Toward eradication: the effect of Mycobacterium bovis infection in wildlife on the evolution and future direction of bovine tuberculosis management in New Zealand.

New Zealand's bovine tuberculosis (TB) control programme has greatly reduced the burden of tuberculosis on the farming industry, from 11% of mature cattle found with TB at slaughter in 1905 to <0.003% in 2012/13. New Zealand implemented TB control measures in cattle from the mid-twentieth century, and later in farmed deer. Control was based on established methods of tuberculin testing of herds, slaughter of suspect cases, and livestock movement control. Unexplained regional control failures and serious disease outbreaks were eventually linked to wildlife-vectored infection from the introduced Australian brushtail possum (Trichosurus vulpecula), which also triggered a wildlife disease complex involving a range of introduced species. This paper reviews the progressive elucidation of the epidemiology of Mycobacterium bovis in New Zealand's wildlife and farmed livestock, and the parallel development of research-led, multi-faceted TB control strategies required to protect New Zealand's livestock industries from damaging infection levels. The adoption of coordinated national pest management strategies, with increasingly ambitious objectives agreed between government and industry funders, has driven a costly but very successful management regime targeted at controlling TB in the possum maintenance host. This success has led to initiation of a strategy designed to eradicate TB from New Zealand's livestock and wildlife, which is considered a realistic long-term prospect.

Epidemiology, diagnostics, and management of tuberculosis in domestic cattle and deer in New Zealand in the face of a wildlife reservoir.

The control of tuberculosis (TB) in cattle and farmed deer in New Zealand has been greatly influenced by the existence of a wildlife reservoir of Mycobacterium bovis infection, principally the Australian brushtail possum (Trichosurus vulpecula). The reduction in possum numbers in areas with endemic M. bovis infection through vigorous vector control operations has been a major contributor to the marked reduction in the number of infected cattle and farmed deer herds in the past two decades. Management of TB in cattle and farmed deer in New Zealand has involved a combination of vector control, regionalisation of diagnostic testing of cattle and deer herds, abattoir surveillance and movement control from vector risk areas. Accurate diagnosis of infected cattle and deer has been a crucial component in the control programme. As the control programme has evolved, test requirements have changed and new tests have been introduced or test interpretations modified. Subspecific strain typing of M. bovis isolates has proved to be a valuable component in the epidemiological investigation of herd breakdowns to identify whether the source of infection was domestic livestock or wildlife. New initiatives will include the use of improved models for analysing diagnostic test data and characterising disease outbreaks leading to faster elimination of infection from herds. The introduction of the National Animal Identification Tracing programme will allow better risk profiling of individual herds and more reliable tracing of animal movements. TB in cattle and farmed deer in New Zealand can only be controlled by eliminating the disease in both domestic livestock and the wildlife reservoir.

Development of integrated surveillance systems for the management of tuberculosis in New Zealand wildlife.

Disease surveillance for the management of bovine tuberculosis (TB) in New Zealand has focussed, to a large extent, on the development of tools specific for monitoring Mycobacterium bovis infection in wildlife. Diagnostic techniques have been modified progressively over 30 years of surveillance of TB in wildlife, from initial characterisation of gross TB lesions in a variety of wildlife, through development of sensitive culture techniques to identify viable mycobacteria, to molecular identification of individual M. bovis strains. Of key importance in disease surveillance has been the elucidation of the roles that different wildlife species play in the transmission of infection, specifically defining brushtail possums (Trichosurus vulpecula) as true maintenance hosts compared to those that are predominantly spillover hosts, but which may serve as useful sentinel species to indicate TB persistence. Epidemiological modelling has played a major role in TB surveillance, initially providing the theoretical support for large-scale possum population control and setting targets at which control effort should be deployed to ensure disease eradication. As TB prevalence in livestock and wildlife declined throughout the 2000s, more varied field tools were developed to gather surveillance data from the diminishing possum populations, and to provide information on changing TB prevalence. Accordingly, ever more precise (but disparate) surveillance information began to be integrated into multi-faceted decision-assist models to support TB management decisions, particularly to provide informed parameters at which control effort could be halted, culminating in the Proof of Freedom modelling framework that now allows an area to be declared TB-free within chosen confidence limits. As New Zealand moves from large-scale TB control to regional eradication of disease in the coming years, further integrative models will need to be developed to support management decisions, based on combined field data of possum and TB prevalence, sentinel information, risk assessment in relation to financial benefits, and changing political and environmental needs.

