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Serological profile of Toxoplasma gondii and Neospora caninum infection in commercial sheep from São Paulo State, Brazil
Veterinary Parasitology 177 (2011) 50–54
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Serological profile of Toxoplasma gondii and Neospora caninum infection in commercial sheep from São Paulo State, Brazil Helio Langoni ∗ , Haroldo Greca Júnior, Felipe F. Guimarães, Leila S. Ullmann, Fernanda C. Gaio, Robson S. Uehara, Eric P. Rosa, Rogério M. Amorim, Rodrigo C. Da Silva School of Veterinary Medicine and Animal Science (FMVZ), São Paulo State University (UNESP), Botucatu Campus, São Paulo State, Brazil
a r t i c l e
i n f o
Article history: Received 23 October 2009 Received in revised form 8 November 2010 Accepted 10 November 2010 Keywords: Toxoplasma gondii Neospora caninum Sheep Infection Serology Public health
a b s t r a c t Neosporosis and toxoplasmosis are two important infections in young and adult sheep, leading to low production and abortion. This study aimed to determine the frequency of antibodies to Toxoplasma gondii and Neospora caninum in sheep from the eastern region of São Paulo State, Brazil. Serum samples (382) were collected from the sheep and assayed for T. gondii through modified agglutination test (MAT) and indirect fluorescent antibody test (IFAT), and for N. caninum antibodies, through IFAT, with cut-off titers equal to 16 (T. gondii) and 25 (N. caninum). All frozen samples were sent to the Center for Zoonoses Research (NUPEZO), Department of Veterinary Hygiene and Public Health (DHSVP), FMVZ, UNESP, for serological tests. A total of 71/382 (18.6%) samples reacted to T. gondii, especially at titers 16 (28; 39.4%), 64 (15; 21.1%), 256 (21; 29.6%) and 1024 (6; 8.5%) by MAT, and 16 (34; 47.9%), 64 (18; 25.4%), 256 (14; 19.7%) and 1024 (5; 7%) by IFAT. As regards N. caninum, 49/382 (12.8%) samples reacted at titers 25 (17; 34.7%), 50 (11; 22.5%), 100 (11; 22.5%), and ≥200 (10; 20.4%). These animals presented infection but no clinical signs. Six and ten animals had high titers for toxoplasmosis and neosporosis. No significant association was observed between antibodies for both parasites (P = 0.535) according to Fisher’s exact test, and no correlation was found between T. gondii (MAT) and N. caninum antibody titers (r = −0.0068; P = 0.895), T. gondii (IFAT) and N. caninum antibody titers (r = −0.0025; P = 0.961). Thus, T. gondii and N. caninum infections were observed in farms located in São Paulo State, where sheep play an important economical role for the national and regional business. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Toxoplasmosis and neosporosis are worldwide infections caused by Toxoplasma gondii and Neospora caninum, respectively, two obligate intracellular protozoan parasites of great importance to ovine production (Dubey, 2009). Toxoplasmosis is the major cause of abortion in sheep,
∗ Corresponding author at: Departamento de Higiene Veterinária e Saúde Pública, Faculdade de Medicina Veterinária e Zootecnia (FMVZ), Universidade Estadual Paulista (UNESP), Distrito de Rubião Júnior s/n, 18618-000 Botucatu, SP, Brazil. Tel.: +55 14 38116270; fax: +55 14 38116075. E-mail address: [email protected] (H. Langoni). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.11.024
besides leading to fetal malformations, preterm deliveries and stillbirths, which result in severe economic losses (Dubey et al., 1990; Bártová et al., 2009). In Brazil, the seroprevalence of both parasites in naturally exposed sheep varies depending on the flock management (Soares et al., 2009). N. caninum causes severe neuromuscular disease and abortion in pets and livestock. It infects many warmblooded animals, similarly to T. gondii and Sarcocystis spp. (Dubey et al., 1988). Its life cycle was established for domestic dogs in 1998 (McAllister et al., 1998) and for the coyote (Canis latrans) in 2004 (Gondim et al., 2004); both are definitive hosts of this parasite. Sheep can be infected by ingesting oocysts eliminated by dogs in their feces
H. Langoni et al. / Veterinary Parasitology 177 (2011) 50–54
