Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in swine from Northeastern Brazil Prevalência de anticorpos anti-Toxoplasma gondii e anti-Neospora caninum em suínos do Nordeste do Brasil

A serologic survey was conducted among 130 swine slaughtered in the public slaughterhouse of the city of Patos, Paraíba State, Northeastern Brazil, to determine the prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies, and to verify possible associations between sex of the animals and antibody prevalence. The sera were analyzed by indirect antibody tests, considering 1:64 (T. gondii) and 1:50 (N. caninum) dilutions as cut-off points. The prevalence of anti-T. gondii antibodies was 36.2% (47/130) (95% CI = 27.9 – 45.0%) with reciprocal titers ranging from 64 to 2,048, and of anti-N. caninum antibodies was 3.1% (4/130) (95% CI = 0.8 – 7.7%) with reciprocal titers ranging from 50 to 6,400. Three of the four N. caninum-positive samples were also positive for T. gondii antibodies. All Neospora and Toxoplasma IFAT-positive animals were also positive for confirmatory immunoblotting techniques using total and purified N. caninum and T. gondii tachyzoite antigens, i.e., p38 (NcSRS2) and p30 (TgSAG1). There was no association between sex of animals and prevalence of anti-T. gondii and anti-N. caninum antibodies. This is the first indication of N. caninum natural infection in pigs from Brazil.


Introduction
The production and productivity indices of swine herds can be influenced by several conditions including genetic, environmental, nutritional, toxic, management, and infectious factors.Among parasitic diseases, toxoplasmosis occupies an important position.Toxoplasmosis is a worldwide zoonosis (ACHA; SZYFRES, 2003), and its control in pigs is important for consumer protection.This infection may also affect the reproduction of sows (DUBEY; URBAN, 1990;GIRALDI et al., 1996;JUNGERSEN et al., 2001) and productivity of pigs in general and thus may play an important role in the swine industry.
It is now accepted that Neospora caninum is a significant cause of bovine abortion worldwide and, with the exception of dogs, the disease has rarely been described in other species (DUBEY; LINDSAY, 1996;DUBEY, 1999).Experimental infection of sows led to transplacental transmission of N. caninum (JENSEN et al., 1998) but naturally acquired porcine infection has not been described and nothing is known on its possible presence in the pig population of Brazil.
Recent surveys conducted worldwide showed that the prevalence of anti-N.caninum antibodies in swine is very low.Using ELISA as screening test and an immunoblotting technique as confirmatory test, one positive animal was detected among 2,041 sows from Hesse, Germany (DAMRIYASA et al., 2004).All 454 breeding sows from England and Wales were negative by IFAT (HELMICK et al., 2002).In Switzerland, seropositive frequencies of 3 and 1% were observed in sows and fattening pigs, respectively, by ELISA, however, no case was confirmed by polymerase chain reaction (PCR) (WYSS et al., 2000).
To date, no published report exists on seropositivity to T. gondii and N. caninum in pigs in the State of Paraíba.This study was conducted to determine the prevalence of anti-T.gondii and anti-N.caninum antibodies in swine slaughtered in the public slaughterhouse of Patos municipality, State of Paraíba, Northeastern Brazil.

Field samples
Sera from swine slaughtered in the public slaughterhouse of Patos municipality (7° 1´ S and 37° 17´ W) were analyzed.The number of samples examined was calculated taking into account the assumed prevalence for anti-T.gondii and anti-N.caninum antibodies close to 50%, to maximize the sample size, and to obtain a minimal confidence of 95% and a statistical error of 10% (THRUSFIELD, 1995).Calculations were executed using EpiInfo version 6.04, resulting in a sample of 96 swine.As a matter of opportunity a total of 130 blood samples were collected during October and November 2006.In the region, swine are confinement-raised; however, in general the conditions of sanitary management are not appropriate.It was not possible to recover the husbandry/history of the pigs tested, as the animals are slaughtered without identification.
Blood was collected before slaughter from the Vena cava cranialis into sterile vacuum tubes, separated after clotting, and the sera were centrifuged and stored at -20 °C until analysis.

IFAT
For the detection of anti-T.gondii antibodies IFAT (CAMARGO, 1974) was performed considering 1:64 dilution as the cut-off point (GARCIA et al., 1999a), using RH strain tachyzoites as antigen.IFAT for N. caninum (PARÉ et al., 1995) was performed using 1:50 dilution as the cut-off point (LINDSAY et al., 1995); NC-1 strain of N. caninum cultured in bovine monocytes was used as the antigen.The conjugate used in both tests was anti-pig IgG (whole molecule, SIGMA, St. Louis, MO, USA).Positive and negative pig sera were used as controls and the positive samples were tested until the maximum dilution titer was reached.

