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Seroprevalence of Neospora caninum in female water buffaloes (Bubalus bubalis) from the southeastern region of Brazil
Veterinary Parasitology 99 (2001) 331–334
Short communication
Seroprevalence of Neospora caninum in female water buffaloes (Bubalus bubalis) from the southeastern region of Brazil T.U. Fujii a , N. Kasai b , S.M. Nishi b , J.P. Dubey c , S.M. Gennari b,∗ a
Laboratório de Sanidade Animal e Vegetal de Registro, Instituto Biológico de São Paulo, São Paulo, Brazil Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Orlando Marques do Paiva, 87, Cidade Universitária, São Paulo, SP, CEP 05508-000, Brazil c US Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Parasite Biology, Epidemiology and Systematics Laboratory, Building 1001, Beltsville, MD 20705, USA
b
Received 19 March 2001; received in revised form 11 June 2001; accepted 12 June 2001
Abstract Antibodies to Neospora caninum were assayed in sera of 222 female water buffaloes from Ribeira Valley of São Paulo State, Brazil, using an indirect fluorescent antibody test (IFAT) and Neospora agglutination test (NAT). IFAT antibodies were found in 64% of buffaloes with titers of 1:25 (42 buffaloes), 1:50 (53 buffaloes), 1:100 (31 buffaloes), 1:200 (10 buffaloes), 1:400 (3 buffaloes), or ≥1:800 (3 buffaloes). NAT antibodies were found in 53% of buffaloes; in titers of 1:40 in 52 buffaloes, 1:80 in 27 buffaloes, 1:160 in 21 buffaloes, and ≥1:320 in 17 buffaloes. Results indicate a high prevalence of N. caninum exposure in water buffaloes in Brazil and warrant an investigation of the role of N. caninum as an abortifacient in water buffaloes. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Water buffaloes; Bubalus bubalis; Seroprevalence; IFAT; Neospora agglutination test
1. Introduction Neospora caninum is now recognized as an important cause of bovine abortion in many countries (Dubey and Lindsay, 1996; Dubey, 1999). Recently, Guarino et al. (2000) detected N. caninum-like tissue cysts in two of four aborted fetuses from water buffaloes (Bubalus ∗ Corresponding author. E-mail address: [email protected] (S.M. Gennari).
0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 4 7 4 - 5
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T.U. Fujii et al. / Veterinary Parasitology 99 (2001) 331–334
bubalis) in Italy. Antibodies to N. caninum were reported in 68% of 75 buffaloes from Egypt (Dubey et al., 1998), 1.5% of 200 buffaloes from Vietnam (Huong et al., 1998) and 34.6% of 1377 buffaloes from 50 farms in southern Italy (Guarino et al., 2000). Water buffaloes are economically important in many developing countries, especially in Brazil, India, and southeastern Asia. Little is known about the causes of abortion in water buffaloes throughout the world. An oral presentation on the prevalence of N. caninum antibodies in buffaloes from the northeastern region of Brazil (Gondim et al., 1999) is the only report of neosporosis in buffaloes from this country. The aim of this study was to determine the seroprevalence of N. caninum in breeding-age buffaloes from Ribeira Valley region of Brazil.
2. Materials and methods 2.1. Experimental animals and blood samples Sera from 222 female cross-breed Murrah water buffaloes, from 3 to 16 years of age were collected from 11 dairy farms from Ribeira Valley area, São Paulo State, southeastern region of Brazil. Blood samples were collected from a jugular vein, using vacuum tubes without anti-coagulant and the serum samples were kept at −20◦ C until analysis. The animals were selected at random and divided into four age groups: 3–5 years (84 animals), 6–8 years (59 animals), 9–11 years (45 animals) and 12–16 years (34 animals) of age. 2.2. Serological analysis N. caninum antibody levels were measured by an indirect fluorescent antibody test (IFAT) and Neospora agglutination test (NAT). For the IFAT, NC-1 tachyzoite slides and positive and negative bovine control sera were obtained commercially (VMRD, Pullman, WA). Rabbit anti-bovine IgG labeled with fluorescein isothiocyanate (Sigma, St. Louis, MO) was used as conjugate. The initial serum test dilution was 1:25, and dilutions were doubled to a titer of ≥1:800, as detailed by Venturini et al. (1999). Continuous peripheral fluorescence was considered specific (Conrad et al., 1993; Paré et al., 1995). The NAT was performed as described by Romand et al. (1998) with NC-1 tachyzoite antigen produced commercially (Vétoquinol, Canada). Sera were screened at a 1:40 dilution and the positive samples were titrated to end points. 2.3. Statistical analysis The samples size was calculated based on the total number of females of reproductive age from 11 farms in the region (905 animals) according to Thrusfield (1995). Due to the lack of information related to N. caninum prevalence in the area, it was estimated to be 1.5% with a confidence limit of 95%. The differences between the proportions of N. caninum positive animals by the NAT and IFAT, by age group, were analyzed by an χ 2 association test with α = 5%. To compare the results obtained by IFAT and NAT, the Kappa concordance test was used (Pereira, 1995).
