Moredun Research Institute, International Research Centre, Pentland Science Park,

Bush Loan, Penicuik EH26 OPZ, UKb Institute of Biomedical and Life Sciences, Division of Infection and Immunity, Joseph Black Building,

University of Glasgow, Glasgow G12 8QQ, UKc 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 ofNeospora caninum in 879 beef and dairy cattle in different locations ofParaguay was determined by an ELISA. In the survey, 262 (29.8%) cattle were positive toN.caninum, and animals with anti-Neospora antibody titre were observed in all the locations testedin the country. Serum samples taken from a herd that exhibited persistent abortion had the highestpercentage of animals being positive to the parasite (17/30, 56.7%). In the same herd, abortion wassignificantly more likely in animals with high anti-Neospora antibody titre. Immunoblot analysisdemonstrated that the banding pattern from positive Paraguayan cattle was similar to that seen withthe positive control sample. In conclusion,N. caninum infection is present among Paraguayan beefand 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 incattle worldwide (Dubey and Lindsay, 1996). Bovine abortions due toNeospora infectionhas been reported to result in substantial economic loss to the cattle industry (Dubey, 1999).Thurmond and Hietala (1997)found thatNeospora-seropositive dairy cows produced less

∗ Corresponding author. Present address: Department of Veterinary Medicine, Faculty of Agriculture, IwateUniversity, Ueda 3-18-8, Morioka 020-8550, Japan. Tel.:+81-19-621-6278; fax:+81-19-621-6278.E-mail address: osawa@iwate-u.ac.jp (T. Osawa).

0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S0304-4017(02)00309-6

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milk and had a shortened production life, compared with seronegative herdmates.Barlinget al. (2000)reported thatNeospora-seropositive status was associated with significantreductions in postweaning weight gain and carcass weight in beef cattle.

Cattle farming is one of the most important industries in South America, and the totalnumber of cattle in this continent is nearly 300 million, more than the total number inEurope and North America (FAO, 1996). There have been several reports onN. caninuminfection 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 understandthe 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). Noreport exists so far on the incidence of neosporosis in Paraguay. We used an enzyme linkedimmunosorbent assay (ELISA) (Osawa et al., 1998) for a serological prevalence survey inthis country, and conducted an immunoblot analysis in order to confirm the validity theELISA.

The objectives of this study were to examine whetherN. caninum infection was present inParaguay 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 tem-peratures reach over 35◦C and mild winters with an average temperature of 18◦C. Paraguayis located at latitude between 19 and 27◦ south and longitude between 55 and 62◦ west, andhas 406,750 km2 (157,055 miles2) of land, a human population of 5.0 million, and a cattlepopulation 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 notavailable, except Farm G that had recorded endemic abortion in the previous 3 years. Thetotal 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 abortedpreviously. One cow had aborted in 1994, three in 1995, eight in 1996 and four in 1997. Someof 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 thathad no history of abortion. The herd was free from brucellosis and there was no evidence ofother potential abortifacient agents such as infectious bovine rhinotracheitis (IBR), bovineviral 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 19

Table 1Bovine serum groups tested for the presence of antibodies toNeospora caninum in Paraguay

Group Region Type ofherd

Breed Age(years)

Herdsize

No. of animalstested

Herd A Alto Paraguay Beef Nelore 3–11 1000a 444Herd B Boqueron Beef Brahman, Charolais,

St. Gertrudis0–9 61 61

Herd C Boqueron Beef nd nd nd 48Herd D Presidente Hayes Beef Nelore, Brahman 4 nd 10Herd E Paraguarı Beef Aberdeen Angus nd nd 19Herd F Central Dairy Holstein 2.5 nd 25Herd G Central Dairy Holstein, Brown Swiss 3–16 70 30Herd H Central Dairy Holstein 4–8 15 8Herd I Concepcion Dairy Crossbred 4–15 23 22Herd J Concepcion Dairy Holstein 3–6 18 15Zone Kb Cordillera,

