Introduction

Leishmaniasis is a group of illnesses transmitted by sand flies that affect domestic and wild animals, as well as human. Dogs are considered the main reservoir of etiologic agent of visceral leishmaniasis (VL) in the urban context and one of the targets in the control strategy. In addition to the great lethality if untreated, Leishmania infantum = L. chagasi, agent of VL, is capable of modulating the lymphocyte function, causing a severe immunosuppression in their hosts (Pinelli et al. 1994). The compromised immune system can facilitate the infection, proliferation and spread of the pathogens after the initial infection or allow the emergence of latent infection by opportunistic agents. Neospora caninum and the related parasite Toxoplasma gondii are coccidians cosmopolitans that cause neurologic disease in dogs, both protozoans using carnivores, respectively the dog and the cat, as the definitive host, which excrete oocysts in their feces, contaminating the environment and infecting a wide range of intermediate hosts. The objective of this study was to analyze the humoral immune response against N. caninum and T. gondii in dogs naturally infected with L. chagasi presenting different clinical conditions, and compare with healthy dogs.

Materials and Methods

The urban dogs used in this study were captured by Zoonosis Control Center in the county of Santa Luzia, metropolitan region of Belo Horizonte, Minas Gerais, Brazil, in September 2005. Sixty dogs from different breeds, of unknown age and naturally infected with L. chagasi were identified during an epidemiological survey of canine visceral leishmaniasis. The infection and serology diagnosis was confirmed by IFAT, with a cut off 1:40, and ELISA. In addition, parasitological diagnosis was reached by observations of parasite forms in cytological examination of bone marrow aspirates. All infected dogs were clinically classified according to as follows (Mancianti et al. 1988): Asymptomatic dogs--apparently healthy animals; Oligosymptomatic dogs--animals exhibiting lymphoid adenopathy, moderate weight loss and/or dull brittle fur; Symptomatic dogs--animals that exhibited the classical signs of the disease such as cutaneous alterations alopecia, exfoliative dermatitis or ulcers, onychogryphosis, keratoconjunctivitis, cachexia and anemia. Other 50 healthy dogs seronegative to L. chagasi, from the same municipality, also participated of the study as VL uninfected control group. Anti-N. caninum and anti-T. gondii antibodies were detected by IFAT, with cutoff titers of 50 and 16 respectively. Statistical analyses were conducted using the SPSS software version 9.0 for Windows. Possible associations between seropositive and seronegative samples for Leishmania, the presence of anti-T. gondii and anti-N. caninum antibodies were analyzed through Fischer test or Chi-square test (p = 0.05). The comparison between T. gondii and N. caninum occurrence in the different VL dog groups asymptomatic, oligosymptomatic and symptomatic were analyzed through Chi-square test (p = 0.05). The comparison between Leishmania spp. titers ranks from asymptomatic, oligosymptomatic and symptomatic dogs were made through the Kruskal Wallis test, using Mann Whitney as postroc test.

Results

Among the 60 naturally infected dogs with L. chagasi, 10 (16.7%) were clinically classified as asymptomatic, 22 (36.7%) as oligosymptomatic and 28 (46.7%) as symptomatic. The titers ranged from 80 to 81920, being the most frequent titer 20480 (14/60). Lymphadenopathy (44/50, 88%) was the most frequent sign, followed by skin disorders, which included in decreasing order of frequency, dull fur (40/50, 80%), exfoliative dermatitis (38/50, 76%), hypotrichosis/alopecia (30/50, 60%) and skin ulcers (16/50, 32%). Ophthalmopathies was observed in 12 of 50 (24%) dogs while onychogryphosis in 8% (4/50) of the animals. The occurrence of anti-N. caninum antibodies was 4.5% (1.5% < IC, 95% < 10.3%) with 5 positive out of 110 dogs, all with titers of 50. For T. gondii, 36 out of 110 dogs (32.7%) had positive results (24.1% < IC, 95% < 42.3%) and titers ranged from 16 to 4096, being the most frequent titer 128 (9/36) (Table 1). The occurrence of anti-N. caninum antibodies was 6% (3/50) in the L. chagasi negative group, while in the L. chagasi seropositive group the occurrence was 3.3% (2/60). No association between the Leishmania infection and N. caninum antibodies was observed (P = 0.657). Anti-T. gondii antibodies in the seronegative and seropositive L. chagasi groups showed an occurrence of 42.0% (21/50) and 25.0% (15/60), respectively. The statistical analyses showed no significant association between the canine leishmaniasis and T. gondii antibodies. However, when presence (oligosymptomatic and symptomatic) and absence (asymptomatic) of clinical signs in VL dogs were evaluated (Table 2), a tendency of association was observed (P = 0.053) with higher occurrence of infection for T. gondii in animals with symptoms of VL. No significant difference was observed between T. gondii titer ranks from the oligosymptomatic and symptomatic groups (p > 0.05). In the VL infected group 3.3% (2/60) of the animals were positive simultaneously for N. caninum and T. gondii and in the Leishmania seronegative group, coinfection was observed in 2.0% (1/50) of the dogs. The proportion test reveal no significant difference for T. gondii and N. caninum coinfections in the positive or negative L. chagasi groups (p > 0.05).