T.U. Fujii et al. / Veterinary Parasitology 99 (2001) 331–334
333
Table 1 Number of buffaloes and the respective anti-Neospora caninum antibodies titer by NAT and IFAT per age group Age group (years)
Total NAT samples Titers
IFAT
40
80
160
Positive Titers (%) (≥40) ≥320 25 50
3–5 6–8 9–11 12–16
84 59 45 34
14 15 14 9
11 9 3 4
8 5 7 1
3 8 3 3
Total
222
52
27
21
17
Total (%)
23.4 12.1
9.4
7.6
42.8 62.7 60.0 50.0
100
200
400 ≥800
11 14 6 11
25 18 7 3
6 12 9 4
2 0 6 2
1 1 1 0
1 1 1 0
42
53
31
10
3
3
18.9 23.8 13.9
4.5 3.3
Positive (%) (≥25) 54.7 77.9 66.6 58.8
1.3
3. Results and discussion The proportion of buffaloes seropositive for N. caninum by NAT and IFAT are shown in Table 1. Antibodies were found in 64% by IFAT and 53% of 222 buffaloes by NAT. Seroprevalence in the four age groups (3–5 years old, 6–8 years old, 9–11 years old and 12–16 years old) were 42.8, 62.7, 60.0 and 50% by NAT and 54.7, 77.9, 66.6 and 58.8% by IFAT, respectively. The χ 2 found an association (P = 0.0345) between age group and antibody level when determined by IFAT, with a higher number of positive animals from 6 to 11 years old. Results obtained by NAT showed no differences among age groups. All 11 farms had N. caninum seropositive buffaloes. The cut-off values of 1:40 (for NAT) and 1:25 (for IFAT) were based on data from naturally-infected cows (Venturini et al., 1999) and in experimentally-infected calves (De Marez et al., 1999). There were no data available with respect to naturally-infected or experimentally-infected buffaloes. Therefore, data are presented in Table 1 for all dilutions tested so that it may be reevaluated in the future. Results in different surveys are difficult to compare because of the differences in tests used, age groups, and breed of buffaloes surveyed. In the present study, both IFAT and NAT were used. Huong et al. (1998) reported IFAT antibodies in 3 of 200 buffaloes with titers of 1:640 or more, and Gondim et al. (1999) and Guarino et al. (2000) screened sera at 1:200 dilution in IFAT. The overall prevalence of N. caninum antibodies by NAT was 53% (≥1:40), similar to the 60% prevalence found by Dubey et al. (1998) in buffaloes from Egypt. The overall prevalence detected by IFAT was 64% (≥1:25), which differs from the finding of Gondim et al. (1999) from northeastern Brazil, who reported that 36.5% of buffalo sera with titers of ≥1:200 were positive. However, if the cut-off value of 1:200 had been used in the present study, prevalence would have been 7.27%. The frequency of occurrence of N. caninum antibodies showed no increase with the age of the animals, when measured by NAT, however, by the IFAT, an association between age and seroprevalence to N. caninum was observed, with the animals from 6 to 11 years presenting a higher prevalence. In Italy, Guarino et al. (2000) found that N. caninum prevalence increased with age of buffaloes, indicating exposure to N. caninum oocysts.