(O’Handley et al., 2002), presenting neurological, dermatological, gastrointestinal, and respiratory disorders. Neosporosis causes economic losses for sheep flocks due to reproductive disorders and abortion (Soares et al., 2009; Ueno et al., 2009). Its zoonotic potential is unknown; however, the human population is in permanent contact with this parasite, presenting high frequency of antibodies, especially immunocompromised patients, like HIV bearers (Lobato et al., 2006). Nevertheless, there are no evidences of the infection zoonotic characteristics (Dubey, 2003). Dubey et al. (1990) reported the first occurrence of N. caninum in a one-week-old lamb, which presented neurological signs and many cysts in the brain tissue and spinal cord. Subsequently, Kobayashi et al. (2001) reported cases of natural infection by N. caninum in sheep fetuses, indicating vertical transmission. Hässig et al. (2003) reported the first association between N. caninum and abortion in naturally infected sheep through polymerase chain reaction (PCR) in the brain of four fetuses from a flock with frequent abortions. This study aimed to investigate the presence of N. caninum and T. gondii antibodies in serum samples of sheep flocks from different farms in the eastern region of São Paulo State. 2. Materials and methods 2.1. Sampling The sampling included 382 sheep from eight farms (A–H), seven of which were located in Botucatu Municipality (22◦ 53 09 S, 48◦ 26 42 W) and the remaining one in Pardinho Municipality (23◦ 04 52 S, 48◦ 22 25 W). The sheep were studied considering 95% confidence interval and 3.0% sampling error, according to Sampaio (1998). All animals were randomly sampled; they were older than two years, and their breeds were “Santa Inês” or “Bergamacia”. Dog access to the flocks was reported by farmers. Seven farms bred “Santa Inês” sheep, male and female, mainly for meat production, and the remaining one bred “Bergamacia” sheep, female, mainly for dairy production. During blood collection, the animals were captured and kept in shaded stalls or paddocks with water ad libitum. Blood (10 mL) was obtained by jugular vein puncture. After centrifugation at 1600 × g for 10 min, serum samples were
51
kept at −20 ◦ C until the serological test in the Center for Zoonoses Research (NUPEZO), Department of Veterinary Hygiene and Public Health (DHVSP), School of Veterinary Medicine and Animal Science (FMVZ), São Paulo State University (UNESP), Botucatu Campus, São Paulo State, Brazil. 2.2. Indirect immunofluorescence test (IFAT) for Neospora caninum Serum samples were assayed for N. caninum antibodies by indirect fluorescent antibody test (IFAT), considering 1:25 as the cut-off value, and titrated doubling dilutions from 1:25 to 1:800. The frequency of N. caninum antibodies in sheep farms was obtained based on the binomial distribution probability for the construction of a 95% confidence interval (Goodman, 1964). 2.3. Serology for Toxoplasma gondii For T. gondii antibody assessment, serum samples were subjected to modified agglutination test (MAT) (Desmonts and Remington, 1980; Dubey and Desmonts, 1987) and to indirect immunofluorescence test (Camargo, 1974), considering 16 as the cut-off value and using homemade antigen, formalin-fixed tachyzoites produced in the laboratory own premises, in 30-day-old, female Swiss mice inoculated with RH strain and Sarcoma TG-180 cells (ATCC CCRFS-180 II). MAT results presenting a clear-cut buttonshaped deposit of parasite suspension at the bottom of the well was interpreted as negative, and a carpet of agglutinated organisms ranging from 50% to 100% was considered positive. For IFAT, positive dilutions were considered when tachyzoites presented at least 50% fluorescent tachyzoites per field. 2.4. Statistical analysis Association and correlation between N. caninum and T. gondii antibodies and titers were analyzed by Chi-square or Fisher’s exact test, and correlation determined by Spearman’s correlation test (r), adopting ˛ = 0.05 (Triola, 2005). 3. Results Table 1 shows results regarding T. gondii and N. caninum antibodies in different farms. For N. caninum the prevalence
Table 1 Frequency of T. gondii (MAT and IFAT) and N. caninum (IFAT) antibodies per farm in sheep from São Paulo State, Brazil. Farm
Number of samples
Toxoplasma gondii
Neospora caninum
Number of positive
Percentage (95% confidence intervals)
Number of positive
Percentage (95% confidence intervals)
11.0 (6.3–18.6) 38.3 (25.8–52.6) 7.3 (3.0–17.3) 9.1 (3.3–23.7) 6.1 (1.9–19.7) 0 40.7 (24.5–59.4) 30.6 (21.1–42.0)
14 10 12 8 1 0 2 2
14.0 (8.6–22.2) 21.3 (12.0–35.0) 21.8 (13.0–34.4) 24.2 (12.9–41.2) 3.0 (0.7–15.3) 0 7.4 (5.5–48.4) 2.8 (0.9–9.6)
18.6 (15.0–22.8)
49
12.8 (09.8–16.6)
A B C D E F G H
100 47 55 33 33 15 27 72
11 18 4 3 2 0 11 22
Total
382
71 2
2
Statistics: association among farms concerning T. gondii ( = 44.9108; P < 0.000) and N. caninum ( = 23.1974; P = 0.002) antibody research.