ParaguarıDairy Crossbred nd nd 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 serumwas 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 an-tibodies toN. caninum by an ELISA (Osawa et al., 1998) using water-soluble fraction ofsonicated tachyzoites of NC-1 isolate (Dubey et al., 1988), as antigen. Control and sam-ple sera were tested in duplicate. The optical density (OD) values of test samples wereexpressed as a percentage of a positive control (percent-positive OD, % OD) using the for-mula described byBuxton et al. (1988). The positive control serum was taken from a calfexperimentally infected withN. caninum NC-1 tachyzoites (Osawa, 1997). The cut-off ODvalue, 0.4 for bovine sera was equivalent to 30% OD. Serum samples showing a percentpositive OD equal to or greater than 30% were regarded as positive to indicate a detectablelevel of antibody titre and those showing equal to or greater than 60% OD were regardedas 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 takenin this study to detect appropriateNeospora-specific antigens. Proteins were separated bySDS-PAGE on a 12% discontinuous polyacrylamide gel under non-reducing conditions.The ELISA antigen was probed with six bovine serum samples, which were collected indifferent locations in Paraguay and were positive by the ELISA. The antigen also wasprobed with two control sera obtained from cattle in Scotland (one positive control serum

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in the ELISA and the other one was negative by the ELISA) as well as one bovine samplecollected 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 seropositiveto N. caninum. In all 10 herds as well as Zone K, animals with anti-Neospora antibody titrewere observed (Table 2). A herd that exhibited persistent abortion (Herd G) showed thehighest percentage (17/30, 56.7%) of animals being seropositive to the parasite.

To evaluate an association between anti-Neospora antibody titre and abortion, the per-centage 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 percentageof animals with an anti-Neospora antibody titre of equal to or more than 30% OD in abort-ing cows was similar to that of non-aborting cows. However, the percentage of animalswith 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 2Seroprevalence ofNeospora caninum in beef and dairy herds in Paraguay

Group Sample size No. of seropositive animals at30% OD (% of sample size)

No. of seropositive animals at60% OD (% of sample size)

Herd A 444 134 (30.2) 58 (13.1)Herd B 61 9 (14.8) 6 (9.8)Herd C 48 8 (16.7) 5 (10.4)Herd D 10 2 (20.0) 1 (10.0)Herd E 19 2 (10.5) 1 (5.3)Herd F 25 5 (20.0) 2 (8.0)Herd G 30 17 (56.7) 10 (33.3)Herd H 8 4 (50.0) 3 (37.5)Herd I 22 5 (22.7) 4 (18.2)Herd J 15 2 (13.3) 2 (13.3)Zone K 197 74 (35.6) 48 (24.4)

Total 879 262 (29.8) 140 (15.9)

OD: optical density.

Table 3Percentage of animals being positive (≥30% OD) and strongly positive (≥60% OD) toN. caninum in abortingand 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 (16) 10 (62.5) 8 (50.0∗)Non-aborting cows (14) 7 (50.0) 2 (14.3∗)

∗ A significant difference in strongly positive rate was observed between aborting cows and non-aborting cowsby Fisher’s exact probability test (P < 0.05).

T. Osawa et al. / Veterinary Parasitology 110 (2002) 17–23 21

Fig. 1.Neospora caninum (NC-1) tachyzoite immunoblots (14.4�g/lane) of sera (diluted 1:500) from Paraguayancattle. Sera were diagnosed as positive (lanes 3–8) and negative (lane 9) by the ELISA. The serum samples werecollected 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 acalf experimentally infected with NC-1 tachyzoites and used as positive control in the ELISA (lane 1). The otherserum 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 inFig. 1. All seropositive sera fromParaguayan cattle showed strong bands at about 19 and 36 kDa. The banding pattern ofthe immunoblots from positive Paraguayan cattle was similar to that seen with the positivecontrol sera.

4. Discussion

The seroprevalence for ten selected farms and one zone from seven different regions ofParaguay varied between 10.5 and 56.7%, and the overall estimated seroprevalence ofN.caninum infection in the herds examined was 29.8%. The results strongly suggest that theparasite is widely spread among beef and dairy cattle in Paraguay. The figure of 29.8% isof 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 overallseroprevalence ofN. caninum in Paraguay since sampling was not strictly random and didnot adequately cover the entire country.

