Summary
The diagnosis of bovine 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 1900s. 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 and made practical for use in cattle in Australia, but specific antigens are required to obtain adequate specificity faeces can be tested quickly and accurately using direct PCR immune suppression and weight loss during bovine Johne's disease may be explained by dysregulation of amino acid (tryptophan) metabolism new antibody and cytokine-based tests on blood appear to have limitations a blood test based on cell proliferation may be predictive of infection in cattle 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 gene expression studies can reveal the dominant features of the early immune response, opening up new avenues for research on diagnosis and prevention.
A further objective of the project was to ensure that there is a credible team of researchers available to Australian beef cattle 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 a diagnostic test which had severe practical and technical limitations can be improved and may be of substantial benefit in the near future. This is the direct faecal PCR test which can provide results to producers within a few days instead of the current 3 months for culture.
In addition there are several new research-level tests of immune function that require further development. Direct faecal PCR test The direct faecal PCR test will be a breakthrough for the beef cattle industry. Previously, faecal samples were collected, sent to a laboratory and 3 months would elapse before negative test results could be confirmed. For cattle sales this meant considerable forward planning and great inconvenience for the producer. Where culture was used to confirm a suspected herd infection, for example after positive or suspect ELISA test results, 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.
Furthermore it is suitable for testing pooled faecal samples, which enables a cheap method of herd testing, either to detect infection or to show that it is not present in herds in the Market Assurance Program. The test will also be suitable for environmental testing. Additional validation of this test was recommended by the JD Research Advisory Group to provide better estimates of sensitivity and specificity. This was due to there being only a low number of samples of suitable quality from infected herds, and few culture positive samples, despite appropriate effort to obtain these.
An unexpected finding of the research was a requirement for faecal samples to be stored at -80C prior to PCR many samples obtained from infected herds had not been stored appropriately at a commercial laboratory. A request for funding to obtain appropriate samples has been submitted to MLA; faecal samples will be obtained from a Financial non Financial Assistance Program beef herd in Tasmania which has a high prevalence of ELISA reactors. Final data will be submitted to the SubCommittee on Animal Health Laboratory Standards (SCAHLS) for approval for use of the test in the National Johne's Disease Program later in 2011. Whole blood interferon gamma and lymphocyte proliferation tests A whole blood interferon gamma assay which was developed to prototype stage for the detection of OJD in a previous project has been modified for cattle 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.
Two blood additives were trialled to extend the life of the blood samples to 48 hours. This would make it possible to ship samples from most places in Australia to a laboratory in time to conduct the test. The additive may have worked, but there was concomitant loss of specificity. Additional research is now required to find a way to make the test more specific, 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.
Also in the basic research program, the ability of white blood cells to remember contact with Mptb has been tested in a proliferation assay in experimentally infected cattle remarkably the response in non-exposed controls remained low while it increased and remained elevated in exposed cattle. Gene expression studies Gene expression studies in infected cattle in the early stages of infection revealed a remarkable pattern of regulation of genes responsible for immunological processing. This uncovered pathways that previously have not been suspected to play much of a role in the early development of Johne's disease. This new knowledge will be applied in a future project.
Mechanism of disease progression
A contributing factor in the weight loss that occurs in bovine Johne's disease may have been found in addition to intestinal malabsorption and diarrhoea, an amino acid deficiency induced by the infection may contribute to wasting. Blood levels of the amino acid tryptophan were shown to plummet as Johne's disease develops in a sheep model. Expression of the enzyme that breaks down this amino acid was shown to be elevated in the blood of cattle exposed to Mptb. This relates to manipulation of the host by the mycobacterium, which induces the cow to destroy its own tryptophan, and impacts the way the immune system of the animal functions. Further research will be conducted across these fundamental discoveries to maximise their potential. New experimental infection model From the basic research program a method was proven for creating experimental bovine Johne's disease in a herd under tightly controlled conditions, leading to realistic and natural outcomes. Surgical biopsy of the intestine on two occasions confirmed that the animals had become infected, were developing Johne's disease at different rates, and did not have unrealistically severe infection which has been so common in overseas studies. 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.