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H. Langoni et al. / Veterinary Parasitology 177 (2011) 50–54
Table 2 Frequency of T. gondii (MAT and IFAT) and N. caninum (IFAT) antibody titers in sheep from São Paulo State, Brazil. Titer
T. gondii (MAT)
T. gondii (IFAT)
Titer
N. caninum (IFAT)
Positive (%; 95% confidence intervals)
Positive (%; 95% confidence intervals)
16 64 256 1024
28 (39.4; 28.9–51.1) 15 (21.1; 13.3–32.0) 21 (29.6; 20.2–41.1) 6 (8.5; 4.0–17.3)
34 (47.9; 36.7–59.4) 18 (25.4; 16.7–36.6) 14 (19.7; 12.2–30.5) 5 (7.0; 3.1–15.5)
25 50 100 ≥200
17 (34.7; 22.9–48.8) 11 (22.5; 13.1–36.0) 11 (22.5; 13.1–36.0) 10 (20.4; 11.5–33.7)
Total
71 (100.0; –)
71 (100.0; –)
Total
49 (100.0; –)
Positive (%; 95% confidence intervals)
Legend: 95% CI = 95% confidence interval.
Table 3 Association between T. gondii (MAT and IFAT) and N. caninum (IFAT) antibodies. N. caninum (IFAT)
T. gondii (MAT and IFAT)
Negative Positive Total Statistic: Fisher’s (0.4176–2.1177).
Negative
Positive
Total
274 37
63 8
337 45
311 exact
test = 0.535
71 (P
value);
382 OR = 0.9404
ranges from 2.8% to 24.2% in all eight farms, among which a significant difference was detected (P = 0.002), while for T. gondii ranges from 6.1% to 40.7% (P < 0.000). MAT and IFAT presented the same results when screening test for toxoplasmosis (Table 2). High frequency of low T. gondii titers was found for both tests, but 27 animals had high titers such as 256 (21; 29.6%) and 1024 (6; 8.4%) by MAT, while only 14 (19.7%) and 6 (7.0%) for titers by IFAT, respectively. For neosporosis, the distribution of titers was regular among the titers, but low titers had higher prevalence again. Independent of the test used for T. gondii, no association was observed for the detection of both parasites according to Fisher’s exact test, with P = 0.535 (Table 3). Analyzing the titers, both tests presented a positive correlation (r = 0.9982; P < 0.000), according to Spearman’s correlation test. No correlation was also observed between MAT-T. gondii and IFAT-N. caninum (r = −0.0068; P = 0.895), neither IFAT-T. gondii and IFAT-N. caninum (r = −0.0025; P = 0.961). 4. Discussion For N. caninum, 49/382 (12.8%) samples reacted at titers 25 (17; 34.7%), 50 (11; 22.5%), 100 (11; 22.5%), and ≥200 (10; 20.4%). These animals presented infection but no clinical signs. Six and ten animals had high titers for toxoplasmosis and neosporosis. The present results show that both infections were present in all the studied farms, except in farm F. Besides, although the frequency was lower in farms E, G and H, at least one positive animal was detected. According to Table 2, titer 100 was found for 11 animals (22.4%) and titer ≥200 for two animals, in which titers were 200 and 400. The variation in diagnostic tests and the different established cut-off points can interfere with the
interpretation of results (Sousa et al., 2009), which impairs the comparison of prevalence among different studies. Sample origin is an important epidemiological data since in some cases samples may come from farms with history of reproductive failures, which may result in high positivity levels. Sousa et al. (2009) used 1:20 as cut-off to indicate the seropositive animal. In this study, 1:16 represent the same point based in previous studies when this same dilution was confirmed by the presence of the parasite in bioassay in mice. These results were higher than the 0.5% (3/660) obtained by Helmick et al. (2002) through ELISA and IFAT and the 9.2% reported by Figlioulo et al. (2004) for a total of 597 serum samples tested in the central region of São Paulo State, confirming the importance of this parasite for sheep breed in the studied region. In addition, low prevalence was observed in the present study compared to that obtained by Hässig et al. (2003), who detected 10.3% positivity through IFAT in a flock with 117 ewes from Switzerland. Also, 8.8% and 1.8% prevalence was detected