Longitudinal dynamics of herd-level Mycobacterium bovis MLVA type surveillance in cattle in Northern Ireland 2003-2016.

Investigating genetically-structured diversity in pathogen populations over time is important to better understand disease maintenance and spread. Herd-level surveillance of Mycobacterium bovis genotypes (multi-locus VNTR analysis types, MLVA types) from all culture-confirmed bovine tuberculosis (TB) herd cases was undertaken in Northern Ireland (NI), generating an unparalleled, longitudinal, population-level 14-year survey for this pathogen. Across this population, 295 genetically-distinct M. bovis MLVA types were identified in the 19,717 M. bovis isolates surveyed. Of these, the most frequent was MLVA type 002 (23.0%); 151 MLVA types were represented more than once, in groups ranging from 2 to 4438 isolates. Only 23 MLVA types were isolated in all 14 years. Investigating inter-annual frequency of M. bovis MLVA types, examples of statistically-significant expansions (MLVA types 002, 004, 006, 009 and 027), contractions (MLVA types 001, 007 and 011) and maintenance (MLVA types 003 and 005) were disclosed, during a period of fluctuating bovine TB herd-level incidence at the NI scale. The fixed period frequency distribution of MLVA types remained highly right-skewed. Novel VNTR copy number variant MLVA types (N = 242; an average of 17 per annum) were identified throughout the survey. The MLVA type distribution in the landscape was not random; MLVA types showed statistically-significant geographical localization and strong spatial associations with Divisional Veterinary Office (DVO) regions. There was also evidence of differential risk of particular MLVA types across breeds (Holstein/Friesian vs. other), age-class, and sex and some evidence of an association between the number of animals testing positive for bovine TB during the disclosing test and particular MLVA types, although there was substantial variation.

Discrimination of isolates of Mycobacterium bovis in Northern Ireland on the basis of variable numbers of tandem repeats (VNTRs).

The ability to reproducibly discriminate Mycobacterium bovis isolates and trace their transmission has the potential to clarify sources of infection and major routes of transmission for bovine tuberculosis (TB). A PCR-based genotyping assay has been developed to discriminate between strains of M bovis by examining multiple sites in its genome that consist of variable numbers of tandem repeats (VNTRS). The discriminatory power and reproducibility of this VNTR typing has been compared with that of the established PCR-based spoligotyping technique by using a panel of 461 isolates of M bovis prevalent in Northern Ireland. The VNTR assay discriminated 40 different profiles, the most prevalent of which constituted 21 per cent of the total, compared with 14 profiles discriminated by spoligotyping, the most prevalent of which constituted 65 per cent. No significant differences were observed between the prevalences of the VNTR profiles in the years from 1999 to 2003. A preliminary evaluation indicated that most genotypes predominated in particular areas of the country. This VTNR typing assay was found to be highly discriminating, with the performance characteristics to support its systematic application to the molecular epidemiology of bovine TB.

Modulation by presynaptic adenosine A1 receptors of nicotinic receptor antagonist-induced neuromuscular block in the mouse.

We have investigated how altering the activation of adenosine A1 receptors modifies nicotinic receptor antagonist-induced fade of tetanic contractions in the mouse isolated hemi-diaphragm. Vecuronium-induced tetanic fade was attenuated by an adenosine A1 receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine, DPCPX, 10(-7) M) and by an inhibitor of the synthesis of extracellular adenosine from ATP (alpha,beta-methylene ADP, MeADP, 5 x 10(-5) M). Conversely, vecuronium-induced tetanic fade was potentiated by an adenosine A1 receptor agonist (N6-cyclohexyladenosine, CHA, 10(-7) M) and an inhibitor of the extracellular destruction of adenosine (erythro-9-[2-hydroxy-3-nonyl]adenine, EHNA, 10(-4) M). The ability of an adenosine A1 receptor antagonist to attenuate vecuronium-induced tetanic fade indicates that a component of this fade is due to endogenous adenosine. Further, the ability of the inhibitor of adenosine synthesis to attenuate vecuronium-induced tetanic fade indicates that this endogenous adenosine is derived from ATP. Hexamethonium-induced tetanic fade was also potentiated by increasing adenosine A1 receptor activation, albeit with a higher concentration of CHA (10(-4) M). However, unlike for vecuronium, hexamethonium-induced tetanic fade was not attenuated by reducing adenosine A receptor activation. This latter observation suggests that the tetanic fade produced by hexamethonium and vecuronium does not share a common mechanism of action.