|Keywords:||Paratuberculosis; Johne's disease; Mycobacterium avium; Quantitative PCR; qPCR; Infection trial; vaccine;|
|Full text PDF:||http://dspace.library.uu.nl:8080/handle/1874/311504|
In an infection trial 16 Holstein-Friesian bull calves were raised in a biosecurity level 2 facility at the University of Calgary. Calves were randomly allocated into two groups and euthanized at two and four months. In each group, six calves were infected with a library of Transposon (Tn) mutants created from a MAP strain named A1-157 located in the dominant clade of isolates obtained from Canadian farms. Two calves in each group received the wildtype (WT) A1-157 strain serving as positive infection ‘controls’. After euthanasia, from each calf tissue samples from various anatomical locations were collected. For this study, the distal jejunum, distal jejunum lymph nodes, the spleen and inguinal lymph nodes were used. Using a self-developed protocol DNA from MAP was isolated from these samples in triplicates. A qPCR procedure targeting the F57 and IS900 loci was performed. qPCR results were then analysed with the help of a F57 plasmid standard curve to translate real time fluorescence measurements to bacterial log copy numbers. All calves, library and WT euthanized at two or four months after inoculation, were successfully infected with MAP. F57 Ct values were all below 40 indicating that the tissues sampled were positive. Significant differences in log copy numbers between calves infected with the library of mutants euthanized at two and four months of age could not be detected in any of the tissues. As for the difference between wildtype calves euthanized at two and four months only in the spleen a significant difference in log copy numbers was noted with the four month old calves being less frequently positive (P=0,01). Furthermore, there was a significant difference in log copy numbers between library and wildtype calves euthanized at two and four months for the distal jejunum and jejunal lymph nodes. Log copy numbers were approximately equally divided for all four tissues in calves euthanized at two months, but in the calves euthanized at four months the distal jejunum and jejunum lymph nodes showed higher log copy numbers. CFU counts from MAP grown on agar were compared to CFU expected calculated from qPCR results of the same tissues. There was up to a 3-fold difference between the CFUs grown and expected in the jejunum and a 20-fold difference for the jejunal lymph nodes. We conclude, therefore, that both the WT and library calves were successfully infected and that the protocol to isolate and quantify MAP worked. Furthermore, more MAP was present in the tissues than grown on the agar plates.