by Ueno et al. (2009) in sera from sheep in the Brazilian Federal District and by Soares et al. (2009) in the state of Rio Grande do Norte, Northeastern Brazil, respectively. In addition, Al-Majali et al. (2008) studied sheep from different herds in southern Jordan and found 4.3% positivity for 320 serum samples. Figlioulo et al. (2004) also reported 3.5% prevalence for toxoplasmosis and neosporosis in the state of São Paulo. In the central western region of Paraná State, Romanelli et al. (2007) detected 29/305 (9.5%) seropositive samples; it was the first report of seroprevalence in sheep from this state. Vogel et al. (2006) found 2/62 (3.2%) positive samples through the indirect ELISA test – CHEKIT – in Rio Grande do Sul State. On the other hand, the present results agree with those obtained by Bártová et al. (2009), who found prevalence ranging from 4% to 21% in nine farms from the Czech Republic, with 63/547 (12%) positive animals, similarly to the 12.9% detected in the present study. Dogs could eliminate N. caninum oocysts and thus contaminate the environment (Buxton et al., 1997). Then, seroprevalence could range according to the presence of infected dogs in farms, if observed in the studied farms. On the other hand, T. gondii antibody frequency was 71/382 (18.6%), especially at titers 16 (28; 39.4%), 64 (15; 21.1%), 256 (21; 29.6%) and 1024 (6; 8.5%) by MAT, and 16 (34; 47.9%), 64 (18; 25.4%), 256 (14; 19.7%) and 1024
H. Langoni et al. / Veterinary Parasitology 177 (2011) 50–54
(5; 7%) by IFAT. This prevalence was lower than those observed by Figlioulo et al. (2004) (207/597; 34.7%) and Ragozo et al. (2008) (120/495; 24.2%) in São Paulo State, Ogawa et al. (2003) (185/339; 54.6%) and Romanelli et al. (2007) (157/305; 51.48%) in Paraná State, Soares et al. (2009) (85/409; 20.8%) in Rio Grande do Norte State, Ueno et al. (2009) (393/1028; 38.2%) in the Brazilian Federal District, and Pinheiro et al. (2009) (142/432; 32.8%) in Alagoas State. High variation on the toxoplasmosis seroprevalence can be observed in all of the world (Dubey, 2009) but new data can supply new information about the dissemination of the parasite among production and pet animals. Only one studied farm was dairy property. The present results show a high impact for economy and public health since sheep can abort and transmit tachyzoites through milk in the acute infection stage, as detected by Fusco et al. (2007) by PCR in 4/117 (3.4%) sheep milk samples. Milk is used for human consumption in most studied farms presenting positive results. This high occurrence in some farms represents a problem in sheep flock management, probably due to environmental contamination through oocysts released by cats that had free access to the flock (Tenter et al., 2000; Al-Majali et al., 2008; Bártová et al., 2009; Ogawa et al., 2003; Dubey, 2009; Soares et al., 2009). In many places, cats are bred near sheep. Thus, the presence of cats is an important risk factor for T. gondii infection, of which cat is the definitive host. Domestic cats release oocysts to the environment (Chiari et al., 1987) and therefore are important in the epidemiological chain regarding parasitic transmission to livestock. Thus, the rural population should be concerned about the importance of toxoplasmosis; besides, some preventive measures must be adopted in the management, especially regarding cat access to the sheep flock, in order to reduce the risks of transmission to humans and animals (Ogawa et al., 2003). In conclusion, the present results showed an evidence of the presence of N. caninum and T. gondii infection in the studied sheep flocks from São Paulo State, where sheep play an important role for the public health and reproductive management. Due to the adoption of sanitary measures, studies involving reproductive and management problems in sheep are needed to obtain a new point of view regarding these infections in Brazilian flocks.
