Introduction

The Toxoplasma gondii and Neospora caninum are obligate parasites that can infect several animal species including humans (Garcia et al. 1999; Dubey and Lindsay 2006; Lobato et al. 2006; Benetti et al. 2009; Dubey 2010). The occurrence of these protozooses has been correlated to management, environment (Modolo et al. 2008a; Modolo et al. 2008b), livestock by-products (Hiramoto et al. 2001), and even dissemination through water (Dubey et al. 2004).

In cats, toxoplasmosis can be associated with the feline immunodeficiency virus (FIV) (Lucas et al. 1998). The occurrence of Leishmania infection in animals without immunossupression suggests that other factors must determine the susceptibility, although the literature has reported cases of leishmaniasis in cats positive for both viruses, whose action is essentially immunosuppressive (POLI et al. 2002; GREVOT et al. 2005). In theory, this favors parasitic infection in these animals, which have high degree of natural immunological resistance (VITA et al. 2005).

Thus, the aim of this study was to investigate the occurrence of antibodies against T. gondii, N. caninum, and Leishmania spp. in cats from Andradina Municipality, São Paulo State, Brazil, and the existence of an association between these parasites and infections by FIV and feline leukemia virus (FeLV). The influence of several factors on the occurrence of these parasites was also evaluated including cat age, sex, breed, origin, access to extra-household environment, and contact with other animal species, especially mice.

Material and methods

Animals and sample collection

The experimental group consisted of 70 male and female cats of different breeds and ages from urban and rural areas of the microregion of the Andradina Municipality (−20.866007° S, −51.307152° W, and hypsometry of 405 m), São Paulo State, Brazil. This study was carried out between 2007 and 2009 with the approval of the Animal Experimentation Ethics Committee of Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista, protocol no. 2007–003276.

Blood (5 ml) was collected by venipuncture in siliconized Vacutainer tubes without an anticoagulant and centrifuged at 3,000 rpm for 5 min. The obtained serum was divided into two aliquots, then transferred to sterile plastic tubes and immediately frozen at −20°C.

Laboratory techniques

Serum samples were analyzed through an indirect immunofluorescence antibody test (IFAT) to investigate the presence of immunoglobulin G against T. gondii and N. caninum. Serological diagnosis was done according to the technique described by Camargo (1964), with cutoffs fixed in the dilutions of 1:64 for T. gondii and 1:16 for N. caninum.

Sera were base two diluted until the last dilution in which fluorescence was observed. The presence of total fluorescence around the tachyzoite surface was considered positive and the reactions of apical or partial fluorescence negative. Positive and negative control sera were included in each slide. Antibodies against FIV and FeLV were detected using the ELISA commercial kit (Snap-Combo®, IDEXX Systems, Portland, USA), following the manufacturer’s recommendations.

The detection of antibodies against Leishmania spp. was also done by ELISA using protein A as the antigen (Lima et al. 2005) in triplicate. The cutoff was established based on the mean added to the standard deviation of the optical density obtained during the assay with serum from animals from a nonendemic area. Also, IFAT was performed by using promastigote forms of Leishmania (L.) chagasi as antigen, being considered seropositive titer ≥40. Animal records included individualized sheets containing species, breed, sex, age, place of origin, diet type, contact with animals, access to external or extra-household environments, and the presence of mice (Mus musculus) and rats (Ratus ratus) on the property.

Statistical analysis

Association between the analyzed variables was determined by the chi-square (χ 2) test using the software Minitab Version 11.

Results

Anti-Leishmania spp. antibodies were observed in 4.2% (3/70) of cats from the urban area by ELISA and all negative by IFAT. All positive animals were of undefined breed including an asymptomatic young female and two adult males. The occurrence of Leishmania spp. in the cats of this study was not correlated to the variables sex, breed, age, FIV, and FelV (P > 0.05).

Of the serum samples, 15.7% (11/70) of cats from the rural area were considered positive or reactive to T. gondii with titers equal to or higher than 1:64. There was no seropositivity for N. caninum with IFAT ≥1:16. As to racial pattern, only animals without a definite breed and those with access to the external environment were reactive to T. gondii without correlation with sex.

A higher tendency of occurrence of T. gondii was noted for young cats, which had access to the external environment, cohabited with mice, and fed on homemade food and raw milk. All animals that received this type of food lived in rural properties and had contact with other pets and livestock (P ≤ 0.0001). The variables and positivity for T. gondii are shown in Table 1.

Table 1 Occurrence of seropositivity for T. gondii in 70 cats from Andradina Municipality, São Paulo State, Brazil according to the analyzed variables
Full size table

None of the 70 analyzed cats presented antibodies against FIV and FeLV. Coinfection by T. gondii, N. caninum, and Leishmania spp. was not detected in the analyzed animals.

Discussion

Of the cat population in this study, 5.76% (3/52) were positive for L. (L.) chagasi, and these animals were not serologically reactive to the viruses FIV and FeLV. Similarly, Martín-Sánchez et al. (2007) carried out an epidemiological study on leishmaniasis in cats from Spain and found no association between Leishmania infection and feline immunodeficiency. Although IFAT has been used in epidemiological studies (Ayllon et al. 2008; Poli et al. 2002; Vita et al. 2005), the positive animals of the present study were not seroreactive by IFAT but by ELISA, results similar to that obtained by Figueiredo et al. (2009), who evaluated antibodies anti-Leishmania in 43 cats from Barra Mansa Municipality, Rio de Janeiro State, Brazil by the methods IFAT and ELISA and detected only one reactive animal, 2.4% (1/43), by the latter technique only.