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T.U. Fujii et al. / Veterinary Parasitology 99 (2001) 331–334
Acknowledgements This study was supported by FAPESP. Prof. S.M. Gennari is a recipient of a scholarship from CNPq. References Conrad, P.A., Sverlow, K.W., Anderson, M., Rowe, J., Bondurant, R., Tuter, G., Breitmeyer, R., Palmer, R., 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. De Marez, T., Liddell, S., Dubey, J.P., Jenkins, M.C., Gasberre, L., 1999. Oral infection of calves with Neospora caninum oocysts from dogs: humoral and cellular immune responses. Int. J. Parasitol. 29, 1647–1657. Dubey, J.P., 1999. Neosporosis in cattle: biology and economic impact. J. Am. Vet. Med. Assoc. 214, 1160–1163. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1–59. Dubey, J.P., Romand, S., Hilali, M., Kwok, O.C.H., Thulliez, P., 1998. Seroprevalence of antibodies to Neospora caninum and Toxoplasma gondii in water buffaloes (Bubalus bubalis) from Egypt. Int. J. Parasitol. 28, 527–529. Gondim, L.F.P., Souza, R.M., Guimarães, J.E., Almeida, M.A.O., 1999. Freqüência de anticorpos contra Neospora caninum em búfalos criados no estado da Bahia. In: Seminário Brasileiro de Parasitologia Veterinária, Salvador, Brazil, 1999, Vol. 11, p. 227. Guarino, A., Fusco, G., Savini, G., Di Francesco, G., Cringoli, G., 2000. Neosporosis in water buffalo (Bubalus bubalis) in southern Italy. Vet. Parasitol. 91, 15–21. Huong, L.T.T., Ljungström, B.L., Uggla, A., Björkman, C., 1998. Prevalence of antibodies to Neospora caninum and Toxoplasma gondii in cattle and water buffaloes in southern Vietnam. Vet. Parasitol. 75, 53–57. Paré, J., Hietala, S.K., Thurmond, M.C., 1995. Interpretation of an indirect fluorescent antibody test for diagnosis of Neospora sp. infection in cattle. J. Vet. Diagn. Invest. 7, 273–275. Pereira, M.G., 1995. Epidemiologia: teoria e prática. Guanabara Koogan, Rio de Janeiro, pp. 584. Romand, S., Thulliez, P., Dubey, J.P., 1998. Direct agglutination test for serologic diagnosis of Neospora caninum. Parasitol. Res. 84, 50–53. Thrusfield, M., 1995. Veterinary Epidemiology, 2nd Edition. Cambridge University Press, Cambridge, pp. 479. Venturini, M.C., Venturini, L., Bacigalupe, D., Machuca, M., Echaide, I., Basso, W., Unzaga, J.M., Di Lorenzo, C., Guglielmone, 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.
Short communication
Seroprevalence of Neospora caninum in female water buffaloes (Bubalus bubalis) from the southeastern region of Brazil T.U. Fujii a , N. Kasai b , S.M. Nishi b , J.P. Dubey c , S.M. Gennari b,∗ a
Laboratório de Sanidade Animal e Vegetal de Registro, Instituto Biológico de São Paulo, São Paulo, Brazil Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Orlando Marques do Paiva, 87, Cidade Universitária, São Paulo, SP, CEP 05508-000, Brazil c US Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Parasite Biology, Epidemiology and Systematics Laboratory, Building 1001, Beltsville, MD 20705, USA
b
Received 19 March 2001; received in revised form 11 June 2001; accepted 12 June 2001
Abstract Antibodies to Neospora caninum were assayed in sera of 222 female water buffaloes from Ribeira Valley of São Paulo State, Brazil, using an indirect fluorescent antibody test (IFAT) and Neospora agglutination test (NAT). IFAT antibodies were found in 64% of buffaloes with titers of 1:25 (42 buffaloes), 1:50 (53 buffaloes), 1:100 (31 buffaloes), 1:200 (10 buffaloes), 1:400 (3 buffaloes), or ≥1:800 (3 buffaloes). NAT antibodies were found in 53% of buffaloes; in titers of 1:40 in 52 buffaloes, 1:80 in 27 buffaloes, 1:160 in 21 buffaloes, and ≥1:320 in 17 buffaloes. Results indicate a high prevalence of N. caninum exposure in water buffaloes in Brazil and warrant an investigation of the role of N. caninum as an abortifacient in water buffaloes. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Water buffaloes; Bubalus bubalis; Seroprevalence; IFAT; Neospora agglutination test
1. Introduction Neospora caninum is now recognized as an important cause of bovine abortion in many countries (Dubey and Lindsay, 1996; Dubey, 1999). Recently, Guarino et al. (2000) detected N. caninum-like tissue cysts in two of four aborted fetuses from water buffaloes (Bubalus ∗ Corresponding author. E-mail address: [email protected] (S.M. Gennari).
