Antibodies to Neospora caninum in sheep from slaughterhouses in the state of São Paulo, Brazil

Anticorpos para Neospora caninum em ovinos de abatedouros do estado de São Paulo, Brasil

Laís Moraes Paiz Rodrigo Costa da Silva Benedito Donizete Menozzi Helio Langoni About the authors

Abstracts

Neosporosis is an emergent disease responsible for considerable economic impact due to reproductive losses. Its zoonotic potential remains unknown. This study involved a survey of antibodies to Neospora caninum in slaughtered sheep and their association with epidemiological variables. Serum samples from 596 sheep from the states of São Paulo and Rio Grande do Sul, Brazil, were collected in two slaughterhouses located in São Paulo and evaluated by indirect fluorescence antibody test (IFAT), using cut-off titers of 25. Among these samples, 353/596 (59.23%; 95%CI 55.23-63.10) were positive and 263/353 (74.50%; 95%CI 69.71-78.77%) were from Rio Grande do Sul. Statistical associations were determined in the univariate analysis between the serological results and sex, breed and municipality of origin. Sheep that came from extensive breeding system showed higher chance (OR=2.09) of presenting antibodies to N. caninum in relation to those from semi-intensive system. Higher chance was also observed for the different studied breeds, except Bergamácia, in relation to Hampshire Down. The results revealed the presence of infection by N. caninum in sheep from slaughterhouses.

Neospora caninum; serology; sheep; slaughterhouses


A neosporose é uma doença emergente responsável por considerável impacto econômico devido a perdas reprodutivas e seu potencial zoonótico permanece desconhecido. Este estudo envolveu a pesquisa de anticorpos para Neospora caninum em ovinos abatidos e sua associação com variáveis epidemiológicas. Amostras de soro sanguíneo de 596 ovinos, procedentes dos estados de São Paulo e Rio Grande do Sul, Brasil, foram colhidas em dois abatedouros localizados em São Paulo, e avaliadas pelo teste de reação de imunofluorescência indireta (RIFI), utilizando-se como ponto de corte o título de 25. Dentre essas amostras, 353/596 (59,23%; IC95% 55,23-63,10) foram positivas e dentre os ovinos positivos 263/353 (74,50%; IC95% 69,71-78,77%) eram procedentes do Rio Grande do Sul. Associações estatísticas foram determinadas na análise univariada entre os resultados sorológicos e o sexo, raça e município de origem. Ovinos oriundos de sistema de criação extensivo demonstraram maior chance (OR=2.09) de apresentarem anticorpos para N. caninum em relação aos de sistema semi-intensivo. Uma maior chance também foi observada para as diferentes raças estudadas, exceto Bergamácia, em relação à raça Hampshire Down. Os resultados revelaram a presença de infecção por N. caninum em ovinos de abatedouros.

Neospora caninum; sorologia; ovinos; abatedouros


Introduction

Neospora caninum is a cosmopolitan obligate intracellular apicomplexan protozoan, whose economic importance is tied to reproductive losses in animals of zootechnical interest. Initially described in Norway (BJERKAS et al., 1984Bjerkås I, Mohn SF, Presthus J. Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Z Parasitenkd 1984; 70(2): 271-274. http://dx.doi.org/10.1007/BF00942230. PMid:6426185
http://dx.doi.org/10.1007/BF00942230...
) as a coccidium similar to Toxoplasma gondii, the species N. caninum was only described (DUBEY et al., 1988aDubey JP, Carpenter JL, Speer CA, Topper MJ, Uggla A. Newly recognized fatal protozoan disease of dogs. J Am Vet Med Assoc 1988a; 192(9): 1269-1285. PMid:3391851.) and isolated four years later (DUBEY et al., 1988bDubey JP, Hattel AL, Lindsay DS, Topper MJ. Neonatal infection in dogs: isolation of the causative agent and experimental transmission. Neospora caninumJ Am Vet Med Assoc 1988b; 193(10): 1259-1263. PMid:3144521.). The domestic dog is the main definitive host of N. caninum, playing an important role in the transmission of infection by eliminating oocysts in its feces (MCALLISTER et al., 1998McAllister MM, Dubey JP, Lindsay DS, Jolley WR, Wills RA, McGuire AM. Rapid communication: dogs are definitive hosts of . Neospora caninumInt J Parasitol 1998; 28(9): 1473-1478. http://dx.doi.org/10.1016/S0020-7519(98)00138-6. PMid:9770635
http://dx.doi.org/10.1016/S0020-7519(98)...
). In infected dogs, clinical neosporosis can occur consistently, causing neuromuscular disorders. Similar disorders have been reported in cattle, which are commonly affected by reproductive problems, such as epidemic or endemic abortion around the fifth month of pregnancy (WOUDA et al., 1999Wouda W, Dijkstra T, Kramer AMH, van Maanen C, Brinkhof JMA. Seroepidemiological evidence for a relationship between infections in dogs and cattle. Neospora caninumInt J Parasitol 1999; 29(10): 1677-1682. http://dx.doi.org/10.1016/S0020-7519(99)00105-8. PMid:10608454
http://dx.doi.org/10.1016/S0020-7519(99)...
; DUBEY, 2003Dubey JP. Review of and neosporosis in animals. Neospora caninumKorean J Parasitol 2003; 41(1): 1-16. http://dx.doi.org/10.3347/kjp.2003.41.1.1. PMid:12666725
http://dx.doi.org/10.3347/kjp.2003.41.1....
).