In this study, strongly positive animals with anti-Neospora antibody titre of equal to ormore than 60% OD were observed in all of the 10 herds and zone. Since the ELISA has beenshown not to detect antibodies toBabesia spp. (Osawa et al., 1998), the high prevalence ofbabesiosis in this country (Payne and Osorio, 1990) should not have confounded the datahere. Moreover, immunoblot analysis revealed that the sera from Paraguayan cattle thatwere diagnosed as positive in the ELISA showed a similar-pattern of response to positivecontrol serum collected from a calf experimentally infected withN. caninum, NC-1 isolatetachyzoites. It has been reported that the antigens with apparent molecular weights of 19and 36 kDa, which were revealed in this study, are likely to correspond to surface antigens

22 T. Osawa et al. / Veterinary Parasitology 110 (2002) 17–23

of Neospora tachyzoites (Hemphill et al., 1997; Schares et al., 1999), and that the moleculeof 36 kDa was present inNeospora, but was absent inToxoplasma tachyzoites (Hemphillet al., 1997). Immunoblot analysis, therefore, confirmed the validity of the ELISA results,indicating that the seropositive animals diagnosed by ELISA had indeed been exposed toN. caninum.

The percentage of animals that were ‘strongly positive’ toN. caninum in aborting cows inHerd 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 equalto or more than 30% OD. In this study ‘strongly positive’ was defined as an anti-Neosporaantibody titre of equal to or more than 60% OD. Although this definition was arbitraryand the selected cut-off point of equal to or greater than 30% OD is likely to indicate theprevious exposure of the animal toN. caninum, the higher level of antibody titre 60% ODmay be more useful as an indicator of disease. A possible explanation for the fact that manynon-aborting cows were seropositive relates to the pathogenesis of the disease and the hostimmune 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 ofN. caninum tachyzoites in the host tissue, may not have been high enough tocause clinical symptoms, i.e. abortion, or it could indicate exposure to infection sometimepreviously 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 stronglypositive toN. caninum, is similar to that obtained in a study examining a dairy herd inScotland (Osawa, 1997), and it gives additional evidence that a high antibody titre may bea good indicator of possible clinical problems. Although no aborted material was examineddirectly for the presence ofN. caninum, the high prevalence of anti-Neospora antibody inthe serum samples collected from animals with an abortion history with no other identifiablecause 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 Facultyof Veterinary Sciences, Concepción, National University of Asunción, Paraguay, Dr. Ni-dia 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. Thisstudy was supported by Japan International Cooperation Agency (JICA), and in part bythe Faculty of Veterinary Medicine, the University of Edinburgh (Birrell-Gray TravellingScholarship).

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 ofabortions in cattle attributable to neosporosis in selected dairy herds in California. J. Am. Vet. Med. Assoc.207, 1206–1210.

T. Osawa et al. / Veterinary Parasitology 110 (2002) 17–23 23

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 forNeospora caninum with postweaning weight gain and carcass measurementsin 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 monensinin 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.Neosporacaninum-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 indairy 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 withnatural or experimentalNeospora infections. J. Vet. Diagn. Invest. 5, 572–578.

Dubey, J.P., 1999. Recent advances inNeospora and neosporosis. Vet. Parasitol. 84, 349–367.Dubey, J.P., Lindsay, D.S., 1996. A review ofNeospora caninum and neosporosis. Vet. Parasitol. 67, 1–59.Dubey, J.P., Hattel, A.L., Lindsay, D.S., Topper, M.J., 1988. NeonatalNeospora 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 ofNeospora 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 onNeospora caninum tachyzoites. Parasitology 115, 371–380.Osawa, T., 1997. Development of an ELISA to detect antibodies toNeospora 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 caprinesera. Vet. Parasitol. 79, 19–34.

Payne, R.C., Osorio, O., 1990. Tick-borne diseases of cattle in Paraguay. Part I. Seroepidemiological studies onanaplasmosis and babesiosis. Trop. Anim. Health Prod. 22, 53–60.

Romero, J.J., Perez, E., Dolz, G., Frankena, K., 2002. Factors associated withNeospora caninum serostatus incattle 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. ASwiss case-control study to assessNeospora caninum-associated bovine abortions by PCR, histopathology andserology. 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 monoclonalantibodies. Exp. Parasitol. 92, 109–119.

Thurmond, M.C., Hietala, S.K., 1997. Effect ofNeospora caninum infection on milk production in first-lactationdairy 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 anddairy cows with abortions in Argentina. Int. J. Parasitol. 29, 1705–1708.