The prevalence of disseminated Mycobacterium avium subsp. paratuberculosis infection in tissues of healthy ewes from a New Zealand farm with Johne's disease present.

AIM: To determine the prevalence of disseminated Mycobacterium avium subsp. paratuberculosis (Map) infection in healthy ewes in a flock with a history of clinical Johne's disease. METHODS: Twenty-four healthy ewes, from a large sheep and cattle farm with a history of clinical Johne's disease in the ewe flock, were randomly selected, euthanased, blood sampled, and examined at necropsy. BACTEC™ radiometric culture for Map was performed on samples of faeces, ileum, mesenteric lymph node, biceps femoris muscle and mononuclear cells in peripheral blood. Serum antibody ELISA tests were performed. Histological sections and Ziehl Neelsen (ZN) stains of impression smears of ileum and mesenteric lymph node were examined for pathological lesions characteristic of Johne's disease and acid fast organisms (AFO). Indirect quantification of Map was performed, using BACTEC radiometric growth indices measuring the time taken for the production of (14)CO(2.) RESULTS: No histological evidence of Johne's disease or AFO was found in the ileum and mesenteric lymph nodes. Twelve of the 24 ewes (50%) had Map cultured from the ileum (n=6) and/or mesenteric lymph nodes (n=8) while none had Map cultured from the faeces, biceps femoris muscle or blood mononuclear cells. One of the 12 Map culture positive ewes was serum ELISA positive. The culture growth rates in liquid medium suggest low numbers of Map were present in the tissues of the culture positive ewes. CONCLUSION: Fifty per cent of clinically healthy ewes exposed to Map within a Johne's infected flock were Map culture positive in the ileum and/or mesenteric lymph node(s), while the ELISA was positive in 8% of those animals (n=1). There was no faecal shedding of Map and no Map was cultured from skeletal muscle or from blood mononuclear cells suggesting that systemic Map infection, defined as positive culture of Map from skeletal muscle and/or blood, may be uncommon in healthy mixed age ewes without clinical Johne's disease. CLINICAL RELEVANCE: ELISA serology detected 1 of 12 ewes infected with Map whilst none were detected from faecal BACTEC radiometric culture, suggesting biosecurity measures used to control the spread of Map may be of limited use. Map was not cultured from blood mononuclear cells or skeletal muscle, indicating that meat from healthy ewes, from farms where Johne's disease is present, is an unlikely source of Map exposure for humans. Further research is warranted to establish the prevalence and dissemination of Map in tissues outside the alimentary tract of healthy ewes from farms throughout New Zealand where Map is present.

Mycobacterium avium subsp. paratuberculosis infection in wildlife on three deer farms with a history of Johne's disease.

AIM: To determine the prevalence of Mycobacterium avium subsp. paratuberculosis (Map) infection in wildlife, in pastoral landscapes with a recent history of clinical Johne's disease in livestock. METHODS: A total of 449 wild mammals and birds from three farms in the South Island of New Zealand with recent histories of clinical Johne's disease in their deer herds were trapped and examined for gross pathological changes in the gastrointestinal tract. Additionally, individual mesenteric lymph nodes from 380 mammals, and segments of gastrointestinal tract from 32 birds were excised, homogenised and cultured for viable Map bacilli. The prevalence of Map infection was then calculated for the various species. Faecal samples from those mammals which had culture-positive tissues were further cultured for the presence of Map. RESULTS: Gross pathological changes were identified in the gastrointestinal tract of four brushtail possums, one cat, six ferrets, 12 hares, six hedgehogs, three rabbits, one stoat, and one paradise shelduck. Infection with Map in the gastrointestinal tract was confirmed in only three of these cases, one each of brushtail possums, hares and hedgehogs. In contrast, Map infection in the absence of gross pathological changes was frequently recorded in enteric tract tissues of mammals and birds. Among mammals, Map infection was recorded in 18/73 (25%) brushtail possums, 4/23 (17%) cats, 15/42 (36%) hedgehogs and 29/113 (26%) rabbits. Among birds, intestinal tract tissue Map infection was recorded in 3/17 (18%) paradise shelducks. Among 64 of the 74 mammals which had Map culture-positive tissues, 38% (n=5) of hedgehogs and 11% (n=3) of rabbits also had culture-positive faecal samples. CONCLUSIONS: This study is the first to identify that Map infection can be prevalent in wildlife in New Zealand. There was a high prevalence of Map infection among both scavenging and grazing wild animals. Both mammals and birds are capable of harbouring viable Map organisms in their gastrointestinal tract; further, viable Map was excreted into the environment via faeces by hedgehogs and rabbits. CLINICAL RELEVANCE: Previous studies overseas have postulated a role of wildlife as reservoirs of Map infection and possible vectors of Johne's disease to livestock. Here, brushtail possums, hedgehogs and rabbits and in particular were identified as potential wildlife hosts for Map infection in New Zealand. This suggests that several wildlife species could contribute to the persistence of Map infection within a wildlife/livestock complex, and potentially, perhaps more importantly, to the spread of infection between farms.

