Development and validation of an ELISA to detect antibodies to Corynebacterium pseudotuberculosis in ovine sera.

Several enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of antibodies to Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis (CLA). However, none are commercially available in the UK. It was therefore necessary to develop a new, economic ELISA for use in a research project studying the epidemiology of CLA in UK sheep. The ELISA with its diagnostic qualities is presented. The ELISA was developed using sonicated C. pseudotuberculosis and optimised to detect total antibody or IgG class antibody in serum. Receiver operating characteristic (ROC) curves were obtained and the area under the ROC curve was used to compare the sensitivity and specificity of the two ELISAs. Both versions of the ELISA were evaluated on a panel of 150 positive reference sera and 103 negative reference sera. Using the test at 100% specificity, the sensitivity of detection of total antibody was 71% (95% confidence interval 63-78%), and the sensitivity of detection of IgG antibody to C. pseudotuberculosis was 83% (76-89%), which compares favourably with other reported ELISA tests for CLA in sheep. The sensitivity of the IgG antibody assay may be higher because of the greater affinity of IgG class antibodies compared with the IgM antibodies also detected by the total antibody ELISA. The results of ROC analysis indicated that the IgG isotype ELISA was more accurate than the total antibody ELISA. The efficiency of the test was greatest when serum samples were run in a dilution series than when any single serum dilution was used. The ELISA is considered to be suitable for application in field studies of CLA in UK sheep.

Distribution of fim3 and flaA TTGE sequence types amongst isolates of Bordetella bronchiseptica from different host animals.

Isolates of Bordetella bronchiseptica associated with different animal hosts were analysed by fim3 and flaA genotyping by temporal temperature gradient gel electrophoresis (TTGE). All the isolates from cats (n = 138), dogs (n = 42) and pigs (n = 13) could be assigned to one of two fim3 and one of three flaA TTGE sequence types, respectively. Two avian isolates and a marmoset isolate exhibited novel fim3 sequence types. Dominant but different TTGE sequence types were apparent in isolates from dogs and pigs for both fim3 (87.5% and 100%, respectively, of isolates were the dominant type) and flaA (95% and 92%, respectively, of isolates were the dominant type). There was a wider distribution of TTGE sequence types amongst cat isolates. As both fimbriae and flagella have been implicated in adherence of bordetellae to host cells, sequence variations in fimbrial proteins and FlaA may have a role to play in host preferences.