Comparison of the BBL mycobacteria growth indicator tube, the BACTEC 12B, and solid media for the isolation of Mycobacterium bovis.

We compared different methods for their ability to isolate Mycobacterium bovis from tissue samples from animals with lesions resembling bovine tuberculosis. In the first trial, M. bovis was isolated from 86 of 200 tissue samples that were cultured using 2 liquid media, BACTEC 12B and BBL mycobacteria growth indicator tube (MGIT), and a solid medium, Middlebrook 7H11 supplemented with pyruvate (7H11P). M. bovis was isolated from 2 samples with MGIT but not BACTEC 12B. M. bovis was isolated from 9 samples with BACTEC but not MGIT; these 9 samples came from the North Canterbury/Marlborough region of New Zealand. The proportion of tissues from which M. bovis was isolated with BACTEC 12B or MGIT and the mean time for isolation was different for samples from the North Canterbury/Marlborough region but not the rest of New Zealand. In the second trial, M. bovis was isolated from 401 of 1,033 tissues that were cultured using MGIT, Middlebrook 7H9 broth, or solid 7H11P. The proportion of isolates of M. bovis and the mean time for their isolation with MGIT was different for the North Canterbury/Marlborough and the rest of New Zealand. The reason for this difference was not determined but may be related to the genotypes present in this region. Genotyping using variable number tandem repeats (VNTRs) of 197 isolates of M. bovis revealed that the 44 isolates from North Canterbury/Marlborough were represented by 2 closely related VNTR types that were not found in 153 isolates from the remainder of New Zealand.

Sustained protection against tuberculosis conferred to a wildlife host by single dose oral vaccination.

BACKGROUND: Vaccination of wildlife against bovine tuberculosis (TB) is being considered by several countries to reduce the transmission of Mycobacterium bovis infection to livestock. In New Zealand, where introduced brushtail possums (Trichosurus vulpecula) are the major wildlife hosts, we have previously shown that repeat applications of a lipid-encapsulated oral bacille Calmette-Guerin (BCG) vaccine reduce the incidence of naturally acquired TB in wild possums. Here we extend this conceptual demonstration to an operational level, assessing long-term protection against TB conferred to free-living possums by a single oral immunisation. METHODS: Possums in a non-TB area were randomly allocated to receive lipid-formulated BCG vaccine or remained unvaccinated. After initial trials to assess vaccine immunogenicity and establishment of protection within the first year post-vaccination, 13 individuals of each treatment group were relocated to a biosecurity facility and challenged (at 28 months post-vaccination) by subcutaneous injection of virulent M. bovis. RESULTS: Vaccine immunogenicity and short-term protection were confirmed at 2 months and 12 months post-vaccination, respectively. In the long-term assessment, vaccinated possums had significantly reduced bacterial counts in peripheral lymph nodes compared to controls, with 0.6-2.3 log(10)-fold reductions in M. bovis burdens. DISCUSSION: The magnitude of protective response by possums to experimental challenge at 28 months post-vaccination is known to equate to a high degree of protection against natural infection in this species. With techniques for oral bait delivery well advanced, the longevity of protection demonstrated here shows that an operable wildlife vaccine against TB is feasible.

Isolation of Mycobacterium bovis and other mycobacterial species from ferrets and stoats.

As part of wildlife surveillance for bovine tuberculosis, pooled lymph nodes from 21,481 ferrets, 1056 stoats and 83 weasels were cultured for mycobacteria. A total of 268 isolates of Mycobacterium bovis were obtained from ferrets, 2 from stoats and none from weasels, demonstrating the presence of a wildlife reservoir of infection in ferrets. DNA typing by restriction endonuclease analysis (REA) of 48 selected isolates of M. bovis revealed 23 REA types. Twenty-one of these types had previously been isolated from cattle and farmed deer, demonstrating a complex cycle of infection involving wildlife and domestic animals. Apart from M. bovis, a further 208 mycobacterial isolates were obtained, the majority of which (178) were members of the M. avium complex. Speciation of the remaining 30 mycobacterial isolates by DNA sequencing of the 16s rRNA gene, identified half the isolates as M. triplex. Other species identified included M. fortuitum, M. florentinum, M. interjectum, M. intracellulare, M. holsaticum, and M. septicum/M. peregrinum.