The study was undertaken during 2013−2017 at Zoonoses Research Laboratory, Tamil Nadu Veterinary and Animal Sciences University, Chennai. Blood smears, soil and environmental samples were collected from 23 sporadic incidences of anthrax reported in different parts of Tamil Nadu from 2013−2017. 11 incidences were presumptively diagnosed as anthrax by detecting capsulated bacilli on polychrome methylene blue staining and microscopy. The spores in soil samples (62) were extracted, cultured and DNA was extracted for molecular method of diagnosis. The molecular method detected twelve incidences as anthrax by amplifying the virulence factors like lef gene (385bp) and capsular gene (264bp) encoded in pXO1 and pXO2 plasmids respectively and chromosomal marker (Ba813) gene (152bp) using multiplex PCR. The partial sequence of cap gene of isolates (Vellore and Nolambur strain) revealed a close relationship with B. anthracis species. The relatedness of the isolated strains to virulent strains such as Ames and Vollum strain on phylogenetic analysis showed the nature of the outbreak. Since the multiplex PCR detects the outbreak with short turn-around time by detecting both chromosomal and Plasmid DNA, considered as a confirmative diagnosis. Further the molecular method of diagnosis precludes the need for culturing thereby avoiding unnecessary occupational risks and environmental contamination.
Background: Canine Leptospirosis is a life-threatening disease and zoonosis. Usually, PCR assay is carried out for early diagnosis but requires a thermal cycler and post-PCR procedures. This limits its use in resource-limited areas. Hence, the isothermal amplification of nucleic acid by recombinase polymerase amplification assay was developed as a versatile alternative for the diagnosis of canine leptospirosis in this study. Methods: The RPA assay to detect Leptospira DNA was optimized with Leptospira reference strains and its performance characteristics such as analytical, diagnostics and reproducibility were assessed. Result: The limit of detection of RPA assay was estimated as 102 copies of genomic DNA and specific to amplify the pathogenic Leptospira. Out of 150 dog samples screened, Leptospira DNA was detected in 64 (42.6%) by RPA assay and 67 (44.6%) by PCR. The diagnostic sensitivity and specificity of the RPA assay were 92.5% and 97.59% respectively. The RPA assay has a good diagnostic agreement with a kappa value of 0.905. The reproducibility assessment with the third-party testing laboratory revealed a better agreement with a kappa value of 0.81. The simplicity, rapid and less expensive enable this assay to perform at resource-limited laboratories or point-of-care testing.
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