Mutations in RpoB Gene and Their Association with Rifampicin-resistance Levels in Clinical Isolates of Mycobacterium Tuberculosis

Present study was aimed to identify most frequent mutations in rpoB gene region and to evaluate the association between mutations in rpoB gene and resistance levels to Rifampicin in clinical isolates of Mycobacterium tuberculosis of different geographical regions of India. A total of 100 clinical isolates of Mycobacterium tuberculosis were included in this study. Drug susceptibility testing against first line anti-tuberculosis drugs was performed on LJ medium by conventional minimal inhibitory concentration (MIC) method and the mutation(s) in rpoB gene of M. tuberculosis isolates were analyzed by sequencing method. Of the 100 M. tuberculosis isolates, 31 (31.0%) and 18 (18.0%) were found resistant and susceptible for all four first-line anti-tuberculosis drugs. The genetic mutations were observed in 96% (72/75) rifampicin-resistant M. tuberculosis isolates, while 4% (3/75) of rifampicin-resistant isolates did not have any mutation in rpoB gene. The mutation TCG531TTG (Ser531Leu) was found as most common and frequent mutation in 69.3% (52/75) of rifampicin-resistant isolates of M. tuberculosis with MIC level (≥ 512mg/l). The mutation at codon 511 was associated with low degree (128mg/l) of rifampicin-resistance, deletions at codons 514-516 or substitution at codon 516 were found to be associated with moderate degree (256mg/l) of rifampicin-resistance and mutations at codon 526, 531 were associated with the high degree (512mg/l) of rifampicin-resistance in M. tuberculosis isolates of Indian origin. The findings of this study will be useful for the development of raid and more specific indigenous molecular tools for the early diagnosis of multidrug-resistant tuberculosis in the country

Immunoprotective Effect of Seabuckthorn (Hippophae rhamnoides) and Glucomannan on T-2 Toxin-Induced Immunodepression in Poultry

The present investigation was undertaken to study the immunoprotective effect of seabuckthorn berries and glucomannan against T-2 toxin-induced immunodepression in 15-day-old chicks. T-2 toxin was produced in the laboratory by growing Fusarium sporotrichioides MTCC 2081 on wheat. T-2 toxin was fed to birds at 1 ppm level of the diet. The powdered seabuckthorn berries were added at 400 and 800 ppm levels, and glucomannan added at 1 g/kg of feed. All the treatments were continued up to 28 days. The immunoprotective effects of seabuckthorn and glucomannan were assessed by evaluating humoral immune reaction against NCD vaccine (haemagglutination test and immunoglobulin estimation), serum immunoglobulin levels, phagocytic index, and DTH reaction against DNFB between day 25 and day 28 of experiment. There was significant (P < .05) decrease in non-specific immunity in T-2 toxin-treated group as evidenced by a reduction in phagocytic index, DTH reaction, HI titer, and total serum Ig compared to the healthy control group. A significant increase (P < .05) in HI titer and total serum Ig was seen in seabuckthorn and glucomannan fed group. A significant (P < .05) increase in DTH reaction and non-specific immune response was seen in seabuckthorn and glucomannan fed birds. The present investigation revealed that the seabuckthorn alone protected the immunosuppressant action of T-2 toxin, but seabuckthorn and glucomannan in combination provided an additive protection against T-2 toxicity

Diagnostic value of in situ polymerase chain reaction in childhood leprosy

Objective: Our aim was to assess the diagnostic value of in situ polymerase chain reaction (PCR) in leprosy, particularly for enhancing histopathological diagnosis. Methods: We prospectively studied 20 children (aged &lt;16 years) with leprosy. Clinical examination of each case was performed, and skin smear for acid-fast bacillus was prepared. A biopsy of the lesion site was performed for histopathological examination and in situ PCR testing. Results: Histopathological examination confirmed the clinical diagnosis in only 45% of the cases; nonspecific histopathology was reported for the remaining 55% of the cases. In situ PCR showed a positivity of 57.1% in the early/localized form of leprosy (indeterminate/borderline tuberculoid) and 61.5% in the borderline borderline/borderline lepromatous group. When compared with the histopathological examination, a significant enhancement of 15% in diagnosis was seen. With in situ PCR, the diagnosis could be confirmed in 4 (36.3%) of 11 cases with nonspecific histopathological features (which is common in early disease) in addition to confirmation of 8 (88.8%) of 9 histopathologically confirmed tissue sections. Histopathology and in situ PCR combined together confirmed the diagnosis in 13 (65%) of the 20 cases. Conclusions: In situ PCR is an important diagnostic tool, especially in early and doubtful cases of leprosy

