Comparative Evaluation of Enzyme-Linked Immunosorbent Assays Based on Crude and Recombinant Leishmanial Antigens for Serodiagnosis of Symptomatic and Asymptomatic Leishmania infantum Visceral Infections in Dogs

Abstract

The diagnosis of visceral leishmaniasis remains difficult in rural areas where the disease is endemic, and serologic methods still need assessment, as they are not very sensitive for the detection of asymptomatic infectious dogs. Here we present data on the development of enzyme-linked immunosorbent assay (ELISA)-based methods for the detection of antibodies against recombinant leishmanial antigens (namely, the recombinant K26 [rK26] and rK39 antigens from Leishmania infantum and the rA2 protein from Leishmania donovani) in comparison to ELISAs employing crude soluble antigen (CSA). The assays utilized sera from known negative controls (n ‫؍‬ 25) and clinically asymptomatic (n ‫؍‬ 50) and symptomatic (n ‫؍‬ 50) dogs with confirmed L. infantum infections. Additional studies were also done using sera from animals harboring other infections (n ‫؍‬ 14) for the evaluation of cross-reactivity. Our study indicated that rK26 and rK39 used in ELISAs provided very high sensitivities for the detection of symptomatic dogs (94% and 100%, respectively), followed by CSA (88%) and rA2 (70%). Conversely, rA2 was more sensitive for asymptomatic dogs (88%) than rK39 and rK26 (both 66%) and CSA (30%). Some cross-reactivity in sera from dogs with other infections (Leishmania braziliensis and Leptospira interrogans) was identified, but the rA2 protein provided the greatest specificity (98%). Data further indicate that all three recombinant proteins must be used in parallel to detect essentially all infected dogs. Efforts should be made to develop a cheap and reliable serologic test based on epitope selection from these diagnostic markers for the sensitive detection of L. infantum-infected dogs.

Expression of a recombinant protein, A2 family, from Leishmania infantum (Jaboticabal strain) and its evaluation in Canine Visceral Leishmaniasis serological test

“…Porrozzi et al (2007) showed that ELISA based on crude antigens shown minor percentage (30%) for asymptomatic dogs than that ELISA based on recombinant leishmanial antigens rA2 (88%); rK39 and rK26 (66%). Thus, many efforts have been done to development more sensitive, specific and rapid tests to detection of CVL helping in epidemiology and to control the disease (ZIJLSTRA et al, 2001;ATTAR et al, 2001;MOHEBALI et al, 2004;AKHOUNDI et al, 2013).…”

“…Additional investigation with symptomatic and asymptomatic dogs, done by parasitological tests, may provide further information for a better understanding of these results. Porrozzi et al (2007) showed that serologic tests using the recombinant protein rK39 and rK26 (both from L. infantum) were more efficient to identify symptomatic dogs while rA2 (from Leishmania donovani) seem to be effective for the serodiagnosis of asymptomatic dogs with the disease. Interestingly, only two of the 29 Leishmune  -vaccinated animals were seropositive by A2_ELISA, and showed ELISA levels near the cut-off point.…”

“…Dogs from non-endemic areas with previously negative results with L. infantum -IFAT and SLA-ELISA studies were also negative by A2_ELISA. Further studies are needed to improve the specificity of the developed assay and more satisfactory results using the A2 protein for serodiagnosis of VL could probably be achieved by using a broader spectrum of proteins instead of a single protein (GHEDIN et al, 1997;PORROZZI et al, 2007;COSTA et al, 2012). Moreover, the high specificity and sensibility of crude antigen compared to the soluble recombinant protein is due to the broad spectrum of protein available for antibody binding.…”

Comparison of recombinant A2-ELISA with rKE16 dipstick and direct agglutination tests for diagnosis of visceral leishmaniasis in dogs in Northwestern Iran

“…This report from Brazil provided a comparison of ELISA using L. infantum rK26 and rK39 antigens and the L. donovani rA2 protein, as well as ELISA with crude soluble antigen (CSA), indicating a high sensitivity for rK26 and rK39 in the detection of symptomatic dogs (94% and 100%, respectively), followed by CSA (88%) and rA2 (70%). Conversely, rA2 was reported to be more sensitive for asymptomatic dogs (88%) than rK39 and rK26 (both 66%) and CSA (30%) (9) .…”

“…Therefore, there is a need to develop a more rapid and simple assay for the diagnosis of VL (9) . Because the elimination of infected dogs is an important mean for controlling the transmission of VL, an ideal diagnostic test for VL in dogs would have to be inexpensive and simple, in addition to being specifi c and sensitive (9) .…”