Early antibody response and clinical outcome in experimental canine leishmaniasis

Abstract

Infected dogs are the main reservoir of zoonotic visceral leishmaniasis, a widespread parasitic disease caused by Leishmania infantum. Therefore, the control of canine infections is required to reduce the incidence of human cases. Disease outcome in dogs depends on the fine balance between parasite virulence and efficacy of the immune system. Thus, knowledge of early response could yield relevant information for diagnosis and follow-up. In our study, 20 Beagle dogs were intravenously infected with 108 amastigotes of a fresh isolate of L. infantum and monitored along 16 weeks post inoculation. Specific antibody response and clinical evolution of infected animals were highly variable. Immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA) were useful to assess infection status, although only ELISA with promastigote-coated plates and, particularly, western blotting (WB) allowed an early diagnosis. Prominent antigens were identified by mass peptide fingerprinting. Chaperonin HSP60, 32 and 30 KDa antigens were recognized by all dogs on week 10 post infection. This suggests that these antigens may be valuable for early diagnosis. Advanced infection showed, in addition, reactivity to HSP83 and HSP70. Disease outcome did not show a clear relationship with ELISA or IFAT titers. Correlation between the clinical status and the combined reactivity to some antigens sustains their use for diagnosis and follow-up.

Infection of dogs by Leishmania infantum elicits a general response of IgG subclasses

“…L. infantum strain was obtained from the spleen of a naturally infected dog from southern Spain (Órgiva, Granada), clinically and serologically diagnosed. Details on the isolation of parasites to inoculate the dogs have been previously published 49 . Briefly, the spleen was cut into small pieces and homogenized in a glass-in-glass tissue grinder.…”

“…Dogs (24 female Beagles) were obtained when they were 4–5 months old (Envigo, France) and housed at the animal facility Nr ES280790000091 of the Faculty of Veterinary Medicine UCM (Madrid). Details of the housing, inoculation and follow-up of the experimental animals have been previously published 49 . Briefly, when the animals reached 10–11 months age, 20 were inoculated (cephalic vein) with 10 8 amastigotes of L. infantum /animal, in 1 mL.…”

“…Follow up included IFAT and infection status of the animals on week 16th pi by popliteal lymph node biopsy. Clinical scoring (CS) of the animals ( Suplementary Information ) included clinical signs and lesions, and biochemical and hematological abnormalities, and was determined following a previous publication 49 .…”

“…Procedures to propagate promastigotes by back transformation of amastigotes from the original isolate and to obtain soluble leishmanial antigen (SLA) have been described previously 49 . Briefly, promastigotes were cultured in RPMI 1640 medium, supplemented with antibiotic–antimycotic mixture (BioWhittaker), l -glutamine (BioWhittaker), 10% fetal calf serum (Gibco) and human urine (1%).…”

“…This is most critical since the inaccurate determination of the immune scenario (cytokines, cells and immunoglobulins, particularly IgG subclasses) in canine resistance, resilience or susceptibility is a strong shortcoming to develop vaccines or to assess the efficacy of chemotherapy or immunotherapy. In our case, the availability of sera from a large number of dogs subjected to an experimental infection with a recently isolated wild strain of L. infantum 49 allowed us to determine the dynamics of production of specific IgG subclasses and their relationship to the clinical course of the experimental animals.…”