0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 4 7 4 - 5
332
T.U. Fujii et al. / Veterinary Parasitology 99 (2001) 331–334
bubalis) in Italy. Antibodies to N. caninum were reported in 68% of 75 buffaloes from Egypt (Dubey et al., 1998), 1.5% of 200 buffaloes from Vietnam (Huong et al., 1998) and 34.6% of 1377 buffaloes from 50 farms in southern Italy (Guarino et al., 2000). Water buffaloes are economically important in many developing countries, especially in Brazil, India, and southeastern Asia. Little is known about the causes of abortion in water buffaloes throughout the world. An oral presentation on the prevalence of N. caninum antibodies in buffaloes from the northeastern region of Brazil (Gondim et al., 1999) is the only report of neosporosis in buffaloes from this country. The aim of this study was to determine the seroprevalence of N. caninum in breeding-age buffaloes from Ribeira Valley region of Brazil.
2. Materials and methods 2.1. Experimental animals and blood samples Sera from 222 female cross-breed Murrah water buffaloes, from 3 to 16 years of age were collected from 11 dairy farms from Ribeira Valley area, São Paulo State, southeastern region of Brazil. Blood samples were collected from a jugular vein, using vacuum tubes without anti-coagulant and the serum samples were kept at −20◦ C until analysis. The animals were selected at random and divided into four age groups: 3–5 years (84 animals), 6–8 years (59 animals), 9–11 years (45 animals) and 12–16 years (34 animals) of age. 2.2. Serological analysis N. caninum antibody levels were measured by an indirect fluorescent antibody test (IFAT) and Neospora agglutination test (NAT). For the IFAT, NC-1 tachyzoite slides and positive and negative bovine control sera were obtained commercially (VMRD, Pullman, WA). Rabbit anti-bovine IgG labeled with fluorescein isothiocyanate (Sigma, St. Louis, MO) was used as conjugate. The initial serum test dilution was 1:25, and dilutions were doubled to a titer of ≥1:800, as detailed by Venturini et al. (1999). Continuous peripheral fluorescence was considered specific (Conrad et al., 1993; Paré et al., 1995). The NAT was performed as described by Romand et al. (1998) with NC-1 tachyzoite antigen produced commercially (Vétoquinol, Canada). Sera were screened at a 1:40 dilution and the positive samples were titrated to end points. 2.3. Statistical analysis The samples size was calculated based on the total number of females of reproductive age from 11 farms in the region (905 animals) according to Thrusfield (1995). Due to the lack of information related to N. caninum prevalence in the area, it was estimated to be 1.5% with a confidence limit of 95%. The differences between the proportions of N. caninum positive animals by the NAT and IFAT, by age group, were analyzed by an χ 2 association test with α = 5%. To compare the results obtained by IFAT and NAT, the Kappa concordance test was used (Pereira, 1995).
T.U. Fujii et al. / Veterinary Parasitology 99 (2001) 331–334
333
Table 1 Number of buffaloes and the respective anti-Neospora caninum antibodies titer by NAT and IFAT per age group Age group (years)
Total NAT samples Titers
IFAT
40
80
160
Positive Titers (%) (≥40) ≥320 25 50
3–5 6–8 9–11 12–16
84 59 45 34
14 15 14 9
11 9 3 4
8 5 7 1
3 8 3 3
Total
222
52
27
21
17
Total (%)
23.4 12.1
9.4
7.6
42.8 62.7 60.0 50.0
100
200
400 ≥800
11 14 6 11
25 18 7 3
6 12 9 4
2 0 6 2
1 1 1 0
1 1 1 0
42
53
31
10
3
3
18.9 23.8 13.9
4.5 3.3
Positive (%) (≥25) 54.7 77.9 66.6 58.8
1.3
