A novel biphasic approach to reactivation of dormant Mycobacterium avium subspecies paratuberculosis.

A new culture technique that involves a potato slice and enriched Middlebrook 7H9 in a two-part glass tube has been developed to revive dormant or persistent Mycobacterium avium subspecies paratuberculosis and acclimate it to Middlebrook 7H9 liquid media. This method is more efficient than directly introducing the bacteria into the liquid medium.

Drug susceptibility testing of Mycobacterium Avium subsp. Avium isolates from naturally infected domestic pigeons to avian tuberculosis.

OBJECTIVE: Avian tuberculosis is one of the most important infections affecting most species of birds. Several mycobacterial species have been identified causing avian tuberculosis, and the organisms confirmed most frequently are Mycobacterium avium and Mycobacterium genavense. Any species of birds can be infected with M. avium. Generally, domesticated fowl or captive wild birds are affected more frequently than those living in the wild. M. avium can not only infect all species of birds, but can also infect some domesticated mammals to cause disease, usually with localized lesion. In immunocompetent individuals, M. avium complex isolates produce localized soft tissue infections, including chronic pulmonary infections in the elderly and cervical lymphadenitis in children, but rarely any disseminated disease. In patients infected with HIV and AIDS or in other immunocompromised individuals, M. avium complex isolates frequently cause severe systemic infections. The importance of avian tuberculosis and the risk of its zoonotic spread motivated our interest to determine the drug susceptibility testing of M. avium subsp. avium isolates from naturally infected domestic pigeons to avian tuberculosis. METHODS: Based on their clinical signs, 80 pigeons suspected with avian tuberculosis were subjected to the study. Out of the 51 identified isolates, 20 M. avium subsp. avium were subjected to the test. Drug susceptibly testing was performed according to the guidelines by Centers for Disease Control and Prevention and using proportional method. RESULTS: In the drug susceptibility testing, all isolates were resistant to streptomycin, kanamycin, ethionamide, and thiophene carboxylic acid hydrazide. Additionally, 3, 2, and 1 isolates were susceptible to isoniazid, rifampin, and ethambutol, respectively. To date, no study has documented the drug susceptibility testing of M. avium isolates from infected birds to avian tuberculosis. Pigeons are extensively kept in urban and rural areas for homing and racing purposes; thus, they can infect people and farm animals exposed to their droppings containing pathogenic M. avium, and the severity of drug resistance of these isolates indicate lethality in immunocompromised individuals and incurable lymphadenitis in immunocompetent individuals. CONCLUSION: We suggest drug susceptibility testing for more nontuberculous mycobateria, particularly M. avium complex isolated from infected birds and humans, as well as molecular basics of drug sensitivity in order to detect resistance genes of pathogenic M. avium subsp. avium.

Rapid identification of Mycobacterium avium ssp paratuberculosis laboratory strains by IS900-Nested polymerase chain reaction.

INTRODUTION: Mycobacterium avium ssp paratuberculosis (MAP) causes paratuberculosis (Johne's disease) in ruminants. As a species, M. avium comprises M. avium subsp. hominissuis and a number of clones that are known to have evolved from this subspecies, namely M. avium subsp. avium (MAA), M. avium subsp. silvaticum, and MAP. Despite the very high genomic similarity of MAP and MAA, the insertion sequence IS900, which is 1,451-bp long, is now understood to be exclusively present in 10-20 copies in the genome of MAP. In the present study, a multidiscipline polymerase chain reaction (PCR)-based algorithm targeting16SrRNA, IS6110, IS901, IS1245, and IS900 markers has been employed to differentiate between six laboratory strains of M. avium complex (including MAP 316F, III&V, and 2e plus MAA D4), Mycobacterium tuberculosis DT, and Mycobacterium bovis AN5 strains used at the Razi Institute (Tehran, Iran) for the preparation of paratuberculin, avian, human, and bovine tuberculin, respectively. MATERILS AND METHODS: Three laboratory strains of III&V, 2e, and 316F were subcultured on Herrold's egg yolk medium, whereas the MAA strain of D4 along with M. bovis AN5 and M. tuberculosis DT were subcultured on Lowenstein-Jensen slopes. All the inoculated culture tubes were incubated for 8weeks at 37°C. Eventually, their genomic DNA was extracted according to the method of van Soolingen. Five individual PCRs were conducted on these templates to amplify 16SrRNA (genus-specific marker shared by all mycobacteria), IS900 (MAP-specific marker), IS901 (MAA-specific marker), IS1245 (M. avium complex (MAC)-specific marker), and IS6110 (M. tuberculosis complex (MTC)-specific marker) loci. RESULTS: Consequently, a 543-bp amplicon was amplified by all the six strains in PCR against 16SrRNA, an indication of their identity as members of Mycobacterium genus. A 245-bp fragment was detected in only IS6110-PCR with M. bovis AN5 as well as M. tuberculosis DT. In the IS1245 assessment, the MAA strain of D4 produced a 427-bp amplicon, whereas none of the other studied strains produced this amplicon. A 1,108-bp amplicon fragment of the IS901 marker was successfully produced by MAA strain, whereas no PCR product was achieved in amplification of all the three MAP strains. In IS900-nested PCR, the three MAP strains produced the expected 400-bp and 298-bp fragments CONCLUTION: However, no amplification was observed with other strains. Two main achievements of this work are the development of an efficient means of differentiation between the six Razi laboratory mycobacterial strains and characterization of the genomic profile of these strains, a capability that is vital when cross contamination is potentially an important concern.

A review of the contemporary knowledge of bovine tuberculosis and government policy in Iran.

In 1931 Carpantier reported bovine TB (BTB) in Iranian cattle. Some eighty years on with a national test-and-slaughter programme in place for over four decades, the efforts to vanquish Mycobacterium bovis (M. bovis) infection in cattle have been in vain as the vast majority of the 30 Iranian provinces still have reports of BTB in their cattle herds every year. This paper reviews the present epidemiology of BTB in Iran and in the region and evaluates the success of government policy in controlling this disease.

Molecular genotyping and epidemiology of Mycobacterium bovis strains obtained from cattle in Iran.

Restriction fragment length polymorphism (RFLP) genotyping was employed to analyze the population genetics of Mycobacterium bovis in Iran. One hundred and twenty-three isolates collected from slaughtered tuberculosis-suspect cattle and one clinically asymptomatic buffalo were subjected to RFLP analysis with probes of the polymorphic GC-rich sequence (PGRS) and the direct repeat sequence (DR) using DNA digested with PvuII and AluI. All these methods detected a large homogeneous population in which only a few isolates had variant genotypes. Only AluI-based RFLPs of both the PGRS and DR sequences were able to clearly differentiate between BCG and field strains of M. bovis. As in previous reports, these findings seem to reflect a recent dispersal of one or a few strains in Iran following the substantial expansion of Holstein-Friesian cattle over the last few decades.