A Novel Exo-antigen-based ELISA for the Detection of Canine Leishmaniasis

Abstract

Dogs which are infected with leishmania parasites serve as major reservoir hosts for zoonotic visceral leishmaniasis. The incidence of zoonotic visceral leishmaniasis is rising in many countries. This may be associated with the continuing drift of people and their pets from rural areas into peri-urban settings, particularly at the fringe of large cities. At the same time, there is evidence of adaptation of sand fly vectors to these urban settings. This has created an alarming situation because, even though domestic and stray dogs may be infected, many remain asymptomatic but are still highly infectious to the sand fly vectors and thus pose a serious threat to human health. Over half of the infected dogs have asymptomatic infections and current assays are not sensitive enough under field conditions to distinguish asymptomatic from symptomatic dogs. There is an urgent need for a specific and sensitive screening tool for use in the field. We have previously demonstrated that promastigote exo-antigen-based ELISAs can be used in the specific diagnosis of human visceral leishmaniasis (HVL). A cocktail of exo-antigens prepared from three species (L. infantum, L. donovani, and L. major) was used to develop and optimize a canine ELISA assay. Serum samples from dogs with a variety of pathological conditions but living in a non-leishmania endemic area were used as negative controls and their reactivity was used to determine a cut-off value for the ELISA. Samples from dogs residing in a leishmania endemic area were tested in parallel using direct agglutination (DAT), immunofluorescence (IFAT), and ELISA. The ELISA results correlated closely (100%) with the clinical symptoms, and were elevated in one asymptomatic dog. This sample was also found to be positive by IFAT. Based on its sensitivity and specificity, the cocktail exo-antigen-based ELISA may prove useful, even at 1:2,000 serum dilutions, for screening dogs in different geographical regions of the world.

Serological Evidence of Infection byLeishmania (Leishmania) infantum(Synonym:Leishmania (Leishmania) chagasi) in Free-Ranging Wild Mammals in a Nonendemic Region of the State of São Paulo, Brazil

“…Serological cross-reactions may also occur with other species of the genus Leishmania, although the antigen used in this study is recommended for the detection of anti-L. donovani complex antibodies. However, the DAT generally provides excellent diagnostic accuracy, with high sensitivity and specificity reported by numerous authors (Silva et al 2005, Sundar et al 2006, Ritmeijer et al 2006, Rajasekariah et al 2008, Sukmee et al 2008, Mahmoudvand et al 2011, Sousa et al 2011.…”

“…In addition, this cutoff point has also been used in several studies involving domestic and/or wild canids, which are the most frequently studied species (De Korte et al 1990, Semião-Santos et al 1995, 1996, Ozbel et al 2000, Mohebali et al 2005, Rajasekariah et al 2008, Mahmoudvand et al 2011, Sharifdini et al 2011, Moshfe et al 2012). …”

Serological diagnosis of canine leishmaniosis: comparison of three commercial ELISA tests (Leiscan®, ID Screen® and Leishmania 96®), a rapid test (Speed Leish K®) and an in-house IFAT

“…A problem associated with this lack of “gold standard” is that a standardization of results is not possible, which means that comparison of results between studies is difficult. In addition, there is a clear tendency in many studies to select only sick dogs with clinical signs and there are limited studies with descriptions of clinical staging and severity of illness, thus, an imbalance between studies on infected sick dogs and subclinical infected dogs exist [19,34]. This type of approach can lead to obtaining higher values of diagnostic performance measures than would be achieved with more heterogenous dog groups.…”

The Efficacy of L. (L.) chagasi Excreted-Secreted Antigens (ESAs) for Visceral Leishmaniasis Diagnosis Is Due To Low Levels of Cross-Reactivity

“…8,11 Leishmania spp. conditioned medium has been used as a potential source of antigens for immunodiagnosis in the detection of cases of human VL, 5,6,12,13 canine leishmaniasis, 12,14 and cutaneous leishmaniasis (CL). 13,15 Although serological tests, such as the direct agglutination test, immunofluorescence test, indirect hemagglutination assay, enzyme-linked immunosorbent assay (ELISA), and Western blotting have been found to display high levels of sensitivity, the specificity indices were not significant.…”