Immunological and pathological responses of red deer resistant or susceptible genotypes, to experimental challenge with Mycobacterium avium subsp. paratuberculosis.

This study aimed to monitor the clinical, immunological and pathological changes in red deer for 49 weeks after experimental oral challenge with Mycobacterium avium subsp. paratuberculosis (MAP) and to assess the heritability of resistance in the offspring of two red stags. Eighteen young deer, which were bred from unselected hinds and sired by two stags resistant (R) or susceptible (S) to paratuberculosis, were challenged with MAP and monitored for 49 weeks. Biopsy samples of the jejunal lymph node were collected at Weeks 4 and 13 and at necropsy after euthanasia of clinically affected animals or when electively killed at Week 49. Three animals (two S and one R) developed clinical disease and were euthanised. The nine S offspring had significantly more severe lesions than the nine R offspring (Mantel-Haenszel Chi-square P=0.017). The average Lesion Severity Score (LSS) of R offspring was 5.9 (mild), and 7/9 had no or very mild lesions. In contrast, the LSS of S offspring averaged 11.7 (severe), and 7/9 had severe lesions. Most of the resistant, but not the susceptible, animals showed evidence of resolving lesions and a reduction in the number of MAP between 13 and 49 weeks after challenge. One R offspring appeared to completely cure itself, and progressed from mild culture-positive paratuberculosis lesions at Week 13 to having no signs of disease or infection 36 weeks later. This study showed significant heritable resistance/susceptibility to paratuberculosis and key differences in immunological responses in the first 3 months after challenge, indicating different paths to relative success or failure to control MAP. In general, R deer had higher IFN-γ levels, low antibody titres and fewer MAP, while S deer had lower IFN-γ levels, higher antibody and more MAP.

A bovine macrophage screening system for identifying attenuated transposon mutants of Mycobacterium avium subsp. paratuberculosis with vaccine potential.

Johne's disease is a chronic granulomatous enteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease is responsible for considerable economic losses in the livestock industry and in particular within the dairy sector. A more effective vaccine against Johne's disease would be of major benefit. In this study, we developed an efficient procedure for identifying mutants of MAP with reduced virulence that are potential live vaccine candidates against Johne's disease. A mariner transposon was used to create random insertional libraries in two different MAP strains (989 and k10), an effective cattle macrophage survival system was developed, and a total of 1890 insertion mutants were screened by using a 96-prong multi-blot replicator (frogger) system. Two of the transposon mutants with poor survival ability in macrophages were tested in mice. These strains were found to be attenuated in vivo, thereby validating the further use of this macrophage screening system to identify MAP mutants with potential as candidate vaccines against Johne's disease.

Mycobacterial diseases of deer.

The most significant mycobacterial diseases of free-living, captive and farmed deer are bovine tuberculosis, caused by Mycobacterium bovis, Johne's disease (paratuberculosis), caused by Mycobacterium avium subsp paratuberculosis (basonym M. paratuberculosis), and avian tuberculosis, caused principally by M. avium subsp avium. The first case of M. bovis infection in farmed deer was identified in New Zealand in 1978. In 1983, a voluntary scheme was introduced in New Zealand to control tuberculosis in farmed deer, followed by a compulsory tuberculosis control scheme in 1990. The primary control measure is the slaughter of infected animals, detected by skin testing and blood testing, together with movement control and vector control. The number of infected deer herds peaked in the mid 1990s at over 160 herds, but by 30 June 2002 this had been reduced to 79 (1.45%), and to 67 (1.23%) by June 2003. Deer-to-deer transmission occurs, but the majority of herd breakdowns are believed to be from infected vectors. Factors likely to affect the susceptibility of deer include age, environment, population density, exposure and genetics. Avian tuberculosis occasionally causes clinical disease in wild, captive and farmed deer in New Zealand and overseas. Mycobacterium intracellulare, and subspecies of M. avium other than M. paratuberculosis, are widespread throughout New Zealand and are thought to be largely responsible for the high level of sensitisation to avian purified protein derivative (PPD), which is used for comparison purposes in tuberculosis skin testing of deer in this country. Infections with these organisms are usually subclinical in farmed deer, although M. avium subsp avium commonly causes lesions in retropharyngeal, mesenteric and ileocaecal lymph nodes. These lesions cause problems because of their gross and microscopic similarity to those due to M. bovis infection. Birds and domestic animals are most likely to become infected via environmental contamination of food, water, bedding litter or soil, while carnivores or scavengers may also become infected by ingesting infected carcasses. Johne's disease has been reported in deer in the wild and in zoos, especially in North America, the United Kingdom (UK) and Europe. Since first being confirmed in farmed deer in New Zealand in 1979, the incidence of Johne's disease has increased steadily. To date, M. paratuberculosis has been identified in >600 farmed deer on 300 properties. The majority of cases have been identified from suspected tuberculous lesions submitted from deer slaughter plants. Clinically, Johne's disease in deer is similar to the disease in sheep and cattle, with typical signs of loss of weight and condition, and diarrhoea. However, outbreaks of Johne's disease frequently occur in young red deer, 8-15 months of age, whereas the clinical disease in sheep and cattle is sporadic and usually affects adults 3-5 years of age. The disease is characterised by a chronic granulomatous enteritis and lymphadenitis, especially affecting the jejunum and ileum and the mesenteric lymph nodes. Deer affected subclinically may have lesions in these lymph nodes at slaughter, which are grossly indistinguishable from those due to bovine tuberculosis. Because of the antigenic similarity between M. intracellulare and all the subspecies of M. avium, including M. paratuberculosis, the diagnostic tests for Johne's disease lack sensitivity and specificity, making control difficult.