Immunoblot analyses
The NC-1 strain of N. caninum (DUBEY et al., 1988) and the RH strain of T. gondii (SABIN, 1941) were maintained in Vero cell cultures and purified as previously described (SCHARES et al., 1998(SCHARES et al., , 1999)).Cell-culture-derived tachyzoites were frozen as a pellet at -80 °C until used for immunoblot or antigen purification.
To detect antibodies against N. caninum tachyzoite antigens, the incubation of westernblot strips was performed as previously described (SCHARES et al., 1998), with a few modifications.Sera were diluted 1:50 in PBS-TG.Serum of an experimentally N. caninum-infected swine (2.5 × 10 6 tachyzoites i.m., isolate SweB1) (JENSEN et al., 1998), kindly provided by Prof. Peter Lind (Department of Veterinary Diagnostics and Research, Section of Immunology and Parasitology, Copenhagen, Denmark), was used as a positive control.
Based on previous studies (reviewed in DUBEY; SCHARES, 2006), only reactions with antigens of 29, 30, 33, and 37 kDa Mr were regarded to be specific for the interpretation of the N. caninum immunoblot using total tachyzoite antigens.In the N. caninum immunoblot using purified p38 (NcSRS2), the reactivity of sera with a single band of 38 kDa Mr was regarded a positive reaction.
To detect antibodies against T. gondii tachyzoite antigens, the incubation of westernblot strips was performed as described for N. caninum.Sera were diluted 1:10 in PBS-TG.We used a serum of a swine experimentally infected with 10 4 T. gondii Me49, i.v.
(kindly provided by Dr. Christian Bauer, Institute for Parasitology, Justus Liebig University Giessen, Germany) as a positive control.Based on the previous results (HUSKINSON et al., 1989) in this test, only the reactions against three immunodominant antigens of about 20, 30, and 35 kDa were regarded to be specific.In the T. gondii immunoblot using purified p30 (TgSAG1), the reactivity of sera with a single band of 30 kDa Mr was regarded as a positive reaction.

Statistics
The prevalence of anti-T.gondii and anti-N.caninum antibodies was estimated from the ratio of positive results to the total number of swine examined, with the exact binomial confidence interval of 95% (THRUSFIELD, 1995), using the program EpiInfo, version 6.04.Positive associations between sex of the animals and T. gondii and N. caninum antibody seroprevalence were verified by Chi-square test (χ 2 ) or by Fisher exact test (ZAR, 1999), with a significance level of 5%.

Discussion
This is the first survey to give indication on the presence of N. caninum antibodies in pigs in Brazil.
The occurrence of T. gondii antibodies of 36.2% is in the range of the values found in other studies from Brazil with adult pigs (8.54 to 90.4%) (GUIMARÃES et al., 1992;GARCIA et al., 1999b;CAVALCANTE et al., 2006;MOURA et al., 2007;MILLAR et al., 2008).
As the samples were obtained from slaughtered animals, the only available information was on the sex of the pigs.No association was observed when sex was compared with seroprevalence of N. caninum and T. gondii antibodies.
Toxoplasma gondii antibody titers were higher than observed in sows from Germany, where 73% of the reactive samples presented   titers of 1:16, just above the cut-off value used in their assay (DAMRIYASA et al., 2004).However, other Brazilian studies using the same method as the present study (IFAT) also found higher titer values (GARCIA et al., 1999b;CAVALCANTE et al., 2006).
In studies conducted in Hesse, Germany (DAMRIYASA et al., 2004), sows responding to Sarcocystis miescheriana antigen were found to be more frequently T. gondii positive (21%) than Sarcocystis-negative animals (16.9%).There were no studies using sera from Brazilian pigs testing the presence of antibodies to other related coccidian parasites and cross reactivity with T. gondii.This might be of epidemiological importance and requires further investigation.
Concerning N. caninum, 3.1% (four samples) were positive by IFAT (≥ 50) and three of them were also T. gondii positive by IFAT.To confirm the specificity of our IFAT results we employed four western blotting techniques, using total and purified antigens of N. caninum and T. gondii.
One N. caninum IFAT-and immunoblot-negative serum also showed clear reactions with immunodominant T. gondii antigens and with the 30 kDa antigen of T. gondii (Figure 1c, 1d -lane 5).However, this serum did not react with either N. caninum total immunodominant antigens or the 38 kDa antigen, thus confirming the specificity of our IFAT results.When the samples were probed with total T. gondii antigen, the sample that was N. caninum IFAT-positive and T. gondii IFAT-negative was the only negative in the immunoblot analyses (Figure 1c, 1d -lane 2), also confirming the specificity of our IFAT results.
Consequently, similar to the findings of others (DAMRIYASA et al., 2004) our serological results suggest that only low numbers of pigs seem to be naturally infected with N. caninum.However, the final proof of natural N. caninum infection in pigs is lacking and can only be achieved by isolating viable N. caninum from swine.Nevertheless, our results show that further studies are necessary to elucidate a potential effect of N. caninum on reproduction in swine.

Table 1 .
Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies by IFAT in 130 swine slaughtered in the municipal public slaughterhouse of Patos, Paraíba State, Northeastern Brazil, by sex.

Table 2 .
Prevalence of anti-T.gondii and anti-N.caninum antibodies by IFAT in 130 sera from swine slaughtered in the municipal public slaughterhouse of Patos, Paraíba State, Northeastern Brazil, by serum dilution.