Different animal species have been reported as susceptible to N. caninum infection, including more recent reports of intermediate hosts of the species Gallus gallus domesticus (COSTA et al., 2008Costa KS, Santos SL, Uzêda RS, Pinheiro AM, Almeida MAO, Araújo FR, et al. Chickens (Gallus domesticus) are natural intermediate hosts of . Neospora caninumInt J Parasitol 2008; 38(2): 157-159. http://dx.doi.org/10.1016/j.ijpara.2007.10.008. PMid:18054356
http://dx.doi.org/10.1016/j.ijpara.2007....
) and Sminthopsis crassicaudata (KING et al., 2011King JS, McAllan B, Spielman DS, Lindsay SA, Hůrková-Hofmannová L, Hartigan A, et al. Extensive production of Neospora caninum tissue cysts in a carnivorous marsupial succumbing to experimental neosporosis. Vet Res 2011; 42(1): 75. http://dx.doi.org/10.1186/1297-9716-42-75. PMid:21635733
http://dx.doi.org/10.1186/1297-9716-42-7...
), as well as definitive canid hosts of the species Canis lupus dingo (KING et al., 2010King JS, Slapeta J, Jenkins DJ, Al-Qassab SE, Ellis JT, Windsor PA. Australian dingoes are definitive hosts of . Neospora caninumInt J Parasitol 2010; 40(8): 945-950. http://dx.doi.org/10.1016/j.ijpara.2010.01.008. PMid:20149793
http://dx.doi.org/10.1016/j.ijpara.2010....
) and Canis lupus (DUBEY & SCHARES, 2011Dubey JP, Schares G. Neosporosis in animals—the last five years. Vet Parasitol 2011; 180(1-2): 90-108. http://dx.doi.org/10.1016/j.vetpar.2011.05.031. PMid:21704458
http://dx.doi.org/10.1016/j.vetpar.2011....
). Serological evidence of human exposure to N. caninum has also been demonstrated in several studies (NAM et al., 1998Nam HW, Kang SW, Choi WY. Antibody reaction of human anti-Toxoplasma gondii positive and negative sera with Neospora caninum antigens. Korean J Parasitol 1998; 36(4): 269-275. http://dx.doi.org/10.3347/kjp.1998.36.4.269. PMid:9868893
http://dx.doi.org/10.3347/kjp.1998.36.4....
; TRANAS et al., 1999Tranas J, Heinzen RA, Weiss LM, McAllister MM. Serological evidence of human infection with the protozoan Neospora caninum. Clin Diagn Lab Immunol 1999; 6(5): 765-767. PMid:10473533.; LOBATO et al., 2006Lobato J, Silva DAO, Mineo TWP, Amaral JDHF, Segundo GRS, Costa-Cruz JM, et al. Detection of immunoglobulin G antibodies to in humans: high seropositivity rates in patients who are infected by human immunodeficiency virus or have neurological disorders. Neospora caninumClin Vaccine Immunol 2006; 13(1): 84-89. http://dx.doi.org/10.1128/CVI.13.1.84-89.2006. PMid:16426004
http://dx.doi.org/10.1128/CVI.13.1.84-89...
); however, the zoonotic potential of the parasite has not been completely elucidated.

