Automated multi-sample DNA extraction for genotyping live Xenopus embryos.

BACKGROUND: Xenopus frogs are used extensively for modeling genetic diseases owing to characteristics such as the abundance of eggs combined with their large size, allowing easy manipulation, and rapid external embryo development enabling the examination of cellular and phenotypic alterations throughout embryogenesis. However, genotyping of mutant animals is currently done either as part of a large group, requiring many embryos, or late in development with welfare effects. Therefore, we adapted the Zebrafish Embryonic Genotyper for rapid genomic DNA extraction from Xenopus tropicalis and Xenopus laevis at early stages. RESULTS: Sufficient and good quality DNA was extracted as early as the Nieuwkoop and Faber stage 19 and, importantly, no detrimental effects of the extraction process on the subsequent tadpole development, behavior, or morphology were observed. Amplicons of up to 800 bp were successfully amplified and used for further analyses such as gel electrophoresis, T7 endonuclease I assay and Sanger sequencing. CONCLUSION: This method allows rapid genotyping during the early stages of Xenopus development, which enables safe identification of mutants. These can be analyzed at early developmental stages or selected for raising without the need for invasive genotyping later, with resource savings and ethical gains in line with the 3Rs principles.

The use of recombinant K39, KMP11, and crude antigen-based indirect ELISA as a serological diagnostic tool and a measure of exposure for cutaneous leishmaniasis in Sri Lanka.

Cutaneous leishmaniasis (CL) in Sri Lanka is caused by Leishmania donovani, a parasite widely known to cause visceral leishmaniasis. Despite the fact that CL is not generally believed to elicit serological immune responses, recent studies show the presence of antibody responses against this atypical form of CL. This study assesses the potential of using recombinant K39 (rK39), KMP11, and crude parasite antigen-based indirect ELISAs as serological diagnostic tools and measures of exposure for CL in Sri Lanka. The study used serum samples from confirmed CL patients (n = 266) and apparently healthy individuals from endemic settings (n = 411). Serum samples from individuals residing in non-endemic areas were used as negative controls. In-house indirect ELISAs were optimized and validated for recombinant antigens. Previously validated crude parasite extract-based indirect ELISA was performed for comparison. The statistical analyses were performed using SPSS v26.0. The rK39 (sensitivity = 71.2%, specificity = 64%) and KMP11 (sensitivity = 79.2%, specificity = 71.4%) based indirect ELISA were shown to be less suitable for the diagnosis of CL, while crude parasite extract-based indirect ELISA (sensitivity = 82.4%, specificity = 85.7%) might be a better method of diagnosis. All 03 ELISAs seemed to be good methods as measures of exposure since correlations were observed between the seropositivity of all 03 ELISAs (rK39: p = 0.037, KMP11: p = 0.007, CrudeAg: p = 0.000) with provincial case incidences. The findings will be important in identifying the disease hotspots in order to design the control measures for CL induced by L. donovani in Sri Lanka.