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Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Serological profile of Toxoplasma gondii and Neospora caninum infection in commercial sheep from São Paulo State, Brazil Helio Langoni ∗ , Haroldo Greca Júnior, Felipe F. Guimarães, Leila S. Ullmann, Fernanda C. Gaio, Robson S. Uehara, Eric P. Rosa, Rogério M. Amorim, Rodrigo C. Da Silva School of Veterinary Medicine and Animal Science (FMVZ), São Paulo State University (UNESP), Botucatu Campus, São Paulo State, Brazil
a r t i c l e
i n f o
Article history: Received 23 October 2009 Received in revised form 8 November 2010 Accepted 10 November 2010 Keywords: Toxoplasma gondii Neospora caninum Sheep Infection Serology Public health
a b s t r a c t Neosporosis and toxoplasmosis are two important infections in young and adult sheep, leading to low production and abortion. This study aimed to determine the frequency of antibodies to Toxoplasma gondii and Neospora caninum in sheep from the eastern region of São Paulo State, Brazil. Serum samples (382) were collected from the sheep and assayed for T. gondii through modified agglutination test (MAT) and indirect fluorescent antibody test (IFAT), and for N. caninum antibodies, through IFAT, with cut-off titers equal to 16 (T. gondii) and 25 (N. caninum). All frozen samples were sent to the Center for Zoonoses Research (NUPEZO), Department of Veterinary Hygiene and Public Health (DHSVP), FMVZ, UNESP, for serological tests. A total of 71/382 (18.6%) samples reacted to T. gondii, especially at titers 16 (28; 39.4%), 64 (15; 21.1%), 256 (21; 29.6%) and 1024 (6; 8.5%) by MAT, and 16 (34; 47.9%), 64 (18; 25.4%), 256 (14; 19.7%) and 1024 (5; 7%) by IFAT. As regards N. caninum, 49/382 (12.8%) samples reacted at titers 25 (17; 34.7%), 50 (11; 22.5%), 100 (11; 22.5%), and ≥200 (10; 20.4%). These animals presented infection but no clinical signs. Six and ten animals had high titers for toxoplasmosis and neosporosis. No significant association was observed between antibodies for both parasites (P = 0.535) according to Fisher’s exact test, and no correlation was found between T. gondii (MAT) and N. caninum antibody titers (r = −0.0068; P = 0.895), T. gondii (IFAT) and N. caninum antibody titers (r = −0.0025; P = 0.961). Thus, T. gondii and N. caninum infections were observed in farms located in São Paulo State, where sheep play an important economical role for the national and regional business. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Toxoplasmosis and neosporosis are worldwide infections caused by Toxoplasma gondii and Neospora caninum, respectively, two obligate intracellular protozoan parasites of great importance to ovine production (Dubey, 2009). Toxoplasmosis is the major cause of abortion in sheep,
∗ Corresponding author at: Departamento de Higiene Veterinária e Saúde Pública, Faculdade de Medicina Veterinária e Zootecnia (FMVZ), Universidade Estadual Paulista (UNESP), Distrito de Rubião Júnior s/n, 18618-000 Botucatu, SP, Brazil. Tel.: +55 14 38116270; fax: +55 14 38116075. E-mail address: [email protected] (H. Langoni). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.11.024
besides leading to fetal malformations, preterm deliveries and stillbirths, which result in severe economic losses (Dubey et al., 1990; Bártová et al., 2009). In Brazil, the seroprevalence of both parasites in naturally exposed sheep varies depending on the flock management (Soares et al., 2009). N. caninum causes severe neuromuscular disease and abortion in pets and livestock. It infects many warmblooded animals, similarly to T. gondii and Sarcocystis spp. (Dubey et al., 1988). Its life cycle was established for domestic dogs in 1998 (McAllister et al., 1998) and for the coyote (Canis latrans) in 2004 (Gondim et al., 2004); both are definitive hosts of this parasite. Sheep can be infected by ingesting oocysts eliminated by dogs in their feces
