Development and evaluation of advanced molecular methods for the veterinary diagnostic bacteriology laboratory.

摘要

This study developed three advanced molecular assays for the veterinary diagnostic bacteriology laboratory. Firstly, the application of 16S rRNA gene sequence analysis for bacterial identification was evaluated using 22 difficult-to-identify veterinary clinical bacterial isolates. Using 16S rRNA full gene sequencing, 95% of the isolates were identified to the genus level and 86% to the species level. Sequencing of the variable regions l, 2, and 3 of the 16S rRNA gene had similar ability to identify bacteria to the genus level but identified only 73% of isolates to the species level. Sequencing of the variable regions 7, 8 and 9 gave more ambiguous identifications. By contrast, phenotypic characterization correctly identified only 55% of the isolates to the genus and 4.5% to the species level. Secondly, a real-time PCR assay was developed for the detection of Mycoplasma bovis from bovine milk and lung tissue samples. The assay also differentiated M. bovis from the closely related species M. agalactiae. In validation testing of 165 individual mastitic milk and 53 pneumonic lung samples, the sensitivity and specificity were 100% and 99.3% for milk, and 96.6% and 100% for lung tissue samples. The amount of culturable M. bovis in milk did not change significantly (p>0.05) after storage at 4°C for 4 days, but was reduced by 24% and 36% (p0.01) after storage at -20°C for 4 and 7 days; no significant difference was found with PCR test results (p>0.05). Isolation showed that the herd prevalence of M. bovis in mastitic milk samples submitted to the Mastitis Laboratory (AHL, Guelph, ON) was 2.4% (5/201). Thirdly, an antibody microarray was developed for Salmonella serotyping. A model array was constructed using 35 antibodies for identification of 20 common Salmonella serovars, and evaluated using 117 target and 73 non-target Salmonella strains. The assay allowed complete serovar identification of 86 target strains, and partial identification of 30 target strains, and allowed exclusion of the 73 non-target strains from the target serovars. The antibody microarray can identify somatic and both phase 1 and 2 flagella antigens simultaneously. The cost advantage of the three molecular assays developed over conventional methods is discussed.