Estudo epidemiológico prospectivo em cães assintomáticos infectados por Leishmania (Leishmania) infantum e identificação de biomarcadores de infecção

Abstract

Over the last three decades, visceral leishmaniasis has spread to a number of metropolitan areas in Brazil. Various factors contribute to the urbanization of the disease including the difficulties of implementing control measures relating to the domestic reservoir (delayed diagnosis and euthanasia, continuous replacement of dogs and lack of sensitivity of the methods of diagnosis used) and to the insect vector. Epidemiological studies using molecular techniques have previously indicated that theprevalence of canine visceral leishmaniasis (CVL) is significantly higher than that detected by serology. The aim of the present study was to determine the prevalence, incidence and the risk factors associated with Leishmania infantum infection among seronegative and polymerase chain reaction (PCR) positive dogs in the northwest sanitary district of Belo Horizonte, Minas Gerais, Brazil. In addition, this study was toperform a detailed analysis of the humoral and cellular immune response investigating in detail the canine asymptomatic infection compared with symptomatic dogs naturally infected by L. infantum, focussing on a possible biomarker that could be used to identifyresistant and susceptible profiles during ongoing CVL. A total of 1.443 dogs were examined and 230 (15.9%) were seropositive in at least one of the enzyme-linked immunosorbent assays applied, whilst PCR amplicons submitted to restriction fragment length polymorphism (RFLP) revealed that 356 animals (24.7%) were positive for L. infantum DNA. A cohort study was carried out follow seronegative dogs to identifyincidence of L. infantum infection. Dogs were followed during 26 months and the incidence rates were 5.8/100 dogs/months by PCR-RFLP and 1.2/100 dogs/months by ELISA. In order to identify the risk factors associated with the infection by L. infantum, only seronegative animals were evaluated in the cross-sectional study. Comparison ofthe two groups of dogs, PCR+ (296) and PCR- (917) with respect to the characteristics of the animals and their owners indicated that the risk factors associated with infection were family income < one minimum salary (OR: 4.1), knowledge of the owner regarding the vector (OR: 1.9), regular permanence of dog in the backyard (OR: 2.3), and lack of examination for CVL (OR: 1.6). Cox models were used to analyse thecohort study. The risk factors associated with infection were: presence of cases of CVL sometime in the past (HR. 1.4), unplastered wall (HR. 3.5), short-haired dog (HR. 1.3), and dogs run way from home (HR. 1.6). Also, the risk factors for seroconversion were: presence of rubble in the backward (HR. 2.8) and large dogs (HR. 2.8). In this study, asymptomatic dogs naturally infected with L. infantum were re-classified by serological and molecular evaluation as follows: Asymptomatic Dogs I (AD-I) with negative serology and positive molecular results and Asymptomatic Dogs II (AD-II) with positive serology and molecular diagnosis for L. infantum. Analysis of humoral immune response (IgG, IgG1, IgG2, IgM, IgA and IgE); immunophenotyping of T-lymphocytes and their subsets, B-cells and monocytes; and synthesis of intracytoplasmic IFN- and IL-4 by CD4+ and CD8+ T-cell subsets before and after antigen-specific stimulation in vitro; were performed in the AD-I and AD-II groups and compared with Symptomatic Dogs (SD) and Control Dogs (CD). AD-I presented immunophenotypic features similar to those detected in CD, including isotype profile and the number of monocytes cells.