G.W. de Lisle

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.

A field evaluation of serological and cellular diagnostic tests for bovine tuberculosis

This paper describes the field evaluation of a serological test and a new in vitro assay for cell-mediated reactivity for the diagnosis of bovine tuberculosis. The use of a Mycobacterium bovis-specific antigen (MPB-70) in an ELISA to test the serological response to tuberculosis infection resulted in a specificity of 96.4% and a sensitivity of 18.1%. The most favourable results were obtained with the interferon gamma (IFN-gamma) assay which had a sensitivity of 81.8% and a specificity of 99.1%. Respective figures for the single intradermal tuberculin test were 68.1% and 96.7%. The use of MPB-70 as the antigen in the IFN-gamma assay reduced the sensitivity of this assay, without producing any useful increase in specificity. The IFN-gamma assay was also demonstrated to be a practical diagnostic test for use with large groups of cattle.

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.

Mycobacterial diseases of deer

Abstract The most significant mycobacterial diseases of free-living, captive and farmed deer are bovine tuberculosis, caused by Mycobacterium bovis, Johnes 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 subdinical 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. Johnes 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 Johnes 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, Johnes 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 Johnes 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 Johnes disease lack sensitivity and specificity, making control difficult.

Detection of Mycobacterium bovis in tissues by polymerase chain reaction.

A polymerase chain reaction (PCR) test was developed to detect Mycobacterium bovis in tissues. The test was based on amplification of a 248 bp segment of the insertion sequence, IS1081, present in six copies in strains of M. bovis and other members of the tuberculosis complex. The procedure involved digestion with proteinase K, lysis with sodium dodecyl sulphate, and extraction with hexadecyl tetramethyl ammonium bromide and phenol:chloroform:iso-amyl alcohol. When agarose gel electrophoresis was used for detection, the method was able to detect 1 fg of pure DNA, or 0.2 genome equivalents. It could also detect as few as 10 organisms from pure cultures and between 200-500 organisms from tissues spiked with cultured organisms. Detection by hybridization was only marginally more sensitive. The method was tested on 110 selected tissues recovered post mortem from a variety of animals. Fifty three of 58 samples diagnosed as M. bovis culture positive, including all samples containing microscopically visible acid-fast bacilli, were positive on duplicate testing by PCR. Five of 52 culture negative samples were also positive by PCR including three which contained large numbers of acid-fast organisms. Ten of the culture negative samples came from animals in a herd known to be free of bovine tuberculosis and all these were negative by PCR.

Comparison of polymerase chain reaction tests and faecal culture for detecting Mycobacterium paratuberculosis in bovine faeces.

A polymerase chain reaction (PCR) test for M. paratuberculosis was developed based on a 218 bp segment of a DNA insertion sequence, IS900, that is specific for this organism. The method involved two consecutive amplification reactions, with the second set of primers being nested inside the first set. The method reliably detected 50 organisms/g faeces. This PCR test was applied to 32 bovine faecal specimens containing high, moderate or low numbers of M. paratuberculosis organisms as determined by culture. The PCR test detected all specimens containing > or = 1600 colony forming units (cfu)/g faeces, six of ten specimens with 160-480 cfu/g faeces but only two of 13 specimens containing < or = 112 cfu/g faeces. The sensitivity of this test was better than that of a commercial PCR test which was carried out on the same faecal specimens.

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.

A study of the environmental survival of Mycobacterium bovis on a farm in New Zealand

Mycobacterium bovis organisms absorbed on cotton ribbons were placed in different natural habitats on a farm in New Zealand. Mycobacterium bovis was not re-isolated from ribbons placed on pasture after 4 days. Survival on ribbons was longest in brushtail possum dens, where the maximum period of survival in dens was less than 7 days in summer and greater than 14 days but less than 28 days in winter and spring. The maximum period of survival on a forest floor was intermediate between pasture and dens less than 4 days in summer and greater than 14 days but less than 28 days in winter. The overall probability of survival was influenced by season and was shortest in summer and longest in spring and winter. Survival time increased as minimum daily temperatures decreased. These studies showed there was a relatively short period of survival of M. bovis outside hosts and support a conclusion that environmental contamination of pasture, particularly in summer months, may be relatively unimportant in the epidemiology of tuberculosis in cattle, deer and possums.

A DNA prime-live vaccine boost strategy in mice can augment IFN-γ responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis

The Mycobacterium bovis bacille Calmette–Guérin (BCG) vaccine has variable efficacy for both human and bovine tuberculosis. There is a need for improved vaccines or vaccine strategies for control of these diseases. A recently developed prime‐boost strategy was investigated for vaccination against M. bovis infection in mice. BALB/c and C57BL/6 mice were primed with a DNA vaccine, expressing two mycobacterial antigens, ESAT‐6 and antigen 85 A and boosted with attenuated M. bovis strains, BCG or WAg520, a newly attenuated strain, prior to aerosol challenge. Before challenge, the antigen‐specific production of interferon‐γ (IFN‐γ) was evaluated by ELISPOT and antibody responses were measured. The prime‐boost stimulated an increase in the numbers of IFN‐γ producing cells compared with DNA or live vaccination alone, but this varied according to the attenuated vaccine strain, time of challenge and the strain of mouse used. Animals vaccinated with DNA alone generated the strongest antibody response to mycobacterial antigens, which was predominantly IgG1. BCG and WAg520 alone generally gave a 1–2 log10 reduction in bacterial load in lungs or spleen, compared to non‐vaccinated or plasmid DNA only control groups. The prime‐boost regimen was not more effective than BCG or WAg520 alone. These observations demonstrate the comparable efficacy of BCG and WAg520 in a mouse model of bovine tuberculosis. However, priming with the DNA vaccine and boosting with an attenuated M. bovis vaccine enhanced IFN‐γ immune responses compared to vaccinating with an attenuated M. bovis vaccine alone, but did not increase protection against a virulent M. bovis infection.

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.