Advances in canine leishmaniosis with emphasis on the use of saliva samples

Abstract

This PhD thesis as a compendium of publications was conceived to go in-depth on the study of the development of assays that could allow the use of saliva samples to diagnose leishmaniosis in dogs. The objectives were: (1) to develop and validate an ultrasensitive assay for anti-Leishmania antibodies quantification that could allow their measurement in serum and saliva of dogs; (2) to evaluate the usefulness of the assay for anti-Leishmania antibodies measurement in both serum and saliva samples as a tool for monitoring the treatment of canine leishmaniosis (CanL); (3) to assess the possible relationship between serum anti-Leishmania antibody levels measured by the new assay and by a commercially-available ELISA kit and the concentration of acute phase proteins; (4) to evaluate and compare the kinetics of anti-Leishmania IgG2 and IgA by the new assay in serum and saliva from experimentally infected dogs with L. infantum during one-year follow-up; (5) to assess the possible circadian rhythm of anti-Leishmania IgG2 and IgA levels in serum and saliva from experimentally infected dogs; and (6) to develop and validate a qPCR for quantification of Leishmania spp. DNA in canine saliva. Precision, accuracy and analytical sensitivity were evaluated by the coefficients of variation intra- and inter-assay, linearity under dilution, percentage of recovery and correlation study, and limits of detection and limits of quantification to validate the new assays. For that, serum and saliva samples from Leishmania-seronegative and Leishmania-seropositive dogs were collected. To evaluate the usefulness of the TR-IFMAs for treatment monitoring, serum samples were obtained from 16 Leishmania-seropositive dogs at the time of diagnosis and after 30 and 180 days of treatment. As well as, to evaluate the utility of the TR-IFMAs for treatment monitoring using saliva samples, serum and saliva samples were collected from 20 dogs with clinical signs compatible with CanL at the time of diagnosis and after 30 days of treatment. Also, 205 serum samples were evaluated to study the correlation between the TR-IFMA, a commercial ELISA and the concentration of acute phase proteins in dogs at the time of diagnosis and during treatment monitoring. To study the kinetics of anti-Leishmania antibody levels, 11 dogs were infected with Leishmania infantum. After, serum and saliva samples were monthly collected during 1-year and analyzed by TR-IFMAs. To assess the possible circadian rhythm in the levels of anti-Leishmania antibodies serum and saliva of 6 dogs experimentally infected presenting clinical signs of CanL were collected during a 16-h period at 4-h intervals on two consecutive days. Finally, to develop and validate a qPCR for the detection of Leishmania spp. DNA in saliva of dogs, saliva samples from 16 dogs experimentally infected were collected pre-infection and at 16-weeks post-infection. The conclusions of this PhD thesis are: 1. The immunofluorometric assays developed in this PhD thesis allow the quantification of anti-Leishmania IgG2 and IgA in canine serum and saliva samples in a reliable way. An overlap between seropositive and seronegative dogs was observed for IgA. 2. The assays developed detect decreases in anti-Leishmania IgG2 and IgA levels in serum and saliva from dogs with clinical leishmaniosis after treatment, which is associated with clinical improvement. 3. The assay developed for anti-Leishmania IgG2 is more correlated with CRP and ferritin concentrations than the ELISA results in treated dogs. 4. Levels of anti-Leishmania IgG2 and IgA in canine serum and saliva increase after experimental infection, surpassing the cut-off approximately one month earlier in serum than in saliva. Anti-Leishmania IgG2 shows better diagnostic value than IgA. 5. No circadian rhythm is observed for anti-Leishmania IgG2 and IgA levels in both serum and saliva samples of dogs with CanL during a 16 h-period. Additionally, higher intra-individual variations in the specific antibody levels are observed in saliva than in serum. 6. The qPCR assay developed allows the quantification of Leishmania kDNA in saliva samples of experimentally infected dogs with L. infantum. However, the sensitivity of this assay in the saliva is lower than in bone marrow.