Neosporosis in sheep was first reported in 1990 (DUBEY et al., 1990Dubey JP, Hartley WJ, Lindsay DS. Congenital Neospora caninum infection in a calf with spinal cord anomaly. J Am Vet Med Assoc 1990; 197(8): 1043-1044. PMid:2243037.), in a lamb born at term with clinical signs of neurological disease. In Brazil, there have been a few reports of natural infections of sheep by N. caninum. Given the importance of seroepidemiological surveys in evaluating the occurrence of infection in this species due to the expansion of sheep farming in the country, economic losses resulting from reproductive problems, and the need to elucidate doubts regarding infection in sheep, this study aimed to determine the presence of antibodies to N. caninum in sheep from slaughterhouses located in the state of São Paulo, Brazil, and their association with the epidemiological variables.

Materials and Methods

Origin of samples

The serum samples were collected from 596 sheep (Ovis aries) in two slaughterhouses located in the Midwestern region of the state of São Paulo, which process sheep from the states of São Paulo and Rio Grande do Sul, Brazil, regardless of sex, breed, age or origin. Data on the sheep's age, breed, sex, breeding system and origin were collected from the slaughterhouses. Blood samples were drawn by jugular venipuncture into sterile glass tubes sealed with rubber stoppers, which were sent to the Zoonosis Research Center (NUPEZO) of the Faculty of Veterinary Medicine and Animal Science (FMVZ) of São Paulo State University (UNESP). The samples were centrifuged at 1600 g for 10 min to obtain the serum, which was stored at -20 ºC until the serological tests were performed.

Antigen

The NC-1 strain of Neospora caninum was used as antigen, which was cultured in vitro in a VERO (African Green Monkey Kidney) cell line and RPMI 1640 (LGCbio®) medium supplemented with 10% antibiotic antimycotic solution (Vitrocell®) containing 100U.mL–1 of penicillin, 100 µg.mL–1 of streptomycin and 0.25 µg.mL–1 of amphotericin B. In the case of VERO cells that were not inoculated with NC-1 tachyzoites, the medium was supplemented with 10% sterile fetal bovine serum free of Mycoplasma (Cultilab®). The 25 cm2 culture flasks were stored in an incubator at 37°C, in 5% CO2 and under controlled humidity.

Indirect Fluorescence Antibody Test (IFAT)

Antibodies to N. caninum were surveyed using the indirect fluorescence antibody test (IFAT). This reference and gold standard test for detecting anti-N. caninum antibodies (BJÖRKMAN & UGGLA, 1999Björkman C, Uggla A. Serological diagnosis of infection. Neospora caninumInt J Parasitol 1999; 29(10): 1497-1507. http://dx.doi.org/10.1016/S0020-7519(99)00115-0. PMid:10608435
http://dx.doi.org/10.1016/S0020-7519(99)...
) was performed according to Dubey et al. (1988b)Dubey JP, Hattel AL, Lindsay DS, Topper MJ. Neonatal infection in dogs: isolation of the causative agent and experimental transmission. Neospora caninumJ Am Vet Med Assoc 1988b; 193(10): 1259-1263. PMid:3144521.. The serum samples were first screened using a dilution of 1:25 (DUBEY et al., 2005Dubey JP, Knickman E, Greene CE. Neonatal Neospora caninum infections in dogs. Acta Parasitol 2005; 50(2): 176-179.), the cut-off adopted, and positive samples were then titrated in duplicate dilutions of up to 1:400. Antibodies were detected using a commercial anti-IgG sheep conjugated to fluorescein isothiocyanate (Sigma-Aldrich®, USA). The slides were analyzed using a fluorescence optical microscope (Zeiss® SH250, Germany) equipped with a 40x lens, and the dilutions were considered positive when complete fluorescence was observed in at least 50% of tachyzoites (BJÖRKMAN & UGGLA, 1999Björkman C, Uggla A. Serological diagnosis of infection. Neospora caninumInt J Parasitol 1999; 29(10): 1497-1507. http://dx.doi.org/10.1016/S0020-7519(99)00115-0. PMid:10608435
http://dx.doi.org/10.1016/S0020-7519(99)...
). This was determined by the reactions of the positive and negative control sera used.

Data analysis

All the data were tabulated on an Excel spreadsheet. The serological results were associated with the epidemiological variables of origin (state and municipality) and sex, age, breed and breeding system, using univariate analysis by the Chi-square test (χ2) and/or Fisher's exact test, adopting a significance level (α) of 5%.