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(O’Handley et al., 2002), presenting neurological, dermatological, gastrointestinal, and respiratory disorders. Neosporosis causes economic losses for sheep flocks due to reproductive disorders and abortion (Soares et al., 2009; Ueno et al., 2009). Its zoonotic potential is unknown; however, the human population is in permanent contact with this parasite, presenting high frequency of antibodies, especially immunocompromised patients, like HIV bearers (Lobato et al., 2006). Nevertheless, there are no evidences of the infection zoonotic characteristics (Dubey, 2003). Dubey et al. (1990) reported the first occurrence of N. caninum in a one-week-old lamb, which presented neurological signs and many cysts in the brain tissue and spinal cord. Subsequently, Kobayashi et al. (2001) reported cases of natural infection by N. caninum in sheep fetuses, indicating vertical transmission. Hässig et al. (2003) reported the first association between N. caninum and abortion in naturally infected sheep through polymerase chain reaction (PCR) in the brain of four fetuses from a flock with frequent abortions. This study aimed to investigate the presence of N. caninum and T. gondii antibodies in serum samples of sheep flocks from different farms in the eastern region of São Paulo State. 2. Materials and methods 2.1. Sampling The sampling included 382 sheep from eight farms (A–H), seven of which were located in Botucatu Municipality (22◦ 53 09 S, 48◦ 26 42 W) and the remaining one in Pardinho Municipality (23◦ 04 52 S, 48◦ 22 25 W). The sheep were studied considering 95% confidence interval and 3.0% sampling error, according to Sampaio (1998). All animals were randomly sampled; they were older than two years, and their breeds were “Santa Inês” or “Bergamacia”. Dog access to the flocks was reported by farmers. Seven farms bred “Santa Inês” sheep, male and female, mainly for meat production, and the remaining one bred “Bergamacia” sheep, female, mainly for dairy production. During blood collection, the animals were captured and kept in shaded stalls or paddocks with water ad libitum. Blood (10 mL) was obtained by jugular vein puncture. After centrifugation at 1600 × g for 10 min, serum samples were
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kept at −20 ◦ C until the serological test in the Center for Zoonoses Research (NUPEZO), Department of Veterinary Hygiene and Public Health (DHVSP), School of Veterinary Medicine and Animal Science (FMVZ), São Paulo State University (UNESP), Botucatu Campus, São Paulo State, Brazil. 2.2. Indirect immunofluorescence test (IFAT) for Neospora caninum Serum samples were assayed for N. caninum antibodies by indirect fluorescent antibody test (IFAT), considering 1:25 as the cut-off value, and titrated doubling dilutions from 1:25 to 1:800. The frequency of N. caninum antibodies in sheep farms was obtained based on the binomial distribution probability for the construction of a 95% confidence interval (Goodman, 1964). 2.3. Serology for Toxoplasma gondii For T. gondii antibody assessment, serum samples were subjected to modified agglutination test (MAT) (Desmonts and Remington, 1980; Dubey and Desmonts, 1987) and to indirect immunofluorescence test (Camargo, 1974), considering 16 as the cut-off value and using homemade antigen, formalin-fixed tachyzoites produced in the laboratory own premises, in 30-day-old, female Swiss mice inoculated with RH strain and Sarcoma TG-180 cells (ATCC CCRFS-180 II). MAT results presenting a clear-cut buttonshaped deposit of parasite suspension at the bottom of the well was interpreted as negative, and a carpet of agglutinated organisms ranging from 50% to 100% was considered positive. For IFAT, positive dilutions were considered when tachyzoites presented at least 50% fluorescent tachyzoites per field. 2.4. Statistical analysis Association and correlation between N. caninum and T. gondii antibodies and titers were analyzed by Chi-square or Fisher’s exact test, and correlation determined by Spearman’s correlation test (r), adopting ˛ = 0.05 (Triola, 2005). 3. Results Table 1 shows results regarding T. gondii and N. caninum antibodies in different farms. For N. caninum the prevalence
Table 1 Frequency of T. gondii (MAT and IFAT) and N. caninum (IFAT) antibodies per farm in sheep from São Paulo State, Brazil. Farm
Number of samples
Toxoplasma gondii
Neospora caninum
Number of positive
Percentage (95% confidence intervals)
Number of positive
Percentage (95% confidence intervals)
11.0 (6.3–18.6) 38.3 (25.8–52.6) 7.3 (3.0–17.3) 9.1 (3.3–23.7) 6.1 (1.9–19.7) 0 40.7 (24.5–59.4) 30.6 (21.1–42.0)
14 10 12 8 1 0 2 2
14.0 (8.6–22.2) 21.3 (12.0–35.0) 21.8 (13.0–34.4) 24.2 (12.9–41.2) 3.0 (0.7–15.3) 0 7.4 (5.5–48.4) 2.8 (0.9–9.6)
18.6 (15.0–22.8)
49
12.8 (09.8–16.6)
A B C D E F G H
100 47 55 33 33 15 27 72
11 18 4 3 2 0 11 22
Total
382
71 2
2
Statistics: association among farms concerning T. gondii ( = 44.9108; P < 0.000) and N. caninum ( = 23.1974; P = 0.002) antibody research.
