Mycobacterium paratuberculosis infection in sheep : aspects of diagnosis and immunity : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy of Veterinary Science, Massey University, 1999

Abstract

Paratuberculosis is a chronic, wasting disease of ruminants caused by Mycobacterium paratuberculosis. Programmes aimed at controlling paratuberculosis are based on either vaccination or detection and culling of infected animals. Because of its chronic nature, and the lack of suitable tests for early diagnosis, control of the disease using the latter approach is difficult. Standard procedures for the isolation of M. paratuberculosis are time-consuming and some strains are difficult or impossible to grow. Using the published sequence data of IS900, an insertion sequence considered unique for M paratuberculosis, a polymerase chain reaction (PCR) assay was developed. With purified extracts of bacterial DNA the PCR assay was found to be highly sensitive and specific. Among 30 bacterial species tested, the assay showed cross-reactivity only with DNA from M. scrofulaceum. The possibility of M. scrofulaceum causing false positive results in clinical samples from sheep was considered remote, and the assay was subsequently applied to clinical samples. In a study involving 20 sheep suspected of having clinical paratuberculosis, M. paratuberculosis DNA was detected in 90% of liver samples and 66% of blood samples from sheep with advanced clinical paratuberculosis. However, in a longitudinal study involving 14 sheep infected experimentally with M. paratuberculosis, the PCR failed to consistently detect the target DNA in liver biopsy specimens and blood samples of subclinically infected and clinically affected sheep with mild or moderate extraintestinal infection. Furthermore, the sensitivity of the PCR on samples of ileum and ileocaecal lymph node was similar to that achieved by histological examination. An experimental model of ovine paratuberculosis, which was developed primarily to validate the PCR assay, created an opportunity to evaluate the diagnostic performance of three commercially available antibody assays for paratuberculosis: complement fixation test (CFT), agar gel immunodiffusion test (AGID), and enzyme-linked immunosorbent assay (ELISA). Two experimental trials demonstrated a limited value of serology for the control of ovine paratuberculosis, as none of the antibody assays was able to detect all sheep with histologically confirmed paratuberculosis. In comparison, the whole-blood interferon-γ (IFN-γ) assay, which was assessed only during the second trial, detected significantly more experimentally infected sheep and over shorter period of time than any of the serological tests. Furthermore, in a pilot study involving 19 sheep infected experimentally with M. paratuberculosis, 18 of the 19 sheep gave positive reactions in the IFN-γ assay on samples of prescapular lymph node (PLN). The PLN-based IFN-γ assay detected significantly more experimentally infected sheep than the CFT, AGID, ELISA or the blood-based IFN-γ assay. Since the specificities of the blood- and PLN-based IFN-γ assay were similar to that of the serological tests, these data indicate the potential utility of this assay, using blood or samples of peripheral lymph nodes, for the detection of sheep exposed to M. paratuberculosis. Interestingly, among the 18 sheep tested positive by PLN-based IFN-γ assay, 13 had no histological evidence of paratuberculosis at the time of collection of the PLN samples. In addition, results obtained in a study involving 14 sheep infected experimentally with M. paratuberculosis suggest a positive relationship between the magnitude of antigen-induced IFN-γ response in blood and animal's ability to control the infection. Thus, attempts to use this assay in control programmes that are based on testing and culling of positive reactors could result in the removal of animals that have successfully mounted an immune response to the infection. Vaccination provides an alternative method to test-and-cull programmes of controlling paratuberculosis. Results of a study involving 28 lambs infected experimentally with M. paratuberculosis, 14 of which were vaccinated against paratuberculosis with a live-attenuated vaccine 2 weeks postinfection, indicate that vaccination of lambs already exposed to the organism triggered early and strong humoral and cell-mediated immune responses and led to a reduced mycobacterial burden.