Bresciani et al. (2010) found 0.7% (2/283) of cats positive for Leishmania spp. by the IFAT technique in the municipality of Araçatuba, São Paulo State, Brazil. In this study, two females were positive, a young mongrel and an adult feline. The occurrence of Leishmania spp. in the cats of the present study was not correlated to the variables sex, breed, and age. The polymerase chain reaction was considered a better diagnostic method than the serology to define Leishmania infection in cats (Martín-Sánchez et al. 2007).

Infection by T. gondii was detected in 15.7% of the cats evaluated in the present study. Higher prevalence of anti-Toxoplasma antibodies in cats, detected by IFAT, was reported by Garcia et al. 1999 in the Paraná State, Langoni et al. (2001) in the states of São Paulo and Paraná, by Pinto et al. (2009) in the state of Rio Grande do Sul, and by Ortolani et al. (2005) in native Indian villages in the state of São Paulo, where occurrences were 19.4% (37/191), 37.9% (93/245), and 46.4% (13/28), respectively, with titers ≥1:16. Different percentages of positivity can be found in several studies on the soroepidemiology, especially due to the cutoffs adopted for IFAT.

Using the IFAT, other researchers adopted the cutoff at 1:64 to investigate seroprevalence of T. gondii in cats and observed a higher (Bresciani et al. 2007) or similar (Dalla Rosa et al. 2010) percentage than that found in our study. Higher percentage of infection by T. gondii was detected through IFAT by Garcia et al. (1999) in cats from rural areas in the state of Paraná. This fact corroborates the findings of the present study.

Garcia et al. 1999 suggest a statistical association for age groups, especially in young animals, as observed in the present study. These findings corroborate the results of Lucas et al. (1998), in which the highest percentage of cats reactive to toxoplasmosis was detected among adult animals, confirming the predominant occurrence of toxoplasmic infection in the postnatal stage. The association between other variables and sex was not detected as to seropositivity for T. gondii in felines (Bresciani et al. 2007) similar to the present study.

As to the racial pattern, only animals without a definite breed and those with access to the external environment were reactive to Toxoplasma. The breed of the evaluated animals had no influence on the occurrence of T. gondii (Langoni et al. 2001; Bresciani et al. 2007; Pinto et al. 2009; Dalla Rosa et al. 2010).

Reports on N. caninum infection in cats still remain scarce. In Araçatuba Municipality, São Paulo State, Brazil, Bresciani et al. (2007) studied anti-N. caninum antibodies in cats and observed that 24.5% (98/400) were reactive, with IFAT ≥1:16, and found a higher occurrence to N. caninum in older cats, whereas in the present study, no animal was infected. This finding is interesting especially due to the proximity between Araçatuba and Andradina Municipalities and the differences in positivity for such a protozoan in this animal species.

Using agglutination test, Dubey et al. (2002) analyzed the serum of Brazilian cats and observed that 11.9% (60/502) were reactive. A higher percentage was detected in Italian cats using the same technique; 24.8% (70/282) reacted positively. The association between the ages of animals infected by N. caninum was not statistically significant (Ferroglio et al. 2005).

Although the cats of the present study did not present antibodies against N. caninum, even cohabiting with mice, several researchers have observed that some mouse species can be positive for such a parasite through serological (Huang et al. 2004) and molecular (Ferroglio et al. 2007; Jenkins et al. 2007) techniques, suggesting thus that mice can serve as a reservoir of this protozoosis for predator animals.

As to immunosuppression, Lucas et al. (1998) studied 115 blood samples from cats negative for FeLV, of which 22 were positive for FIV; there was a strong association between occurrence of the immunodeficiency virus and anti-T. gondii antibodies, probably due to the reactivation of a latent infection. This finding was not observed in our study, as well the coinfection by Leishmania, Toxoplasma, and Neospora, which suggests a good specificity of this technique to diagnose these parasites minimizing the possibility of cross-reactions.

Among the 11 seropositive cats, 13.1% (5/38) received homemade food with commercial cat food and 27.2% (6/22) received homemade food and raw milk. The 22 animals from the countryside ingested viscera of livestock eventually slaughtered in the property and 27.2% of them were positive for T. gondii. On the other hand, animals fed exclusively with commercial cat food presented no immunoglobulins against T. gondii and N. caninum. This highlights the importance of adopting sanitary–hygienic measures for the preparation of meat by-products from the several livestock categories that can serve as a source of infection when improperly cooked (Dubey and Lindsay 2006) and handled (Millar et al. 2007), so that Toxoplasma may remain infectious even in bovine milk and cheese (Hiramoto et al. 2001).

Conclusion

Based on the current study, we can conclude that the infection by T. gondii and Leishmania spp. present in the cats was not associated with immunosuppression caused by the viruses; in addition, there was a trend of correlation between seropositivity by T. gondii and cat access to the external environment, the presence of rats, as well as diet type.