Longevity of Mycobacterium bovis in brushtail possum (Trichosurus vulpecula) carcasses, and contact rates between possums and carcasses.

AIM: To determine, for a variety of environmental conditions, how long Mycobacterium bovis might remain viable inside the carcass of a brushtail possum (Trichosurus vulpecula) that died of bovine tuberculosis (Tb), and to measure the rate of contact between free-ranging possums and possum carcasses. METHODS: Lesions of M. bovis were simulated by inoculating excised spleens weighing 0.5-1 g with 0.2 mL liquid culture containing approximately 5 x 10(7) cfu M. bovis/mL. Simulated lesions were inserted into possum carcasses (n=48) at the peripheral lymph nodes. Carcasses were placed in the field at two sites (a tussock grassland and a podocarp-broadleaved forest site) and in two seasons (summer and winter) for up to 62 days. Survival rates of M. bovis were estimated by sampling the simulated lesions over time, and culturing the recovered lesion to determine if any viable M. bovis bacteria were present. The time taken for a free-ranging possum to first encounter a dead possum in its home range was estimated by live-trapping possums and fitting them with proximity loggers (n=13). A 'contact' was recorded if these possums came within 40-50 cm of proximity loggers fitted to possum carcasses. RESULTS: There were strong seasonal and site effects in the survival rate of M. bovis in possum carcasses. In the grassland habitat, no viable bacilli were cultured from any carcass after 3 days in summer, whereas in winter all samples were culture-positive for the first 20 days, and some were still positive after 27 days. The survival rates for forest habitat were intermediate between the results for grassland, and there were no culture-positive carcasses after 9 days in summer or 27 days in winter. In summer, infected carcasses (n=6) were first encountered by possums a mean 1.9 (range 0.4-6.7) days after placement. CONCLUSIONS: Possum carcasses were contacted by free-ranging possums within the period that viable M. bovis were shown to survive in a carcass. The risk of such infection is likely to be most significant in winter or in areas with microhabitats where the survival of M. bovis is high. However, the generally low survival rate of M. bovis in possum carcasses and the low frequency of possum-to-carcass contacts indicate this route of transmission alone could not maintain Tb in a possum population.

Detection of Mycobacterium avium subsp. paratuberculosis in skeletal muscle and blood of ewes from a sheep farm in New Zealand.

AIM: To determine whether viable Mycobacterium avium subsp. paratuberculosis (Map) is present in skeletal muscle and blood in ewes with and without Johne's disease confirmed histologically. METHODS: A total of 51 mixed-aged ewes in poor body condition from a farm with a history of clinical Johne's disease were culled and examined at necropsy. BACTEC radiometric culture was performed on samples of skeletal muscle from the biceps femoris, mononuclear cells in peripheral blood (hereafter referred to as blood), and ileum. Histological sections and Ziehl-Neelsen (ZN)-stained impression smears of terminal ileum and mesenteric lymph nodes were examined. Ewes were defined as having confirmed Johne's disease if there was histopathological evidence typical of the disease within the ileum and adjacent lymph nodes. RESULTS: Eighteen of 21 (86%) ewes with confirmed clinical Johne's disease were culture-positive for Map from sites peripheral to the alimentary tract, comprising 15 from skeletal muscle and 13 from blood. Five of 30 (17%) ewes that did not have Johne's disease were culture-positive, with four from skeletal muscle and one from blood. The likelihood that ewes with confirmed Johne's disease had systemic Map infection compared with ewes without was determined as OR=30 (95% CI=6.3-142.0; p<0.001). CONCLUSION: The prevalence of Map infection of skeletal muscle and blood in ewes with confirmed Johne's disease was 71% and 62% respectively, and in unaffected ewes was 13% for muscle and 3% for blood. CLINICAL RELEVANCE: Skeletal muscle and blood are potential sources of exposure of humans to Map, and the risk appears higher from sheep with Johne's disease.

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.

Serological survey of Toxoplasma gondii and Campylobacter fetus fetus in sheep from New Zealand.