Characterization of catalase by micro-immunoprecipitation in tissue-derived cells of Mycobacterium lepraemurium TMC 1701

Cell-free extracts of tissue-derived cells of Mycobacterium lepraemurium TMC 1701 have been found to have mycobacterial catalase which is of the T type. Immunological distance measurements of this catalase against three reference mycobacterial systems, as determined by a micro-immunoprecipitation technique, showed that catalase from M. lepraemurium TMC 1701 is most closely related to that of Mycobacterium avium but is still distinct from it. In this respect, this strain is uniquely positioned between Mycobacterium tuberculosis and M. avium

A sequential study of circulating immune complexes, complement mediated IC solubilisation and immunoglobulins in borderline tuberculoid patients with and without reactions

Sequential estimates of the levels of circulating immune complexes (CIC), complement catabolic fragment C3d, complement-mediated immune complex solubilization (CMS) and immunoglobulins were made in 24 newly diagnosed patients with borderline tuberculoid leprosy over a 20 month period after initiation of chemotherapy. Fourteen of these patients had not suffered from reversal reactions either at the time of presentation or during the follow-up period. The levels of CIC were elevated in them from the third to the eleventh month after starting chemotherapy and immunoglobulin G (IgG) levels were elevated upto eight months. The concentrations of C3d and immunoglobulins A (IgA) and M (IgM) were normal in these patients. The other ten patients had reversal reaction at the time of diagnosis which subsided by the third month after starting treatment. They did not have reversal reactions later. The levels of CIC and IgG were elevated and those of CMS were depressed throughout the study period. Serum C3d level was initially elevated but came down to normal by the third month while IgA and IgM levels were within normal limits. The relevance of these findings to the genesis of reversal reaction is discussed in this communication

Genetic Diversity of Genus \u3cem\u3eAvena\u3c/em\u3e in North Western-Himalayas assessed by Morphological Traits

Oat (Avena sativa L.) is a cool season, annual crop grown mainly in moist areas of temperate climates of the world serving as a food for mankind and forage for cattle. Oat is an important rabi fodder crop in India.In India, oat is also cultivated in Himalayan states like Kashmir, Himachal Pradesh and Uttarakhand. Oat in these regions have a wider adaptability, because of its excellent growing habitat, quick re-growth and better nutritional value (Misri, 2004). Oat breeding programme in Indian regions has not achieved much impetus due to a narrow genetic base of cultivated gene pool within the regionally adapted germplasm. The competition for utilization of land for food grains and fodder necessitates intensified efforts towards more efficient forage research and production, for which it is imperative to characterize and evaluate Avena species in order to identify donors for different traits and diversify primary oat gene pool. Historically, morphological traits have been important in the diversity analysis of crop species. The characterization of germplasm using morphological traits help the plant breeders to select the accessions to be utilized in hybridization programme. Considering the potential forage value of oats and limited genetic information available at morphological level, present study was aimed to assess the genetic diversity of genus Avena using morphological characterization. The information generated from this study will be helpful in characterizing the genus Avena germplasm and in the selection and utilization of diverse genotypes to enhance variability and productivity of commercial oat for future crop improvement endeavors in the Indian North-Western Himalayan region

Identification of a repetitive sequence belonging to a PPE gene of Mycobacterium tuberculosis and its use in diagnosis of tuberculosis

A repetitive sequence specific to Mycobacterium tuberculosis was isolated from a gt11 library of M. tuberculosis by DNA-DNA hybridization using genomic DNA of M. tuberculosis as probe followed by subtractive hybridization with a cocktail of other mycobacterial DNA. This led to identification of CD192, a 1291 bp fragment of M. tuberculosis containing repetitive sequences, which produced positive hybridization signals with M. tuberculosis DNA within 30 min. Nucleotide sequencing revealed the presence of several direct and inverted repeats within the 1291 bp fragment that belonged to a PPE family gene (Rv0355) of M. tuberculosis. The use of CD192 as a DNA probe for the identification of M. tuberculosis in culture and clinical samples was investigated. The 1291 bp sequence was present in M. tuberculosis, Mycobacterium bovis and M. bovis BCG, but was not present in many of the other mycobacterial strains tested, including M. tuberculosis H37Ra. More than 300 clinical isolates of M. tuberculosis were probed with CD192, and the presence of the 1291 bp sequence was observed in all the clinical strains, including those lacking IS6110. The sequence displayed RFLP among the clinical isolates. A PCR assay was developed which detected M. tuberculosis with 100 % specificity from specimens of sputum, cerebrospinal fluid and pleural effusion from clinically diagnosed cases of tuberculosis