F.E. Aldwell

Immunological responses and protection against Mycobacterium bovis in calves vaccinated with a low dose of BCG

Groups of calves (20 per group) were vaccinated subcutaneously with a single dose of BCG Pasteur (6 x 10(4) or 6 x 10(6) colony forming units) and two months later, 15 calves from each group were challenged intratracheally with virulent Mycobacterium bovis. Vaccination with either dose of BCG induced significant protection against the development of tuberculous lesions compared to non-vaccinated controls. Seven months after BCG vaccination, many of the vaccinated animals which had no lesions and were M. bovis culture-negative at necropsy showed positive reactions for M. bovis in three assays for measuring cellular immune responses (comparative intradermal test, interferon-gamma assay and lymphocyte proliferation assay). This effect was most noticeable in the BCG-vaccinated calves which had been challenged with M. bovis rather than in the non-challenged animals. Antibody responses to M. bovis were very low or absent in the calves during the study. The kinetics of the interferon-gamma response of peripheral blood lymphocytes cultured in vitro with bovine PPD showed that BCG vaccination induced a rapid rise in the response followed by a sharp decline, while infection with virulent M. bovis resulted in an increase in the interferon-gamma response by four weeks after challenge and this response remained high through the study.

Protection of cattle from bovine tuberculosis by vaccination with BCG by the respiratory or subcutaneous route, but not by vaccination with killed Mycobacterium vaccae

Groups of cattle were vaccinated either with BCG Pasteur by the intratracheal or subcutaneous route or with killed Mycobacterium vaccae by the intradermal route and challenged intratracheally 54 days later with Mycobacterium bovis. Vaccination with BCG resulted in fewer animals developing tuberculous lesions and in a reduction in the number of lesions in the diseased animals compared with the unvaccinated group and the group vaccinated with M vaccae. None of the nine animals vaccinated intratracheally with BCG developed any tuberculous lung lesions after challenge with M bovis, but two of the nine animals from each of the groups dosed subcutaneously with low and medium doses of BCG developed lung lesions. There was little difference in protection against the M bovis challenge between the animals receiving the low dose (10(3) colony forming units, cfu) or medium dose (10(5) cfu) of subcutaneous BCG, but the medium dose of BCG produced stronger cell-mediated immune responses to bovine purified protein derivative (PPD) after vaccination. Vaccination intradermally with 10(9) heat-killed M vaccae did not protect cattle against an experimental challenge with M bovis and induced only weak cell-mediated immune responses to bovine PPD.

Influence of sensitisation to environmental mycobacteria on subsequent vaccination against bovine tuberculosis.

Bacille Calmette-Guerin (BCG) is the worlds most widely used vaccine, but there are concerns that it provides little protection against pulmonary tuberculosis of humans in countries that have a high prevalence of environmental mycobacteria. Experiments in cattle provide a model to investigate this situation and to develop an improved tuberculosis vaccine. In the third of a series of BCG vaccination trials, calves had high interferon-gamma (IFN-gamma) responses to purified protein derivative (PPD) from Mycobacterium avium prior to vaccination, indicating that infection with environmental mycobacteria had occurred. The calves vaccinated with BCG had minimal protection against an experimental intratracheal challenge with virulent Mycobacterium bovis. In comparison, calves vaccinated with either of two newly-derived attenuated M. bovis strains had significantly better but not complete protection against the development of tuberculous lesions compared to both BCG-vaccinated and non-vaccinated animals. Vaccination with the newly-derived attenuated M. bovis strains induced strong IFN-gamma and interleukin-2 (IL-2) responses to PPD from M. bovis at 2 weeks after vaccination, while BCG vaccination induced only a weak response at this time. In association with the previous two trials, the results suggest that sensitisation of the calves to environmental mycobacteria adversely affected subsequent protective efficacy of BCG. However, the results of vaccination with the other two attenuated M. bovis strains indicated that improved tuberculosis vaccines could be developed for such sensitised animals.

Oral vaccination reduces the incidence of tuberculosis in free-living brushtail possums.

Bovine tuberculosis (Tb) caused by Mycobacterium bovis has proved refractory to eradication from domestic livestock in countries with wildlife disease reservoirs. Vaccination of wild hosts offers a way of controlling Tb in livestock without wildlife culling. This study was conducted in a Tb-endemic region of New Zealand, where the introduced Australian brushtail possum (Trichosurus vulpecula) is the main wildlife reservoir of Tb. Possums were trapped and vaccinated using a prototype oral-delivery system to deliver the Tb vaccine bacille Calmette–Guerin. Vaccinated and control possums were matched according to age, sex and location, re-trapped bimonthly and assessed for Tb status by palpation and lesion aspiration; the site was depopulated after 2 years and post-mortem examinations were conducted to further identify clinical Tb cases and subclinical infection. Significantly fewer culture-confirmed Tb cases were recorded in vaccinated possums (1/51) compared with control animals (12/71); the transition probability from susceptible to infected was significantly reduced in both males and females by vaccination. Vaccine efficacy was estimated at 95 per cent (87–100%) for females and 96 per cent (82–99%) for males. Hence, this trial demonstrates that orally delivered live bacterial vaccines can significantly protect wildlife against natural disease exposure, indicating that wildlife vaccination, along with existing control methods, could be used to eradicate Tb from domestic animals.

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.