Posteriorly, all the variables that were directly related to sheep that showed P < 0.25 in the univariate analysis were then included in the logistic regression model by multivariate analysis, excluding the variables that only described the location where the animals came from (state and municipality). The serological result was considered the dependent variable, positive or negative for N. caninum. Statistically significant difference was determined when the results showed P<0.05 in the univariate and multivariate analysis (TRIOLA, 2005Triola MF. Introdução à estatística. 9th ed. Rio de Janeiro: LTC; 2005.; SOUZA et al., 2012Souza ME, Porto WJN, Albuquerque PPF, Souza OL No, Faria EB, Pinheiro JW Jr, et al. Seroprevalence and risk factors associated with infection by of dairy cattle in the state of Alagoas, Brazil. Neospora caninumPesqui Vet Bras 2012; 32(10): 1009-1013. http://dx.doi.org/10.1590/S0100-736X2012001000011.
http://dx.doi.org/10.1590/S0100-736X2012...
). The statistical tests were performed with EpiInfoTM version 3.5.1 and IBM® SPSS® Statistics version 21 software.

Results

Table 1 lists the results of the survey of antibodies to N. caninum and Table 2 lists their association with the epidemiological variables, by univariate analysis. Among the tested samples, 353/596 (59.23%; 95%CI 55.23-63.10%) were positive, with titers ranging from 25 (129/353, 36.54%; 95%CI 31.69-41.69%) to 400 (8/353, 2.27%; 95%CI 1.17-4.40%).

Table 1
Occurrence of positive sheep among the 596 animals of this study, based on neosporosis serological titers by IFAT.
Table 2
Association (univariate analysis) between epidemiological data and serological test results for Neospora caninum infection by the indirect fluorescence antibody test (IFAT).

Among the seropositive sheep, 74.50% (263/353; 95%CI 69.71-78.77%) came from Rio Grande do Sul. The highest percentage of seropositive sheep (25/30, 83.33%) was originated from the municipality of Pirajuí, in the state of São Paulo. Statistically significant differences were observed between the seropositivity and municipality (P=0.00) of origin.

Animals from farms using semi-intensive livestock breeding systems showed a lower percentage of seropositivity, with 27/60 (45.00%) positive sheep, compared to those from intensive (27/45, 60.00%) and extensive (299/491, 60.90%) breeding systems. Additionally, no statistically significant differences were observed between breeding system and seropositivity (P=0.06), but it was observed when extensive and semi-extensive systems were compared in the univariate analysis (P=0.02).

With regard to sex, ewes presented higher seropositivity, with 220/349 (63.04%), while 133/247 (53.84%) rams were positive. Significant associations (P=0.03; OR=1.46) were observed between seropositivity and sex in the univariate analysis. No significant differences were observed in relation to age (P=0.17) and all three sheep between 2 and 3 years of age were seropositive in the present study.

The Santa Ines breed showed the highest occurrence of antibodies, with 25/37 (67.57%) seropositive sheep, and statistically significant difference was determined in the univariate analysis in relation to breed (P=0.02). Most breeds presented statistically significant differences in the seropositivity in relation to Hampshire Down breed in the univariate analysis and in the multivariate logistic regression (P<0.05; OR>1.00) (Table 3), except Bergamácia (P=0.36). Additionally, the multivariate analysis confirmed the significant differences observed in the univariate analysis between the semi-intensive and extensive systems (P=0.02; OR=2.09).

Table 3
Multivariate logistic regression analysis of epidemiological variables.

