Summary
Background
The diagnosis of ovine Johne's disease remains a problem because until recently there has been very little basic research conducted anywhere in the world, and most of the current knowledge is based on the study of human tuberculosis from the early 1900's. Practical applications of this include tests used today: culture, histopathology and intradermal skin tests, none of which currently are very sensitive. The lack of basic knowledge has severely limited the development of new diagnostic tests and vaccines. Therefore this project included components of basic research, as well as translational research aimed at delivering practical tools for industry in the near term. This project was designed to use basic research and combine it with strategic elements to discover new test options and to improve existing tests. The project involved state of the art methods in microbiology, immunology, molecular biology and genomics in a multidisciplinary team with international collaborations to achieve its objectives.
Major outcomes As a result of the project it is now known that:
interferon gamma tests can be improved for use in sheep in Australia
faeces can be tested quickly and accurately using direct PCR
it is possible to reproduce the disease in a natural form in a controlled experimental situation, opening up options for test evaluation, vaccine development and other studies
new antigens are available for evaluation to improve test specificity and sensitivity
new cytokine-based tests appear to be useful
immune suppression and weight loss during ovine Johne's disease may be explained by dysregulation of amino acid metabolism detection of the organism in blood is not a useful diagnostic approach
a blood test based on cell proliferation may be predictive of disease susceptibility in sheep
A further objective of the project was to ensure that there is a credible team of researchers available to Australian sheep producers, and this also was achieved. Some of the findings of this project have been published already and the project team has an international reputation. As a result of the project it is now clear that two existing diagnostic tests which had severe practical and technical limitations can be improved and may be of substantial benefit in the near future. These include a direct faecal PCR test which can provide results to producers within a few days instead of the current 3 months for culture and an interferon gamma blood test which can be made practical for use in sheep in Australia.
In addition to these there are several new research-level tests of immune function that require further development. Further information on the two most promising tests follows. Direct faecal PCR test The direct faecal PCR test is a breakthrough for the sheep industry. Previously, faecal samples were collected, sent to a laboratory and 3 months would elapse before negative test results could be confirmed. For sheep sales this meant considerable forward planning and great inconvenience for the producer. Where culture was used to confirm a suspected flock infection, the long delay caused considerable additional anxiety for the producer.
The new test overcomes these problems because it can provide results within a few days of receipt of samples at a laboratory. It will cost no more than culture, and will be of similar accuracy. Of 65 culture positive samples, 62 were positive in the new test. Of 140 culture negative samples, 12 were positive in the new test. As no samples from flocks known to be free of OJD tested positive in either test, we believe that the new faecal test is slightly more sensitive than culture. Furthermore it is suitable for testing pooled faecal samples, which enables a cheap method of flock testing, either to detect infection or to show that it is not present in flocks in the Market Assurance Program.
Additional validation of this test has been recommended by the JD Research Advisory Group, after which the data will be submitted to the SubCommittee on Animal Health Laboratory Standards (SCAHLS) for approval for use in the National Johne's Disease Program. A submission on the test will be made to SCAHLS in 2011. Whole blood interferon gamma assay A whole blood interferon gamma assay which was developed to prototype stage in a previous project has been modified and improved in this project. Previously it was necessary to ship blood samples to a laboratory and test them within 8 hours of collection something that usually was impossible and prevented validation of the test.
Two blood additives were discovered which extend the life of the blood samples to 48 hours. Now it is possible to ship samples from most places in Australia to a laboratory in time to conduct the test. Additional research is now required to find a way to make the test more specific, as some uninfected sheep react, and this will be done in project P.PSH.0576, with a goal of validating the new procedure within the life of that project. Interferon gamma detection assays offer the potential to detect more infected animals at an earlier stage of the disease compared to an antibody ELISA or direct detection of Mptb in the faeces. This may provide opportunity for control strategies aimed at removal of young infected animals before they start shedding bacteria into the environment. Experimental infection model From the basic research program a method was proven for creating experimental ovine Johne's disease in a flock under tightly controlled conditions, leading to realistic and natural outcomes.
This approach will be invaluable for diagnostic test and vaccine development studies and has already been adopted by overseas researchers we expect that Australian producers will benefit from such collaborative international studies. Mining the genome of Mptb for better tests Also in the basic research program, computer-based methods were used to mine the DNA sequence of the causative bacterium Mycobacterium paratuberculosis (Mptb) to identify new components to include in future diagnostic tests with the objective of greater accuracy than current tests. Mptb is not found in blood very often It was shown that Mptb does not circulate very often in blood at detectable levels, which is of great significance for diagnosis and has positive implications for public health.
Proliferating cells for detecting exposure to Mptb
The ability of white blood cells to remember contact with Mptb has been tested in a proliferation assay in experimentally infected sheep the response in non-exposed controls remained low while it increased and remained elevated in exposed sheep. Furthermore, the proliferative response varied with disease status and may be predictive of resistance. Mechanisms of disease progression and weight loss in OJD Finally, an explanation for the weight loss that occurs in ovine Johne's disease may have been found rather than intestinal malabsorption, it is possibly due to an amino acid deficiency induced by the infection. Blood levels of the amino acid tryptophan were shown to plummet as ovine Johne's disease develops, and this was due to a trick played by the mycobacterium to induce the sheep to destroy its own tryptophan. Further research will be conducted across these fundamental discoveries to maximise their potential.