Experimental Mycobacterium bovis infection of cattle: Effect of dose of M. bovis and pregnancy on immune responses and distribution of lesions

Groups of 18-month-old cattle were inoculated intratracheally with 5 x 10(5) colony forming units (high dose) or 500 colony forming units (low dose) of Mycobacterium bovis to determine an appropriate dose to induce lesions similar to those seen in the natural disease. An additional group of 21-28 weeks pregnant cattle were inoculated with the high dose of M. bovis to determine if pregnancy increased the susceptibility of cattle to M. bovis infection. By 23-24 weeks after challenge, the high dose of M. bovis had induced extensive lung lesions, and tuberculous lesions were observed in the lymph nodes of the head, neck, and thoracic and abdominal cavities. In contrast, the low dose of M. bovis induced predominantly small lesions (< 1 cm diameter) which were localised to the lungs and pulmonary lymph nodes. The lesions induced by the low dose were similar to those seen in the natural disease in cattle. The majority of the high dose group cattle produced strong antibody responses to M. bovis culture filtrate, while only one low dose animal produced a detectable response. All of the M. bovis-inoculated cattle produced strong cellular immune responses to bovine PPD (skin test and interferon-gamma responses). Pregnancy did not appear to affect the susceptibility to M. bovis infection, and immune responses of the cattle in this group at the end of the study were similar to those in the high dose non-pregnant group. However, from the first test after calving, the interferon-gamma responses of peripheral blood cultures to bovine PPD were low compared with the responses prior to calving.

Effectiveness of BCG vaccination in protecting possums against bovine tuberculosis.

Three groups of eight possums were vaccinated with BCG Pasteur by the subcutaneous, intratracheal or intragastric routes, with a fourth group serving as unvaccinated controls. Forty-two days after the start of vaccination, five possums from each group were challenged intratracheally with virulent Mycobacterium bovis. The vaccination by the subcutaneous or intratracheal routes resulted in a marked reduction in the severity of disease compared with the unvaccinated animals or the animals vaccinated intragastrically. The severity of the disease was assessed by changes in bodyweight, pathological changes in the lungs and bronchial nodes and the number of acid-fast bacilli in the lesions. Before the challenge, lymphocyte blastogenic responses to bovine PPD were observed in the eight animals vaccinated subcutaneously and in two of the animals vaccinated intratracheally.

Route of BCG administration in possums affects protection against bovine tuberculosis.

The Australian brushtail possum (Trichosurus vulpecula) is the major wildlife reservoir of Mycobacterium bovis in New Zealand. Control of bovine tuberculosis in farmed animals requires measures to reduce the transmission of M. bovis from wildlife. Possums were vaccinated with BCG intranasally by aerosol spray, orally or subcutaneously to compare the efficacy of these three routes on protection against challenge with virulent M. bovis. Possums vaccinated with BCG by the intranasal or subcutaneous routes had a marked reduction in severity of disease compared to possums which had been unvaccinated or orally vaccinated. The severity of the disease was assessed by changes in body weight and pathology. BCG vaccination by all three routes resulted in reduced dissemination of M. bovis to the spleen and liver following challenge. Intranasal and oral BCG vaccination induced lower mean peripheral blood lymphocyte blastogenic responses to bovine PPD than subcutaneous vaccination, indicating that these responses did not correlate well with protection from the disease. Given a suitable delivery system, aerosol vaccination of possums, used in conjunction with other control measures, may be a suitable method of reducing the spread of M. bovis from wildlife to domestic animals.

Evaluation of three serological assays for the diagnosis of Mycobacterium bovis infection in brushtail possums

Three serological tests for the diagnosis of Mycobacterium bovis infection were evaluated on 29 possums (Trichosurus vulpecula) with tuberculosis and on 100 possums from a tuberculosis-free area. An indirect ELISA using M. bovis culture filtrate as the antigen had a sensitivity of 45% and a specificity of 96%, while an indirect ELISA using a M. bovis specific antigen (MPB70) had a sensitivity of 21% and a specificity of 98%. A blocking ELISA which utilised a monoclonal antibody against MPB70 had a sensitivity of 28% and a specificity of 99%. Combination of the test results of the three ELISAs resulted in an increase in sensitivity to 51% and a decrease in specificity to 93%. A previous study has shown that possums experimentally infected with M. bovis produced cellular responses to M. bovis antigens relatively early in the infection, but these responses decreased in the terminal stages of the disease. In contrast, analysis of serological responses in the current study from sequentially collected sera of possums experimentally and naturally infected with M. bovis showed that antibody was first detected late in the disease.

Antigen-induced interferon-γ and interleukin-2 responses of cattle inoculated with Mycobacterium bovis

Bovine purified protein derivative (PPD)-induced interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) mRNA expression was measured in peripheral blood lymphocyte cultures of cattle inoculated with Mycobacterium bovis and compared to cytokine protein levels as measured by IFN-gamma enzyme-linked immunosorbent assay and IL-2 bioassay. For individual animals, positive correlations were observed between mRNA and protein levels of bovine PPD-induced IFN-gamma and IL-2, although the correlations were stronger for IFN-gamma. Expression of these two cytokines also correlated with responses from a comparative intradermal test and a M. bovis antibody test. At 7 and 20 weeks after inoculation, bovine PPD-induced IFN-gamma and IL-2 mRNA expression was detected in all animals with tuberculous lesions and in a proportion of the M. bovis-inoculated animals with no lesions. Correlation of antigen-induced IFN-gamma and IL-2 with other immune parameters suggests that these two cytokines play an important role in the immune response to bovine tuberculosis.