A.V. Singh

Genotype diversity in Indian isolates of Mycobacterium avium subspecies paratuberculosis recovered from domestic and wild ruminants from different agro-climatic regions

Fecal samples of 708 domestic and 27 wild ruminants from seven districts of three states submitted to Microbiology Laboratory from 2004 to 2008 were screened for the presence and genotype diversity of Mycobacterium avium subspecies paratuberculosis (MAP) stusing modified microscopic examination and culture tests. MAP colonies were characterized and genotyped by IS900 PCR and IS1311 PCR-REA, respectively. In microscopic examination and culture, 18.7 and 31.2% sampled were positive, respectively. Percent positivity varied in different species and agro-climatic regions. Of the 151 DNA recovered from 230 MAP isolates, 94.7% (143) and 5.2% (8) were genotyped as Indian Bison type and Cattle type, respectively. Cattle and buffaloes were infected with both the genotypes, whereas goat and sheep were infected exclusively by Indian Bison type. Study showed low diversity in MAP genotypes and Indian Bison type was the predominant genotype shared by different species, breeds and agro-climatic regions.

Superoxide dismutase as a regulatory switch in mammalian testicular steroidogenesis

The delta 4-pathway of testosterone biosynthesis in leydig cells, widely believed to proceed through pregnenolone--greater than pregnenedione--greater than progesterone route catalyzed by 5 delta-3 beta-hydroxysteroid dehydrogenase and delta 5-delta 4-isomerase respectively is shown to pass through an alternate pathway mediated by superoxide dismutase and peroxidase. A built-in regulatory switch is incorporated in this route, with the superoxide dismutase inducible upon LH-stimulation of the leydig cells.

Wildlife (Boselaphus tragocamelus)-small ruminant (goat and sheep) interface in the transmission of 'Bison type' genotype of Mycobacterium avium subspecies paratuberculosis in India.

Information on Mycobacterium avium subspecies paratuberculosis (MAP) genotypes infecting different animal species in India is limited. Presence of MAP was investigated in free ranging antelopes (locally known as Nilgai/blue bulls/Boselaphus tragocamelus) using direct microscopy, culture, IS900 PCR and IS1311 PCR-REA. IS900 elements of MAP from Nilgai and previously isolated from goats were sequenced and compared to establish inter-species transmission between free ranging Nilgai and closed farm herds and flocks of goats and sheep sharing common grazing and water resources. Fecal samples were collected from two geographical regions (Mathura and Kanpur Dehat districts) separated by 300km, in North India. Of the 42 fecal samples cultured, MAP colonies were recovered from 23.8% samples (Nilgai). Of the 10 positive fecal samples, two were in Super shedder (>1000cfu/g) category and rest were moderate (<10-100cfu/g) shedders. None of the Nilgai from Kanpur Dehat was positive in culture. The 229bp fragment targeting specific IS900 sequence was amplified from template DNA isolated from all the positive MAP cultures of Nilgai. Using IS1311 PCR-REA, MAP colonies were genotyped as Bison type. Goatherds and a sheep flock located at Central Institute for Research on Goats (CIRG), shared 303.52ha of land (Mathura district of Uttar Pradesh) with Nilgai and were endemic for MAP infection. MAP strains isolated from goats and sheep have been genotyped as Bison type. Nucleotide sequence of the insertion elements (900) from MAP Bison type strain (S5) of goat origin and MAP (B42) from Nilgai showed difference of 2 (1%) base pairs at the 11th and 12th position (Genbank accession number EU130943). Study is first report on sharing (inter-species transmission) of a new Bison type genotype of MAP between free ranging wildlife (Nilgai population) and domestic animals (farm goatherds and sheep flocks) in India.

Molecular identification and characterization of Mycobacterium avium subspecies paratuberculosis in free living non-human primate (Rhesus macaques) from North India

In recent years, Mycobacterium avium subspecies paratuberculosis (MAP) has emerged as major animal pathogen with significant zoonotic concerns, worldwide. MAP infection is endemic in domestic and wild ruminant population in India. However, information on MAP infection in free ranging animal species and non human primates is limited. Present study aimed to estimate the status of MAP infection in free living Rhesus macaques suffering with multiple clinical conditions (coughing and loose stool). A total of 25 stool samples were collected from six colonies of Rhesus macaques from Mathura region (North India) and screened for the presence of MAP, using microscopic examination and IS900 PCR, directly from stool samples. PCR positive DNA samples were further genotyped using IS1311 PCR-restriction enzyme analysis. Of the 25 stool samples, 10 (40.0%) and 2 (8.0%) were positive for MAP using microscopic examination and direct IS900 PCR, respectively. IS900 PCR positive DNA samples were genotyped as Indian Bison type, which is a major MAP genotype infecting domestic and wild ruminant species and human beings in India. Prevalence of MAP in Rhesus macaques (Indian monkeys) was moderately high and confirmed interspecies sharing of MAP between domestic livestock and non-human primates. Presence of MAP in non-human primates, support the etiological role of MAP in inflammatory bowel disease patients. Indian monkeys may serve as model for understanding the role of non-human primates in sustenance, transmission and pathogenesis of MAP infection.

Discovery of a novel and alternate hydrogen peroxide generation machinery in the mammalian thyroid gland that modulates iodination of tyrosine

The hydrogen peroxide-dependent and peroxidase-mediated iodination of tyrosine, a pivotal step in the thyroxine biosynthesis in thyroid glands of mammals, has always been thought to be dependent on a glucose-glucose oxidase system to meet its demand for hydrogen peroxide. This report documents a novel and probably kinetically faster hydrogen peroxide-generating system operating in the thyroid gland cells involving the superoxide dismutase and superoxide anion radical. In the female cycling mice, the superoxide radical and superoxide dismutase activity are shown to be at persistently high levels, quite likely acting as a potent hydrogen peroxide synthesizer in the mammalian thyroid gland. The significance of this discovery in relation to the thyroid physiology is discussed.

Activation of iodine into a free-radical intermediate by superoxide: A physiologically significant step in the iodination of tyrosine

A pivotal biochemical event in the thyroid physiology is identified unravelling a superoxide anion radical-mediated activation of iodine into an active I.- form, which could be the intermediate that is incorporated onto tyrosine. This active iodine species gives fairly stable spin-adducts with PBN that could be characterized using EPR spectroscopy. Thus, a long-lasting puzzle regarding the iodine intermediate formed before iodination of tyrosine seems to be solved.

Superoxide dismutase activity regulation by spermine: A new dimesion in spermine biochemistry and sperm development

Amines are a group of highly important compounds of biological importance; they are known promoters of cell growth, can complex with nucleic acids and can stimulate DNA-primed RNA polymerase activity. Spermine, a polyamine abundantly present in the secretions of the male accessory sex organs, has received no functional attributes till date. This study had been a pioneer attempt to validate the hypothesis of a metalloenzyme activity modulation by spermine and implicit correlations have been drawn.

Superoxide anion radical induces lipid phase transitions that mimic temperature-induced phase-transition phenomenon of membrane lipids.

A biological membrane is shown to undergo lipid-phase transition leading to increased membrane fluidity when exposed to extraneously generated superoxide anion radical. This phase-transition is several folds higher in magnitude when compared to the temperature-induced fluidity change at the transition temperatures. This finding could have significant importance since an altered membrane configuration may involve aspects of biochemistry, biophysics or physiology.