Study of cross-reactivity in serum samples from dogs positive for Leishmania sp., Babesia canis and Ehrlichia canis in enzyme-linked immunosorbent assay and indirect fluorescent antibody test

Abstract

RESUMOPara verificar a existência de reação cruzada entre leishmaniose visceral, erliquiose e babesiose, nos testes sorológicos utilizados em programas de controle da leishmaniose visceral humana, amostras de soro canino provenientes de áreas endêmicas e não endêmicas para essa enfermidade, foram testadas pela Reação de Imunofluorescência (RIFI) e Ensaio imunoenzimático (ELISA). Todos os soros provenientes de área endêmica foram positivos para Leishmania sp pelo ELISA e RIFI, 51% para Babesia canis e 43% para Ehrlichia canis pela RIFI. Pela RIFI, nenhum dos soros provenientes de área não endêmica foi positivo para Leishmania sp, sendo 67% positivos para B. canis e 78% para E. canis pelo mesmo teste. Quando testados pelo ELISA para Leishmania sp., quatro soros da área não endêmica foram positivos. Os cães foram localizados e nenhum sinal clínico, parasito ou anticorpo foi detectado em novos exames realizados ao longo de seis meses. Os resultados desse trabalho sugerem portanto, a presença de uma co-infecção entre os três parasitos citados nas áre-as endêmicas e não a reação cruzada entre eles, nos testes sorológicos de RIFI e ELISA descritos. To verify the presence of cross-reaction among leishmaniosis, ehrlichiosis and babesiosis in serological diagnostics used in human visceral leishmaniasis control programs, serum samples from leishmaniasis endemic and non-endemic areas were collected and tested by Indirect Fluorescent Antibody (IFAT) and Enzyme-linked immunosorbent assay (ELISA). All serum samples from endemic areas were positive for Leishmania sp., by ELISA and IFAT, 51% positive for Babesia canis and 43% for Ehrlichia canis by IFAT. None of the serum samples from non-endemic areas were positive for Leishmania sp., by IFAT, but 67% were positive for B. canis and 78% for E. canis using the same test. When tested by ELISA for Leishmania sp., four samples from non-endemic area were positive. These dogs were then located and no clinical signs, parasites or antibody was detected in new tests for a six month period. Only one of these 4 samples was positive for B. canis by IFAT and ELISA and three for E. canis by IFAT. The results of the work suggest a co-infection in the endemic area and no serological cross-reaction among these parasites by IFAT and ELISA.

Serodiagnosis of visceral and cutaneous leishmaniasis in human and canine populations living in Indigenous Reserves in the Brazilian Amazon Region

“…After the antigen had been obtained, the ELISA test was performed as described previously 16 for Babesia bovis, with small modifications 17 …”

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs

“…These negative reference sera were collected during a rabies vaccination program and were immunologically tested for IgG antibodies anti-Leishmania sp. by IFAT and ELISA (Oliveira et al, 2008). The results have demonstrated that a soluble antigen of L. chagasi prepared from in vitro culture of the parasite could be used in a sensitive and specific ELISA to detect IgM antibodies for Leishmania sp.…”

“…The IFAT was carried out according to Oliveira et al (2008) for IgG antibodies in order to screen positive IgM control serum samples for the ELISA test. Promastigotes of L. chagasi, for antigen preparation, were washed three times in PBS with 1% BSA and resuspended in PBS-buffered formalin for 30 minutes.…”

“…The ELISA was originally described by Oliveira et al (2008). To summarize, 100mL of L. chagasi total soluble antigen diluted in a sodium bicarbonate-carbonated 0.05M buffer (pH 9.6) was added per well to an ELISA plate (Nunclon TM surface; Nunc, Denmark) and protein concentration was adjusted to 5μg/mL, 10μg/mL, 20μg/mL and 40μg/mL.…”

Rural area of the Brazilian Pantanal wetlands associated with the occurrence of anti-<i>Leishmania</i> spp. antibodies in dogs

“…Anti-Leishmania spp. antibodies were evaluated by immunofluorescence assay (IFA), as described by Oliveira et al (2008), using the promastigote forms of isolate CBT 96 (Coleção Brasileira de Tripanossomatídeos) of L. infantum chagasi (COSTA et al, 2015) with a cut-off point at the initial dilution of 1:40 (BRASIL, 2006). Each slide contained sera previously known to be non-reactive (negative control) and reactive (positive control).…”