AIM: To determine the prevalence of antibody titres to Toxoplasma gondii and Campylobacter fetus fetus in sheep from New Zealand. METHODS: As part of a free screening service, unsolicited blood samples were supplied by veterinarians wishing to gauge the exposure of their clients' ewe flocks to T. gondii and C. fetus fetus. Blood samples were submitted from mixed-age ewes throughout New Zealand, from 2006 to 2009, that had not been vaccinated for T. gondii and C. fetus fetus. A total of 2,254 sera were serologically titrated for T. gondii and 3,429 for C. fetus fetus. A latex agglutination kit available commercially was used to quantify antibodies to T. gondii, and an agglutination test developed in-house was used for C. fetus fetus. For T. gondii, titres of ≥1:16 and ≥1:64 were used to define a positive response, and for C. fetus fetus a titre of ≥1:10 was defined as positive. A flock was defined as positive if ≥1 ewe had a positive titre. RESULTS: Of the sera tested for T. gondii, 1,917/2,254 (85%) were positive, using a titre of ≥1:16, and 1,384/2,254 (61%) with a titre of ≥1:64. All 198 ewe flocks tested were seropositive to T. gondii, at a titre of ≥1:16, and all but three were at a titre of ≥1:64. A bimodal distribution was evident in the prevalence of titres to T. gondii suggesting that a percentage of titres ≤1:64 may have been non-specific. Of the sera tested for C. fetus fetus, 1,644/3,429 (48%) were positive to at least one of the four test antigens at titre of ≥1:10. Only 34/298 (11%) flocks tested for C. fetus fetus were completely seronegative. The percentage of seropositive ewes to both T. gondii and C. fetus fetus was significantly higher in the North Island than the South Island. CONCLUSIONS: The study demonstrated that exposure to these two important infectious abortifacients was both considerable and widespread. Minimum titres were postulated to establish a 'cut-off' for a positive result and to allow comparison with past and future studies. The bimodal distribution evident for T. gondii suggested a titre of 1:64 may be an appropriate cut-off. The widespread on-farm exposure probably stimulates the immune response of vaccinated ewes. CLINICAL RELEVANCE: Further studies are required to confirm the clinical significance of flock-based antibody responses, and to validate their use in identifying recently aborted ewes, especially where there are no aborted fetuses for examination.

Newly attenuated Mycobacterium bovis mutants as vaccines against bovine tuberculosis, particularly for possums.

Bovine tuberculosis costs New Zealand more than $80 million per year, mostly because extensive areas of the country are occupied by brushtail possums infected with Mycobacterium bovis. AgResearch has a major programme to produce new live tuberculosis vaccines that can be delivered to possums. Primary work involved development of molecular biological methods to enable genetic manipulation of M. bovis, including the production of random and specific mutants. Many avirulent mutants of M. bovis have been produced and their vaccine efficacy has been compared to BCG in guinea pigs. Selected mutants that perform at least as well as BCG are retested in guinea pigs using an extended vaccination protocol in which animals are pre-sensitized to environmental mycobacteria to mimic natural exposure. Ten candidate vaccines that have induced good protection in guinea pigs have been subsequently tested as vaccines in possums. While the protective efficacy of an M. bovis mutant inoculated into guinea pigs reliably indicated that some protection would be induced in possums, the most protective mutant in guinea pigs was different from that in possums. This illustrates the importance of testing in the target species as part of new vaccine development. An important outcome of this work was the identification of an operon in M. bovis whose inactivation produced an avirulent M. bovis vaccine candidate that was better than BCG in protecting possums from experimental tuberculosis. Allelic exchange methods are now being used to produce vaccine strains with multiple specific mutations to improve safety and immunological characteristics.

The evolution and distribution of phage ST160 within Salmonella enterica serotype Typhimurium.