Construction and screening of Mycobacterium paratuberculosis insertional mutant libraries.

Mycobacterium paratuberculosis causes Johne's disease, a common wasting disease in ruminants. As a first step in studying virulence mechanisms, libraries of random mutants were produced in two M. paratuberculosis strains by using the conditionally replicating shuttle phasmid phAE94 which contains the transposon Tn5367. Two thousand mutants were screened for auxotrophy, carbon source preference, and altered cell wall. Genes interrupted by insertion were identified for seven mutants isolated from the screening process. Two mutants had insertions in putative genes involved in synthesis of the cell wall.

Intraduodenal vaccination of brushtail possums with bacille Calmette-Guérin enhances immune responses and protection against Mycobacterium bovis infection.

SETTING: An effective oral bacille Calmette-Guérin (BCG) vaccine would have advantages for use in humans and as an oral bait vaccine for protecting wild-life against bovine tuberculosis. OBJECTIVE: To compare the level of protection against tuberculosis in intraduodenally BCG-vaccinated possums with those vaccinated intragastrically in order to determine whether degradation of BCG in the stomach lowers vaccine efficacy. DESIGN: Three groups of five possums were vaccinated with BCG by the intraduodenal, intragastric or subcutaneous routes, with a fourth group serving as unvaccinated controls. The animals were later challenged intratracheally with a low dose of virulent Mycobacterium bovis. RESULTS: Possums vaccinated intraduodenally with BCG had significantly greater lymphocyte blastogenic responses to bovine purified protein derivative (PPD) and lower lung bacterial counts in comparison with intragastrically vaccinated animals. In comparison with unvaccinated animals, all of the BCG-vaccinated groups had significant protection against M. bovis infection as assessed by changes in body weight, lung weight and reduction in numbers of mycobacteria and granulomas in the spleen. CONCLUSION: The enhanced immune responses and protection against bovine tuberculosis observed in the intraduodenally BCG-vaccinated possums indicated that if BCG vaccine is protected from degradation in the stomach its efficacy should improve.

Experimental Mycobacterium bovis infection in the brushtail possum (Trichosurus vulpecula): pathology, haematology and lymphocyte stimulation responses.

Groups of adult male brushtail possums (5 per group) were inoculated intratracheally with a high (2 x 10(5) colony forming units (cfu)), medium (2 x 10(3) cfu) or low (approximately 20 cfu) dose of Mycobacterium bovis. Two sham-inoculated groups acted as in-contact controls or controls kept in a separate room. Possums in the high and medium dose groups became clinically affected 3-5 weeks post-inoculation (PI) and all possums were euthanased between 5-9 weeks PI. Grossly visible tuberculous lesions were found in the lungs and associated lymph nodes of all possums from the high, medium and low dose groups. No lesions were observed in possums from the two control groups. Histopathologically, two characteristic types of lesions were observed; microscopic aggregates of macrophages with few acid-fast organisms, and larger lesions with limited granulomatous reaction, extensive necrosis and the presence of numerous acid-fast organisms. M. bovis was isolated from the lungs and lymph nodes of all of the possums from the high, medium and low dose groups and from the lungs of one of the in-contact controls. Changes in the haematological profile of the M. bovis-inoculated possums included lymphocytopaenia and eosinopaenia, together with raised fibrinogen levels. The onset of these changes was dependent on the size of the challenge dose. Lymphocyte stimulation responses to M. bovis tuberculin purified protein derivative were detected in 14 of 15 M. bovis-inoculated possums.