Discussion

Neospora caninum infection has been reported in different regions of the world and it is currently observed on all five continents (DUBEY, 1999Dubey JP. Recent advances in and neosporosis. NeosporaVet Parasitol 1999; 84(3-4): 349-367. http://dx.doi.org/10.1016/S0304-4017(99)00044-8. PMid:10456423
http://dx.doi.org/10.1016/S0304-4017(99)...
). Seroepidemiological studies in sheep are scarce, with seropositivity (titers ≥ 50) of 0.45% (3/660) reported in England (HELMICK et al., 2002Helmick B, Otter A, McGarry J, Buxton D. Serological investigation of aborted sheep and pigs for infection by . Neospora caninumRes Vet Sci 2002; 73(2): 187-189. http://dx.doi.org/10.1016/S0034-5288(02)00093-0. PMid:12204640
http://dx.doi.org/10.1016/S0034-5288(02)...
), while the prevalence in Brazil ranges from 1.8% in the state of Rio Grande do Norte (SOARES et al., 2009Soares HS, Ahid SMM, Bezerra ACDS, Pena HFJ, Dias RA, Gennari SM. Prevalence of anti-Toxoplasma gondii and anti- antibodies in sheep from Mossoró, Rio Grande do Norte, Brazil. Neospora caninumVet Parasitol 2009; 160(3-4): 211-214. http://dx.doi.org/10.1016/j.vetpar.2008.10.102. PMid:19091473
http://dx.doi.org/10.1016/j.vetpar.2008....
) to 64.2% in Pernambuco (TEMBUE et al., 2011Tembue AASM, Ramos RAN, Sousa TR, Albuquerque AR, da Costa AJ, Meunier IMJ, et al. Serological survey of in small ruminants from Pernambuco State, Brazil. Neospora caninumRev Bras Parasitol Vet 2011; 20(3): 246-248. http://dx.doi.org/10.1590/S1984-29612011000300013. PMid:21961757
http://dx.doi.org/10.1590/S1984-29612011...
).

The seropositivity determined in this study was 59.23%, with titers ≥ 25, differing significantly from those of 8.0% and 12.8% previously reported by Machado et al. (2011)Machado GP, Kikuti M, Langoni H, Paes AC. Seroprevalence and risk factors associated with neosporosis in sheep and dogs from farms. Vet Parasitol 2011; 182(2-4): 356-358. http://dx.doi.org/10.1016/j.vetpar.2011.05.021. PMid:21676548
http://dx.doi.org/10.1016/j.vetpar.2011....
and Langoni et al. (2011)Langoni H, Greca H Jr, Guimarães FF, Ullmann LS, Gaio FC, Uehara RS, et al. Serological profile of and infection in commercial sheep from São Paulo State, Brazil. Toxoplasma gondiiNeospora caninumVet Parasitol 2011; 177(1-2): 50-54. http://dx.doi.org/10.1016/j.vetpar.2010.11.024. PMid:21256676
http://dx.doi.org/10.1016/j.vetpar.2010....
, respectively, in the state of São Paulo and using the same cut-off. Some studies in Brazil have reported low percentages of seropositive sheep with titers ≥ 50, as 8.1% (SALABERRY et al., 2010Salaberry SRS, Okuda LH, Nassar AFC, Castro JR, Lima-Ribeiro AMC. Prevalence of antibodies in sheep flocks of Uberlândia county, MG. Neospora caninumRev Bras Parasitol Vet 2010; 19(3): 148-151. http://dx.doi.org/10.1590/S1984-29612010000300004. PMid:20943017
http://dx.doi.org/10.1590/S1984-29612010...
) in Minas Gerais, 9.2% (FIGLIUOLO et al., 2004Figliuolo LPC, Kasai N, Ragozo AMA, de Paula VSO, Dias RA, Souza SLP, et al. Prevalence of anti- and anti- antibodies in ovine from São Paulo State, Brazil. Toxoplasma gondiiNeospora caninumVet Parasitol 2004; 123(3-4): 161-166. http://dx.doi.org/10.1016/j.vetpar.2004.06.006. PMid:15325042
http://dx.doi.org/10.1016/j.vetpar.2004....
) and 13.9% (MUNHÓZ et al., 2010Munhóz KF, Luca M No, Santos SMA, Garcia JL, Guimarães JS Jr, Vidotto O, et al. Occurrence of anti- antibodies in sheep from farms located in northern Parana, Brazil. Neospora caninumSemina: Ciênc Agrár 2010; 31(4): 1031-1040.) in São Paulo and 9.5% (ROMANELLI et al., 2007Romanelli PR, Freire RL, Vidotto O, Marana ERM, Ogawa L, De Paula VSO, et al. Prevalence of and in sheep and dogs from Guarapuava farms, Paraná State, Brazil. Neospora caninumToxoplasma gondiiRes Vet Sci 2007; 82(2): 202-207. http://dx.doi.org/10.1016/j.rvsc.2006.04.001. PMid:17266999
http://dx.doi.org/10.1016/j.rvsc.2006.04...
) in Paraná. In this study, considering as cut-off the titer of 50, the seropositivity decrease to 37.58% (224/596; 95%CI 33.79-41.45%), but stills remains higher than the other studies cited above. However, a high prevalence has been reported in the southeastern region, e.g., by Rossi et al. (2011)Rossi GF, Cabral DD, Ribeiro DP, Pajuaba ACAM, Corrêa RR, Moreira RQ, et al. Evaluation of and infections in sheep from Uberlândia, Minas Gerais State, Brazil, by different serological methods. Toxoplasma gondiiNeospora caninumVet Parasitol 2011; 175(3-4): 252-259. http://dx.doi.org/10.1016/j.vetpar.2010.10.017. PMid:21075529
http://dx.doi.org/10.1016/j.vetpar.2010....
, who described 78.0% (titers ≥ 50) in the state of Minas Gerais.