3. Results and discussion The proportion of buffaloes seropositive for N. caninum by NAT and IFAT are shown in Table 1. Antibodies were found in 64% by IFAT and 53% of 222 buffaloes by NAT. Seroprevalence in the four age groups (3–5 years old, 6–8 years old, 9–11 years old and 12–16 years old) were 42.8, 62.7, 60.0 and 50% by NAT and 54.7, 77.9, 66.6 and 58.8% by IFAT, respectively. The χ 2 found an association (P = 0.0345) between age group and antibody level when determined by IFAT, with a higher number of positive animals from 6 to 11 years old. Results obtained by NAT showed no differences among age groups. All 11 farms had N. caninum seropositive buffaloes. The cut-off values of 1:40 (for NAT) and 1:25 (for IFAT) were based on data from naturally-infected cows (Venturini et al., 1999) and in experimentally-infected calves (De Marez et al., 1999). There were no data available with respect to naturally-infected or experimentally-infected buffaloes. Therefore, data are presented in Table 1 for all dilutions tested so that it may be reevaluated in the future. Results in different surveys are difficult to compare because of the differences in tests used, age groups, and breed of buffaloes surveyed. In the present study, both IFAT and NAT were used. Huong et al. (1998) reported IFAT antibodies in 3 of 200 buffaloes with titers of 1:640 or more, and Gondim et al. (1999) and Guarino et al. (2000) screened sera at 1:200 dilution in IFAT. The overall prevalence of N. caninum antibodies by NAT was 53% (≥1:40), similar to the 60% prevalence found by Dubey et al. (1998) in buffaloes from Egypt. The overall prevalence detected by IFAT was 64% (≥1:25), which differs from the finding of Gondim et al. (1999) from northeastern Brazil, who reported that 36.5% of buffalo sera with titers of ≥1:200 were positive. However, if the cut-off value of 1:200 had been used in the present study, prevalence would have been 7.27%. The frequency of occurrence of N. caninum antibodies showed no increase with the age of the animals, when measured by NAT, however, by the IFAT, an association between age and seroprevalence to N. caninum was observed, with the animals from 6 to 11 years presenting a higher prevalence. In Italy, Guarino et al. (2000) found that N. caninum prevalence increased with age of buffaloes, indicating exposure to N. caninum oocysts.
334
T.U. Fujii et al. / Veterinary Parasitology 99 (2001) 331–334
Acknowledgements This study was supported by FAPESP. Prof. S.M. Gennari is a recipient of a scholarship from CNPq. References Conrad, P.A., Sverlow, K.W., Anderson, M., Rowe, J., Bondurant, R., Tuter, G., Breitmeyer, R., Palmer, R., 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. De Marez, T., Liddell, S., Dubey, J.P., Jenkins, M.C., Gasberre, L., 1999. Oral infection of calves with Neospora caninum oocysts from dogs: humoral and cellular immune responses. Int. J. Parasitol. 29, 1647–1657. Dubey, J.P., 1999. Neosporosis in cattle: biology and economic impact. J. Am. Vet. Med. Assoc. 214, 1160–1163. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1–59. Dubey, J.P., Romand, S., Hilali, M., Kwok, O.C.H., Thulliez, P., 1998. Seroprevalence of antibodies to Neospora caninum and Toxoplasma gondii in water buffaloes (Bubalus bubalis) from Egypt. Int. J. Parasitol. 28, 527–529. Gondim, L.F.P., Souza, R.M., Guimarães, J.E., Almeida, M.A.O., 1999. Freqüência de anticorpos contra Neospora caninum em búfalos criados no estado da Bahia. In: Seminário Brasileiro de Parasitologia Veterinária, Salvador, Brazil, 1999, Vol. 11, p. 227. Guarino, A., Fusco, G., Savini, G., Di Francesco, G., Cringoli, G., 2000. Neosporosis in water buffalo (Bubalus bubalis) in southern Italy. Vet. Parasitol. 91, 15–21. Huong, L.T.T., Ljungström, B.L., Uggla, A., Björkman, C., 1998. Prevalence of antibodies to Neospora caninum and Toxoplasma gondii in cattle and water buffaloes in southern Vietnam. Vet. Parasitol. 75, 53–57. Paré, J., Hietala, S.K., Thurmond, M.C., 1995. Interpretation of an indirect fluorescent antibody test for diagnosis of Neospora sp. infection in cattle. J. Vet. Diagn. Invest. 7, 273–275. Pereira, M.G., 1995. Epidemiologia: teoria e prática. Guanabara Koogan, Rio de Janeiro, pp. 584. Romand, S., Thulliez, P., Dubey, J.P., 1998. Direct agglutination test for serologic diagnosis of Neospora caninum. Parasitol. Res. 84, 50–53. Thrusfield, M., 1995. Veterinary Epidemiology, 2nd Edition. Cambridge University Press, Cambridge, pp. 479. Venturini, M.C., Venturini, L., Bacigalupe, D., Machuca, M., Echaide, I., Basso, W., Unzaga, J.M., Di Lorenzo, C., Guglielmone, 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.