title: The use of real-time PCR for the diagnosis of Johne's disease in cattle and sheep
Creators: name: Nelli, Rahul Kumar
Publisher: name: University of Glasgow
Year: 2007
Subjects: subject: Animal diseases, paratuberculosis, polymerase chain reaction, diagnostic use, cattle, sheep.

description: This study was aimed at the rapid and early detection of Mycobacterium avium paratuberculosis (Map), which is the causative organism for Johne's disease in animals. The history of Johne's disease and its epidemiology, clinical disease, pathology and diagnosis of Johne's disease was reviewed. The characteristics of Mycobacterium avium paratuberculosis and its detection in clinical material were reviewed. The modern diagnostic methods for the identification of pathogens were evaluated and real-time polymerase chain reaction (PCR) was considered worthy of an appropriate tool for the diagnosis of the disease. The animals used in this study were animals suspected of having Johne's disease which had been admitted to the Farm animal clinic. Six cows and 5 ewes were used in this study. Five of the six cows were antibody positive by Enzyme linked immuno sorbent assay (ELISA), faecal smear, intestinal smear and histopathology. Sera of the five sheep were not examined by ELISA but all were positive for acid fast organisms by intestinal smear and post-mortem examination, confirming the presence of the disease. One of the ewes was pregnant and her uterus contained dead twin foetuses. Real-time PCR for Map was developed using a Map specific primer/probe set based on IS900 from a published source (SF214/PR265/SR289). A further real-time PCR was developed for the 18S rRNA house keeping gene, using a primer/probe set designed in this study. Optimisation and validation of these primers and probes was performed. The primer/probe set used for detecting Map was able to detect 1 log copy of Map IS900 DNA with a PCR efficiency of 0.986706 and low standard deviation. The real-time PCR for Map detected single log copies of IS900 and calculations confirmed the Ct values (results of real-time PCR) 39.87, 39.07 and 39.65, represented a single cell of Map. Gene cloning and sequencing of the PCR product was performed to confirm that the target sequence was present, thereby validating the real-time PCR results. Sequencing of 18S PCR product was not successful and requires further work. The cloned product of Map was also used to plot a standard curve for use in quantitation. The real-time PCR assay of the samples examined in this study was carried out in triplicate and the Ct values obtained had a coefficient of variation of less than 10%. DNA was extracted from faeces/tissue samples, milk and blood from the 6 bovine and 5 ovine cases of suspected Johne's disease using standard protocols (QIAGEN Ltd, Crawley, UK) Mycobacterium avium paratuberculosis DNA was detected in five of the 6 bovine faecal samples by real-time PCR but results for the sixth animal were equivocal. Mycobacterium avium paratuberculosis was detected in a wide range of bovine tissues. Map organisms were demonstrated in blood, lymph nodes, tonsil, mammary tissue, mammary secretions, uterus, liver, spleen, kidney and muscle. Similar' results were obtained for the ovine samples where one faecal sample also gave an equivocal result. No organisms were demonstrated in the spleens of adult sheep and retropharyngeal lymph nodes were not examined in this species. The real-time PCR for Map produced in this study successfully detected Map in faeces, blood, mammary secretion and tissues. In some cases it appeared to be superior to other tests. It allowed the enumeration of Map organisms in the samples and will provide a guide to the safety of bovine and ovine tissues in veterinary public health and food safety. It will also allow the early detection of Map infected animals, the original aim of the study. (Abstract shortened by ProQuest.).