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Table 2 Frequency of T. gondii (MAT and IFAT) and N. caninum (IFAT) antibody titers in sheep from São Paulo State, Brazil. Titer
T. gondii (MAT)
T. gondii (IFAT)
Titer
N. caninum (IFAT)
Positive (%; 95% confidence intervals)
Positive (%; 95% confidence intervals)
16 64 256 1024
28 (39.4; 28.9–51.1) 15 (21.1; 13.3–32.0) 21 (29.6; 20.2–41.1) 6 (8.5; 4.0–17.3)
34 (47.9; 36.7–59.4) 18 (25.4; 16.7–36.6) 14 (19.7; 12.2–30.5) 5 (7.0; 3.1–15.5)
25 50 100 ≥200
17 (34.7; 22.9–48.8) 11 (22.5; 13.1–36.0) 11 (22.5; 13.1–36.0) 10 (20.4; 11.5–33.7)
Total
71 (100.0; –)
71 (100.0; –)
Total
49 (100.0; –)
Positive (%; 95% confidence intervals)
Legend: 95% CI = 95% confidence interval.
Table 3 Association between T. gondii (MAT and IFAT) and N. caninum (IFAT) antibodies. N. caninum (IFAT)
T. gondii (MAT and IFAT)
Negative Positive Total Statistic: Fisher’s (0.4176–2.1177).
Negative
Positive
Total
274 37
63 8
337 45
311 exact
test = 0.535
71 (P
value);
382 OR = 0.9404
ranges from 2.8% to 24.2% in all eight farms, among which a significant difference was detected (P = 0.002), while for T. gondii ranges from 6.1% to 40.7% (P < 0.000). MAT and IFAT presented the same results when screening test for toxoplasmosis (Table 2). High frequency of low T. gondii titers was found for both tests, but 27 animals had high titers such as 256 (21; 29.6%) and 1024 (6; 8.4%) by MAT, while only 14 (19.7%) and 6 (7.0%) for titers by IFAT, respectively. For neosporosis, the distribution of titers was regular among the titers, but low titers had higher prevalence again. Independent of the test used for T. gondii, no association was observed for the detection of both parasites according to Fisher’s exact test, with P = 0.535 (Table 3). Analyzing the titers, both tests presented a positive correlation (r = 0.9982; P < 0.000), according to Spearman’s correlation test. No correlation was also observed between MAT-T. gondii and IFAT-N. caninum (r = −0.0068; P = 0.895), neither IFAT-T. gondii and IFAT-N. caninum (r = −0.0025; P = 0.961). 4. Discussion For N. caninum, 49/382 (12.8%) samples reacted at titers 25 (17; 34.7%), 50 (11; 22.5%), 100 (11; 22.5%), and ≥200 (10; 20.4%). These animals presented infection but no clinical signs. Six and ten animals had high titers for toxoplasmosis and neosporosis. The present results show that both infections were present in all the studied farms, except in farm F. Besides, although the frequency was lower in farms E, G and H, at least one positive animal was detected. According to Table 2, titer 100 was found for 11 animals (22.4%) and titer ≥200 for two animals, in which titers were 200 and 400. The variation in diagnostic tests and the different established cut-off points can interfere with the
interpretation of results (Sousa et al., 2009), which impairs the comparison of prevalence among different studies. Sample origin is an important epidemiological data since in some cases samples may come from farms with history of reproductive failures, which may result in high positivity levels. Sousa et al. (2009) used 1:20 as cut-off to indicate the seropositive animal. In this study, 1:16 represent the same point based in previous studies when this same dilution was confirmed by the presence of the parasite in bioassay in mice. These results were higher than the 0.5% (3/660) obtained by Helmick et al. (2002) through ELISA and IFAT and the 9.2% reported by Figlioulo et al. (2004) for a total of 597 serum samples tested in the central region of São Paulo State, confirming the importance of this parasite for sheep breed in the studied region. In addition, low prevalence was observed in the present study compared to that obtained by Hässig et al. (2003), who detected 10.3% positivity through IFAT in a flock with 117 ewes from Switzerland. Also, 8.8% and 1.8% prevalence was detected by Ueno et al. (2009) in sera from sheep in the