ISMap02 element targeted nested polymerase chain in the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples of cattle and buffaloes
Background and Aim: Johne’s disease is chronic granulomatous enteritis which affects ruminants. There are many diagnostic approaches for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) of which molecular detection methods using various elements are less time consuming and more accurate. The present study was conducted using ISMap02 element for nested polymerase chain reaction (nPCR) based detection of MAP in fecal samples. The aim was to test the sensitivity and specificity of the ISMap02 element and also to use this element for the detection of MAP in fecal samples of cattle and buffaloes. Materials and Methods: A total of 211 fecal samples of cattle and buffaloes from different herds around Ludhiana aged between 2 and 13 years were collected, and DNA extraction was done from these samples. The nPCR was carried out for the detection of MAP in fecal samples. Results: The ISMap02 element was specific for the detection of MAP only and showed a sensitivity of detection of 7.6 fg/µL of the standard genomic DNA. Among the 211 fecal samples of cattle and buffaloes tested for the ISMap02 element, 18 samples (8.5%) were positive for MAP. Conclusion: The ISMap02 element is specific and sensitive for the detection of MAP in various samples, and when used in nPCR format, it can increase the sensitivity of detection.
Complete Genome Sequence of Ovine Mycobacterium avium subsp. paratuberculosis Strain JIII-386 (MAP-S/type III) and Its Comparison to MAP-S/type I, MAP-C, and M. avium Complex Genomes
Mycobacterium avium (M. a.) subsp. paratuberculosis (MAP) is a worldwide-distributed obligate pathogen in ruminants causing Johne’s disease. Due to a lack of complete subtype III genome sequences, there is not yet conclusive information about genetic differences between strains of cattle (MAP-C, type II) and sheep (MAP-S) type, and especially between MAP-S subtypes I, and III. Here we present the complete, circular genome of MAP-S/type III strain JIII-386 (DE) closed by Nanopore-technology and its comparison with MAP-S/type I closed genome of strain Telford (AUS), MAP-S/type III draft genome of strain S397 (U.S.), twelve closed MAP-C strains, and eight closed M.-a.-complex-strains. Structural comparative alignments revealed clearly the mosaic nature of MAP, emphasized differences between the subtypes and the higher diversity of MAP-S genomes. The comparison of various genomic elements including transposases and genomic islands provide new insights in MAP genomics. MAP type specific phenotypic features may be attributed to genes of known large sequence polymorphisms (LSPSs) regions I–IV and deletions #1 and #2, confirmed here, but could also result from identified frameshifts or interruptions of various virulence-associated genes (e.g., mbtC in MAP-S). Comprehensive core and pan genome analysis uncovered unique genes (e.g., cytochromes) and genes probably acquired by horizontal gene transfer in different MAP-types and subtypes, but also emphasized the highly conserved and close relationship, and the complex evolution of M.-a.-strains.
Molecular diagnosis of leishmaniosis in dogs. Comparative application of traditional diagnostic methods and the proposed assay on clinical samples.
Leishmaniosis is a zoonotic, parasitic disease caused by members of the genus Leishmania. The disadvantages of the traditional methods have currently rendered the polymerase chain reaction (PCR), the most reliable alternative for the laboratory diagnosis of this disease. Several relevant protocols have been described in the past but their application is in most cases limited to research use. The latter combined with the diagnostic problems that can be caused by the genetic variability of the different Leishmania strains or the presence of PCR inhibitors, indicate that an alternative approach should be followed for the development of a standard diagnostic tool for leishmaniosis. In the present study, we have evaluated several PCR-based protocols, in order to identify a primer combination that would allow the reliable detection of Leishmania DNA from clinical material and the verification of its results, in a manner that could be applicable even for routine use. The evaluation consisted of a BLAST verification of the specificity of the previously described primers, PCR testing, and optimisation of the reaction conditions. Our assessment was completed with the comparative evaluation of the results produced by the proposed PCR assay, light microscopy, and indirect fluorescent antibody technique (IFAT), on clinical samples collected from dogs suspected of leishmaniosis. The proposed assay which consists of a combination of two pairs of primers, targeted to different areas of the kinetoplast DNA of Leishmania spp., specific for Leishmania infantum, Leishmania donovani and Leishmania chagasi, showed optimum performance on our test samples, and detected 41.9% Leishmania-positive dogs from our 160 clinical cases. From the same number of cases, 46.25% were positive by IFAT (titre > or =200), and 19% by microscopic examination of lymph node aspirates.