Evaluation of the toxicogenetic potential of visceral leishmaniasis in naturally infected dogs and salivary samples for definitive disease diagnosis

Abstract

Visceral leishmaniasis is an infectious and zoonotic disease caused by the protozoan Leishmania infantum chagasi, transmitted through the bite of the sandfly Lutzomyia longipalpis, with dogs serving as the primary reservoir. Given the increasing spread of this disease in dogs and its epidemiological and public health significance, this study aimed to investigate whether salivary samples from naturally infected dogs could serve as a diagnostic tool, attempting to replace invasive conventional methods. Additionally, the study analyzed the potential toxicogenetic effects of the disease. Ten adult dogs, both males and females, intact and neutered, of mixed breed, from Rifaina (SP) and attended at the Veterinary Hospital of the University of Franca, were included. These dogs exhibited various clinical signs suggestive of visceral leishmaniasis, with five of them confirmed through serological and parasitological examinations. Salivary samples were collected using sterile swabs from the five seropositive dogs, placed on glass slides, and stained with hematoxylin-eosin for subsequent analysis of amastigote forms of the protozoan using bright-field microscopy; the results were descriptive. Furthermore, bone marrow samples from the sternum were collected from the five seropositive dogs and compared with those from the five seronegative dogs to investigate the toxicogenetic potential of the disease through the micronucleus test. To assess possible genotoxicity, each slide made from bone marrow was analyzed for 2000 immature erythrocytes (IE), totaling 4000 IE/animal, and those containing micronuclei (MN) were identified (IE-MN). Cytotoxicity was determined by analyzing 1000 erythrocytes/slide, totaling 2000 erythrocytes/dog, and subsequently calculating the ratio of IE/IE+NCE (normochromatic erythrocytes). Toxicogenetic results of seropositive dogs were compared with seronegative dogs and statistically verified by simple analysis of variance (ANOVA). The protozoan Leishmania was not detected in any of the salivary samples from seropositive dogs. The mean frequency of IE-MN in seropositive dogs was statistically higher compared to seronegative dogs (p<0.0001), indicating the genotoxicity of the disease. In two of the five seropositive dogs, a decrease in erythrocyte production suggested cytotoxicity of the disease. Given the established methodology, it can be inferred that simple saliva analysis, without more specific assays, does not detect amastigote forms of the protozoan in dogs seropositive for visceral leishmaniasis and should not replace established diagnostic methods. Additionally, the micronucleus test suggested the genotoxicity and cytotoxicity of the disease.