Comparisons of percentages of seropositivity should be cautious, because numerous differences may occur between studies, as a result of different sample sizes, laboratory equipment, cut-off points, and test sensitivity and specificity (BJÖRKMAN & UGGLA, 1999Björkman C, Uggla A. Serological diagnosis of infection. Neospora caninumInt J Parasitol 1999; 29(10): 1497-1507. http://dx.doi.org/10.1016/S0020-7519(99)00115-0. PMid:10608435
http://dx.doi.org/10.1016/S0020-7519(99)...
; MODOLO et al., 2008Modolo JR, Stachissini AVM, Gennari SM, Dubey JP, Langoni H, Padovani CR, et al. Frequência de anticorpos anti- em soros de caprinos do estado de São Paulo e sua relação com o manejo dos animais. Neospora caninumPesqui Vet Bras 2008; 28(12): 597-600. http://dx.doi.org/10.1590/S0100-736X2008001200006.
http://dx.doi.org/10.1590/S0100-736X2008...
). In addition, factors intrinsic to the technique, such as the specific characteristics of the secondary antibody, alterations in the conditions of antigen-antibody reaction, the diversity of antigen preparations and the degree of subjectivity of the IFAT results, given that the interpretation of serological reactions is performed visually (BJÖRKMAN & UGGLA, 1999Björkman C, Uggla A. Serological diagnosis of infection. Neospora caninumInt J Parasitol 1999; 29(10): 1497-1507. http://dx.doi.org/10.1016/S0020-7519(99)00115-0. PMid:10608435
http://dx.doi.org/10.1016/S0020-7519(99)...
). Furthermore, larger numbers of seropositive sheep may be detected in studies using samples from problem herds (VOGEL et al., 2006Vogel FSF, Arenhart S, Bauermann FV. Anticorpos anti-Neospora caninum em bovinos, ovinos e bubalinos no Estado do Rio Grande do Sul. Cienc Rural 2006; 36(6): 1948-1951. http://dx.doi.org/10.1590/S0103-84782006000600048.
http://dx.doi.org/10.1590/S0103-84782006...
) and although N. caninum is closely related to Toxoplasma gondii, Sarcocystis spp.,and other apicomplexans, cross-reactivity has not been a major issue in serologic assays (DUBEY & LINDSAY, 2006Dubey JP, Lindsay DS. Neosporosis, toxoplasmosis, and sarcocystosis in ruminants. Vet Clin North Am Food Anim Pract 2006; 22(3): 645-671. http://dx.doi.org/10.1016/j.cvfa.2006.08.001. PMid:17071358
http://dx.doi.org/10.1016/j.cvfa.2006.08...
).

Higher occurrence of seropositive sheep were observed in this study in the state of Rio Grande do Sul, in strong contrast to the study by Vogel et al. (2006)Vogel FSF, Arenhart S, Bauermann FV. Anticorpos anti-Neospora caninum em bovinos, ovinos e bubalinos no Estado do Rio Grande do Sul. Cienc Rural 2006; 36(6): 1948-1951. http://dx.doi.org/10.1590/S0103-84782006000600048.
http://dx.doi.org/10.1590/S0103-84782006...
, who found a prevalence of only 3.2% in sheep by ELISA. The municipalities with higher occurrences of seropositive sheep (Bagé/Uruguaiana, Sant'Ana do Livramento, São Manuel and Pirajuí) were statistically significant different from the other studided municipalities and from each other when analyzed in pairs (2×2) in the univariate analysis, except São Manuel and Sant'Ana do Livramento. These results may be ascribed to the different farming systems adopted in different states and municipalities, as well as the presence of domestic and wild canids and other risk factors to N. caninum infections.