Brazilian Federal District and by Soares et al. (2009) in the state of Rio Grande do Norte, Northeastern Brazil, respectively. In addition, Al-Majali et al. (2008) studied sheep from different herds in southern Jordan and found 4.3% positivity for 320 serum samples. Figlioulo et al. (2004) also reported 3.5% prevalence for toxoplasmosis and neosporosis in the state of São Paulo. In the central western region of Paraná State, Romanelli et al. (2007) detected 29/305 (9.5%) seropositive samples; it was the first report of seroprevalence in sheep from this state. Vogel et al. (2006) found 2/62 (3.2%) positive samples through the indirect ELISA test – CHEKIT – in Rio Grande do Sul State. On the other hand, the present results agree with those obtained by Bártová et al. (2009), who found prevalence ranging from 4% to 21% in nine farms from the Czech Republic, with 63/547 (12%) positive animals, similarly to the 12.9% detected in the present study. Dogs could eliminate N. caninum oocysts and thus contaminate the environment (Buxton et al., 1997). Then, seroprevalence could range according to the presence of infected dogs in farms, if observed in the studied farms. On the other hand, T. gondii antibody frequency was 71/382 (18.6%), especially at titers 16 (28; 39.4%), 64 (15; 21.1%), 256 (21; 29.6%) and 1024 (6; 8.5%) by MAT, and 16 (34; 47.9%), 64 (18; 25.4%), 256 (14; 19.7%) and 1024
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(5; 7%) by IFAT. This prevalence was lower than those observed by Figlioulo et al. (2004) (207/597; 34.7%) and Ragozo et al. (2008) (120/495; 24.2%) in São Paulo State, Ogawa et al. (2003) (185/339; 54.6%) and Romanelli et al. (2007) (157/305; 51.48%) in Paraná State, Soares et al. (2009) (85/409; 20.8%) in Rio Grande do Norte State, Ueno et al. (2009) (393/1028; 38.2%) in the Brazilian Federal District, and Pinheiro et al. (2009) (142/432; 32.8%) in Alagoas State. High variation on the toxoplasmosis seroprevalence can be observed in all of the world (Dubey, 2009) but new data can supply new information about the dissemination of the parasite among production and pet animals. Only one studied farm was dairy property. The present results show a high impact for economy and public health since sheep can abort and transmit tachyzoites through milk in the acute infection stage, as detected by Fusco et al. (2007) by PCR in 4/117 (3.4%) sheep milk samples. Milk is used for human consumption in most studied farms presenting positive results. This high occurrence in some farms represents a problem in sheep flock management, probably due to environmental contamination through oocysts released by cats that had free access to the flock (Tenter et al., 2000; Al-Majali et al., 2008; Bártová et al., 2009; Ogawa et al., 2003; Dubey, 2009; Soares et al., 2009). In many places, cats are bred near sheep. Thus, the presence of cats is an important risk factor for T. gondii infection, of which cat is the definitive host. Domestic cats release oocysts to the environment (Chiari et al., 1987) and therefore are important in the epidemiological chain regarding parasitic transmission to livestock. Thus, the rural population should be concerned about the importance of toxoplasmosis; besides, some preventive measures must be adopted in the management, especially regarding cat access to the sheep flock, in order to reduce the risks of transmission to humans and animals (Ogawa et al., 2003). In conclusion, the present results showed an evidence of the presence of N. caninum and T. gondii infection in the studied sheep flocks from São Paulo State, where sheep play an important role for the public health and reproductive management. Due to the adoption of sanitary measures, studies involving reproductive and management problems in sheep are needed to obtain a new point of view regarding these infections in Brazilian flocks.
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