Introduction

With the introduction of a vaccine against canine leishmaniasis in Brazil, customary serological tests fail in differentiate a dog showing symptoms of the disease from a vaccinated one that is not healthy. In case of development of any symptom of the disease in the latter, visceral leishmaniasis must be included in the differential diagnosis, since the vaccine does not give a 100% immunity and the animal may have been vaccinated after infection and before seroconversion. Several studies with two IgG subclasses (IgG1 and IgG2) have been performed in order to determine whether their response can be used as a marker of the resistance or susceptibility to visceral leishmaniasis, so that the treatment's efficacy can be assessed and vaccinated dogs can be differentiated from sick ones (Deplazes et al. 1995, Bourdoiseau et al. 1997, Cavaliero et al. 1999, Nieto et al. 1999, Boceta et al. 2000, Solano-Gallego et al. 2000, Leandro et al. 2001, Solano-Gallego et al. 2001, Cordeiro Da Silva et al. 2003, Mendes et al. 2003, Almeida et al. 2005, Iniesta et al. 2005, Reis et al. 2006, Cardoso et al. 2007, Da Costa-Val et al. 2007, Iniesta et al. 2007, Rodríguez-Cortés et al. 2007a). However, the results are very conflicting. According to Day (2007), it is clear that there is no consistent pattern related to a correlation between IgG1 or IgG2 and symptomatic or asymptomatic status in canine visceral leishmaniasis. In this light, the purpose of the present study was determine the profile of four IgG subclasses (IgG1, IgG2, IgG3 and IgG4) in asymptomatic dogs naturally infected with Leishmania chagasi and in dogs vaccinated against visceral leishmaniasis, in an attempt to differentiate serologically both animal groups.

Materials and Methods

Three groups of dogs from both sexes, several breeds and ages were used. The first group was composed of 45 clinically healthy animals, coming from nonendemic areas for visceral leishmaniasis (control), the second was composed of 45 symptomatic dogs with visceral leishmaniasis and the third was composed of 37 clinically healthy dogs which had been vaccinated against canine visceral leishmaniasis. The sample collection from animals of the third group happened in two moments: before the application of the first vaccine dose and 90 days after the application of the first vaccine dose, when the total IgG results indicated that seroconversion had already happened. The presence of anti-Leishmania chagasi IgG in serum was determined by the ELISA technique, as described by Lima et al. (2003). The presence of IgG1, IgG2, IgG3 and IgG4 in serum was determined by the ELISA technique, as described by Quinnell et al. (2003). The ELISA technique's cutoff determination was performed by using the sera from healthy dogs from a non-endemic area for the disease. To serve the purpose, it was considered the group mean added of three standard deviations. Therefore, the cutoffs used for the total anti-Leishmania chagasi IgG detection, IgG1, IgG2, IgG3 and IgG4 were 0.218; 0.110; 0.202; 0.088 and 0.041, respectively.

Results

The results of this study verified that 90 days after the application of the first vaccine dose all analyzed variables (total IgG, IgG1, IgG2, IgG3 and IgG4) showed statistically significant differences in relation to the initial moment. Despite the increase of all studied subclasses, IgG2 had median values below cutoff (0.166), even after seroconversion. The most stimulated classes after vaccination were IgG1 (from 0.044 to 0.813), IgG3 (from 0.027 to 0.497) and lastly IgG4 (from 0.015 to 0.138). Statistically significant differences between symptomatic and vaccinated dogs were observed, considering total IgG, IgG1, IgG3 and IgG4 in the two dog groups. The median values were 1.162 and 0.754 for total IgG; 1.677 and 0.813 for IgG1; 1.242 and 0.497 for IgG3; and 0.431 and 0.138 for IgG4, respectively, in symptomatic and vaccinated dogs. The optical density medians of IgG2 in symptomatic dogs and in vaccinated dogs did not show statistically significant differences, being 0.206 and 0.166, respectively.

Discussion and Conclusions

Despite statistically significant differences were observed between vaccinated dogs and symptomatic animals regarding total IgG, IgG1, IgG3 and IgG4, the values raised above the respective cutoffs in both dog groups. Therefore, it was not possible to differentiate a vaccinated dog having symptoms of any other disease from an animal naturally infected with Leishmania chagasi with symptoms of the disease. IgG2 could not be differentiated among groups either, since the optical density medians of symptomatic and vaccinated dogs do not show statistically significant differences. In this study, IgG1 showed a higher stimulation than IgG2, both in symptomatic dogs and vaccinated animals, thus disagreeing with the results of Mendes et al. (2003), who described, while working with polyclonal antibodies, that the IgG1 increase is associated to a natural infection, whereas the IgG2 is associated to vaccination. In vaccinated animals, the predominant subclass was IgG1, followed by IgG3, IgG2 and finally IgG4. In animals whose clinical picture was due to visceral leishmaniasis, the predominant subclass was IgG1, followed by IgG3, IgG4 and IgG2, corroborating the findings of Quinnell et al. (2003), in studies of symptomatic dogs with visceral leishmaniasis. The results obtained in this study's conditions led to the conclusion that the evaluation of the four IgG subclasses does not differentiate dogs vaccinated against visceral leishmaniasis from dogs naturally infected and bearing a clinical status of the disease.

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