The occurrence of infections by N. caninum is not influenced by breed, age or the number of animals in the herd (BARBER, 1998Barber JS. Canine neosporosis. Waltham Focus 1998; 8(1): 25-29.; BECK et al., 2010Beck R, Marinculic A, Mihaljevic Z, Benic M, Martinkovic F. Seroprevalence and potential risk factors of infection in dairy cattle in Croatia. Neospora caninumVet Arhiv 2010; 80(2): 163-171.) and no evidence of any association was observed between these variables and the seropositivity in the studies conducted by Figliuolo et al. (2004)Figliuolo LPC, Kasai N, Ragozo AMA, de Paula VSO, Dias RA, Souza SLP, et al. Prevalence of anti- and anti- antibodies in ovine from São Paulo State, Brazil. Toxoplasma gondiiNeospora caninumVet Parasitol 2004; 123(3-4): 161-166. http://dx.doi.org/10.1016/j.vetpar.2004.06.006. PMid:15325042
http://dx.doi.org/10.1016/j.vetpar.2004....
, Romanelli et al. (2007)Romanelli PR, Freire RL, Vidotto O, Marana ERM, Ogawa L, De Paula VSO, et al. Prevalence of and in sheep and dogs from Guarapuava farms, Paraná State, Brazil. Neospora caninumToxoplasma gondiiRes Vet Sci 2007; 82(2): 202-207. http://dx.doi.org/10.1016/j.rvsc.2006.04.001. PMid:17266999
http://dx.doi.org/10.1016/j.rvsc.2006.04...
, Soares et al. (2009)Soares HS, Ahid SMM, Bezerra ACDS, Pena HFJ, Dias RA, Gennari SM. Prevalence of anti-Toxoplasma gondii and anti- antibodies in sheep from Mossoró, Rio Grande do Norte, Brazil. Neospora caninumVet Parasitol 2009; 160(3-4): 211-214. http://dx.doi.org/10.1016/j.vetpar.2008.10.102. PMid:19091473
http://dx.doi.org/10.1016/j.vetpar.2008....
, Ueno et al. (2009)Ueno TEH, Gonçalves VSP, Heinemann MB, Dilli TLB, Akimoto BM, de Souza SL, et al. Prevalence of and infections in sheep from Federal District, central region of Brazil. Toxoplasma gondiiNeospora caninumTrop Anim Health Prod 2009; 41(4): 547-552. http://dx.doi.org/10.1007/s11250-008-9220-8. PMid:18726165
http://dx.doi.org/10.1007/s11250-008-922...
, Salaberry et al. (2010) and Rossi et al. (2011)Salaberry SRS, Okuda LH, Nassar AFC, Castro JR, Lima-Ribeiro AMC. Prevalence of antibodies in sheep flocks of Uberlândia county, MG. Neospora caninumRev Bras Parasitol Vet 2010; 19(3): 148-151. http://dx.doi.org/10.1590/S1984-29612010000300004. PMid:20943017
http://dx.doi.org/10.1590/S1984-29612010...
. However, in this study, a statistically significant difference was found between the serological results and breed, but not age. Although the percentage of positive sheep sampled increased from the lowest to the highest age, no statistically significant difference was determined. Furthermore, because it is a cross-sectional study, the serological results refer to the age at which sheep were slaughtered, not infected.

Ewes presented more frequent positive serological reactions than rams, which is similar to the findings of Ueno et al. (2009)Ueno TEH, Gonçalves VSP, Heinemann MB, Dilli TLB, Akimoto BM, de Souza SL, et al. Prevalence of and infections in sheep from Federal District, central region of Brazil. Toxoplasma gondiiNeospora caninumTrop Anim Health Prod 2009; 41(4): 547-552. http://dx.doi.org/10.1007/s11250-008-9220-8. PMid:18726165
http://dx.doi.org/10.1007/s11250-008-922...
, but in disagreement with those of Soares et al. (2009)Soares HS, Ahid SMM, Bezerra ACDS, Pena HFJ, Dias RA, Gennari SM. Prevalence of anti-Toxoplasma gondii and anti- antibodies in sheep from Mossoró, Rio Grande do Norte, Brazil. Neospora caninumVet Parasitol 2009; 160(3-4): 211-214. http://dx.doi.org/10.1016/j.vetpar.2008.10.102. PMid:19091473
http://dx.doi.org/10.1016/j.vetpar.2008....
. In the present study, ewes showed a 1.46-fold greater chance of presenting antibodies to N. caninum, without taking into account the risk factors to which they may have been exposed.