Discussion and Conclusions

Studies with dogs infected with Leishmania spp. and the associations with other coccidian parasites have been conducted, however opposite results were observed. The coinfection between this three parasites in dogs have been studied (Gennari et al. 2006) also in dogs from Brazil, Araçatuba SP, however, the clinical status of the animals related to VL was not reported. In this study the prevalence of N. caninum (17.6%) and T. gondii (36.8%) was higher than the values found in the present study and an association between the presence of L. chagasi and N. caninum antibodies was observed. Other two studies conducted in Mato Grosso do Sul, Brazil (Andreotti et al. 2006) and in Italy (Cringoli et al. 2002) observed, respectively, a non association and an association between dogs L. chagasi and N. caninum positive. Also in Italy, a description of a single case of a simultaneous infection with L. infantum and N. caninum in a dog with a skin infection was reported and the authors concluded that the immunosuppressive effect of L. infantum may be contributed to the development of the N. caninum infection, however, based in histological features, the skin lesions observed were most likely due to Leishmania (Tarantino et al. 2001). In the present study N. caninum seroprevalence was 4.5% and the occurrence in Leishmania positive (3.3%) and negative (6%) dogs were lower than the values observed in dogs from other regions of Brazil (Andreotti et al. 2006; Gennari et al. 2006), indicating a low rate of environmental exposure of the dogs to this agent in the region. All dogs with anti-N. caninum antibodies present titers of 50 and against T. gondii, that most frequent titer was 128. In a cross-section study conducted in apparently healthy dogs from state of Paraíba, Northeast region of Brazil, 50% of the seropositive dogs presented low titers; however some very high titers were also observed for both coccidian parasites (Azevedo et al. 2005). T. gondii occurrence was significantly higher when compared to N. caninum in tested dogs from both groups, what is expected based on observations by others regions from Brazil (Azevedo et al. 2005; Dubey et al. 2007) reflecting the high exposure of this host species to T. gondii in Brazil and the fact that dogs are considered good sentinels for T. gondii environmental contamination. The coinfection between coccidians in the dogs was 2% (1/50) and 3.3% (2/60) of animals were positive simultaneously for all three parasites. This data was similar as previous studies, where coinfection for N. caninum and T. gondii in dogs vary between 1% and 3% and different from the 17.3% (Gennari et al. 2006). The number of positive dogs for N. caninum in each L. chagasi clinical group was not significantly different, suggesting that VL, independently of the clinical form, not influence of presence of antibodies anti-N. caninum. When VL dogs were analyzed as a single group, no association between the infection for Leishmania and the presence of antibodies anti-N. caninum was observed. Similar results were found in asymptomatic dogs from Campo Grande (Andreotti et al. 2006). However, others authors (Gennari et al. 2006) verified a significant association between dogs naturally infected with L. chagasi and high occurrence of N. caninum antibodies 8.6 times greater than VL negative dogs. Working with 1058 asymptomatic dogs from south of Italy (Cringoli et al. 2002) also reported the same association and suggested that immunosuppression is likely the cause of the greater prevalence of N. caninum in Leishmania seropositive dogs. Toxoplasmosis is a disease usually associated with immunosuppressive disorders. In the present survey, among dogs infected with L. chagasi, verified a tendency of association between infection for T. gondii and animals with symptoms, suggesting that clinical form in the canine leishmaniasis appear to enhance susceptibility to T. gondii. Is expected that the immunodeficiency caused by Leishmania helped underlying agents and subsequently the immune system was active. In humans, mice and dogs it was demonstrated that the protective immunity against leishmaniasis is mediated by T cells and associate to the production of IFNγ. The effectiveness of the cellular immunity is a basic aspect in pathogenesis of the illness and its progression. Due the ineffectiveness of the cellular immunity, its likely that dogs with VL that manifesting symptoms facility more the multiplication and spread of the parasites controlled primarily by cellular defenses, for example T. gondii, when compared to asymptomatic condition that present protective cellular immunity. Some authors had affirmed that the detection of antibodies indicate the presence of the parasite, but are not correlated with the severity of the clinical signals or activity of the illness in canine VL, however, in this study, significant levels of antileishmanial antibodies were observed in different clinical groups, with higher titers in the symptomatic dogs. Others publications also registered correlation between the levels of immunoglobulin isotypes and different clinical forms of the canine VL (CostaVal et al. 2007). The clinical signs verified in the dogs of this experiment corroborated the results of the majority of authors (Amusategui et al. 2003; CostaVal 2007), and demonstrated the lymphadenopathy with the more common clinical manifestation of the disease. Further studies involved different immunological aspects should be developed with dogs infected with Leishmania exhibited all clinical forms to determine the role of the immune system in the coinfection by other parasites, especially the opportunists.

Table 1. Occurrence of antibodies against Neospora caninum and Toxoplasma gondii in dogs positive (n = 60) and negative (n = 50) to Leishmania chagasi from the municipality of Santa Luzia, Minas Gerais, Brazil.

a Chi-square Test
b Fisher Test

Table 2. Occurrence of antibodies against Neospora caninum and Toxoplasma gondii in dogs infected with Leishmania chagasi with different clinical forms of canine leishmaniasis (n=60) and in no infected control dogs (n = 50) from the municipality of Santa Luzia, Minas Gerais, Brazil.

Comparisons between letters on the same column
a. P > 0.05 (Chi)
b. P = 0,053 (Fisher Test)
c. P = 0.657 (Fisher Test)
d. P = 0.058 (Chi-square Test)

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