Farms where intensive or semi-intensive breeding systems are adopted usually have lower occurrences of neosporosis, in view of their better sanitation, separation of the sheep from the herd according to age, sex and production purpose, as well as their more skilled workforce (BARLING et al., 2001Barling KS, McNeill JW, Paschal JC, McCollum FT 3rd, Craig TM, Adams LG, et al. Ranch-management factors associated with antibody seropositivity for in consignments of beef calves in Texas, USA. Neospora caninumPrev Vet Med 2001; 52(1): 53-61. http://dx.doi.org/10.1016/S0167-5877(01)00233-1. PMid:11566378
http://dx.doi.org/10.1016/S0167-5877(01)...
). On the other hand, extensive breeding can expose sheep to greater contact with canids, the definitive hosts (DUBEY et al., 2007Dubey JP, Schares G, Ortega-Mora LM. Epidemiology and control of neosporosis and . Neospora caninumClin Microbiol Rev 2007; 20(2): 323-367. http://dx.doi.org/10.1128/CMR.00031-06. PMid:17428888
http://dx.doi.org/10.1128/CMR.00031-06...
).

Nevertheless, a higher seropositivity was observed in this study in sheep from breeding systems with distinct characteristics, extensive and intensive, and no statistically significant difference was determined between seropositivity and breeding system. However, analyzing the breeding systems in pairs (2×2) in the univariate analysis, the seropositivity in sheep from extensive breeding system was statistically significant different from those of semi-intensive system, with a 2.09-fold greater chance of presenting serological evidence of infection by N. caninum, demonstred by the multivariate logistic regression.

This results indicates that exposure of sheep to different epidemiological conditions and risk factors for N. caninum infections can occur in the different breeding systems, although it is known that the extensive system can expose sheep to risk factors more frequently and the adoption of sanitary measures may be necessary to control and prevent N. caninum infections.

Sheep of the Santa Ines breed presented higher seropositivity, though similar values were observed in crossbred sheep and in the Ideal breed. It is known that sheep from these breeds were mostly raised in the extensive breeding systems of Sant'Ana do Livramento, a municipality in the state of Rio Grande do Sul, where a higher percentage of seropositive sheep was determined.

On the other hand, sheep of the Hampshire Down breed presented lowest seropositivity, and the other studied breeds, except Bergamácia, showed significant differences in the serological results in relation to this breed, with higher chance (OR>1.00) of presenting antibodies to N. caninum. Sheep of Santa Inês breed presented 8.91-fold greater chance of presenting positive serological results in relation to Hampshire Down.

Cross-sectional studies may reflect the distribution of a disease and other relevant conditions, including biological factors in the population (GREINER & GARDNER, 2000Greiner M, Gardner IA. Epidemiologic issues in the validation of veterinary diagnostic tests. Prev Vet Med 2000; 45(1-2): 3-22. http://dx.doi.org/10.1016/S0167-5877(00)00114-8. PMid:10802331
http://dx.doi.org/10.1016/S0167-5877(00)...
). Thus, a statistical association was determined in this study between the serological results and epidemiological variables sex, breed and municipality of origin. However, it is noteworthy that risk factors for N. caninum infection can only be determined using a study design that includes the systematic collection of samples and epidemiological information about the herd.

Conclusions

An analysis of the findings revealed serological evidence of N. caninum infection in sheep herds from the states of São Paulo and Rio Grande do Sul, Brazil, which were destined for slaughter. Statistical associations were determined between the serological results and certain epidemiological variables. Sheep that came from extensive breeding system showed higher chance of presenting antibodies to N. caninum in relation to those from semi-intensive system. Higher chance was also observed for the different studied breeds, except Bergamácia, in relation to Hampshire Down. However, it is noteworthy that risk factors for N. caninum infection can only be determined using another study design. Evidence of the occurrence of neosporosis in humans has not yet been proven, but seroconversion in this species has been reported and additional studies involving animals destined for slaughter are particularly important for public health, given the relatively little information available about N. caninum infection in sheep and the fact that their meat is intended for human consumption. Furthermore, studies to investigate the possible zoonotic potential of neosporosis are equally important.

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Publication Dates

  • Publication in this collection
    Jan-Mar 2015

History

  • Received
    02 Sept 2014
  • Accepted
    01 Dec 2014
Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
E-mail: cbpv_rbpv.fcav@unesp.br