Salmonellosis is an internationally important disease of mammals and birds. Unique epidemics in New Zealand in the recent past include two Salmonella serovars: Salmonella enterica subsp. enterica serovar Typhimurium definitive type (DT) 160 (S. Typhimurium DT160) and S. Brandenburg. Although not a major threat internationally, in New Zealand S. Typhimurium DT160 has been the most common serovar isolated from humans, and continues to cause significant losses in wildlife. We have identified DNA differences between the first New Zealand isolate of S. Typhimurium DT160 and the genome-sequenced strain, S. Typhimurium LT2. All the differences could be accounted for in one cryptic phage ST64B, and one novel P22-like phage, ST160. The majority of the ST160 genome is almost identical to phage SE1 but has two regions not found in SE1 which are identical to the P22-like phage ST64T, suggesting that ST160 evolved from SE1 via two recombination events with ST64T. All of the New Zealand isolates of DT160 were identical indicating the clonal spread of this particular Salmonella. Some overseas isolates of S. Typhimurium DT160 differed from the New Zealand strain and contained SE1 phage rather than ST160. ST160 was also identified in New Zealand isolates of S. Typhimurium DT74 and S. Typhimurium RDNC-April06 and in S. Typhimurium DT160 isolates from the USA. The emergence of S. Typhimurium DT160 as a significant pathogen in New Zealand is postulated to have occurred due to the sensitivity of the Salmonella strains to the ST160 phage when S. Typhimurium DT160 first arrived.

Age susceptibility of red deer (Cervus elaphus) to paratuberculosis.

The aim of this study was to measure the relative susceptibility of three age classes of red deer (Cervus elaphus) to the various manifestations of infection and disease states due to Mycobacterium avium subsp. paratuberculosis (MAP) after experimental oral challenge. Three groups of seronegative female deer (30 three-month-old weaners, 20 fifteen-month-old yearlings and 20 adults) received four oral doses of approximately 10(9) colony forming units (cfu) of a bovine strain of MAP. They were monitored for 50 weeks by weighing, blood sampling for immunological assays, skin testing and faecal culturing. Clinically affected animals were promptly euthanised and the remaining deer were killed at the end of the study. Necropsies were carried out and samples of intestine and associated lymph nodes were taken for culture and histopathology from all deer. Ten weaners developed clinical paratuberculosis and were euthanased 20-28 weeks post-challenge (pc). No clinical cases occurred in the yearlings or adults. All 10 clinically affected weaners had severe gross and histopathological lesions typical of paratuberculosis (Johne's disease). At slaughter, gross lesions were seen in the jejunal lymph nodes of 8/17 weaners, 2/19 yearlings, and 0/20 adults. MAP was cultured from samples of the intestine and/or lymph nodes from all 10 clinical cases and from 16/17 weaners, 19/19 yearlings and 18/20 adult hinds at slaughter. Lesion Severity Scores of deer slaughtered 50 weeks pc averaged 4.9, 3.5 and 1.1 for the weaner, yearling and adult groups, respectively. At some time over the course of the trial, 24/28 weaners were antibody positive and immediately prior to slaughter, 13/17 weaners, 15/19 yearlings and 3/20 hinds were antibody positive. There is a strong age-related resistance against clinical disease and subclinical disease, but not to infection with MAP, after heavy oral challenge.

Advances in ante-mortem diagnosis of tuberculosis in cattle.

The tuberculin skin test is effective in the early detection of pre-clinical cases of Mycobacterium bovis infection in cattle. This allows the rapid removal of infected animals, thus limiting transmission of the disease, and has resulted in the eradication of bovine tuberculosis (Tb) from many countries. This test is very likely to remain the primary screening test for M. bovis infection in cattle as it is a simple, robust and inexpensive test. However, a number of ancillary tests are being used, or are currently being validated. These ancillary tests are likely to provide a more accurate diagnosis following skin-testing. The blood-based BOVIGAM interferon-gamma (IFN-gamma) test is a cellular immune assay which can detect early infection, and has become the main ancillary test in New Zealand. It can be used for re-testing skin test-positive animals, to improve specificity and minimise wastage from slaughtering animals with false-positive tests. Alternatively, it can be used in locations of increased risk of infection in parallel with skin-testing, for examining skin test-negative animals for pre-movement testing or in problem herds to identify M. bovis-infected animals that do not respond to the skin test. Several modifications of the test are now being used to improve specificity by altering the cut-off or using specific antigens present in virulent mycobacteria such as the 6 kDa early secreted antigenic target (ESAT-6) and 10 kDa culture filtrate protein (CFP-10). While antibody based tests generally lack sensitivity, as high levels of antibodies tend to occur late in the disease process, they may have unique desirable properties such as the ability to be used as a cow-side test. The use of these new ancillary tests in association with skin-testing will improve the detection of M. bovis-infected cattle and reduce the unnecessary slaughter of false-positive reactors.

Isolation of Mycobacterium bovis from brushtail possums with non-visible lesions.

AIM: To determine the prevalence of Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula) that did not have macroscopic lesions of bovine tuberculosis, and to evaluate culture of pooled tissues from multiple possums as a method for determining the M. bovis-infection status of wildlife populations in New Zealand. METHODS: Pools of selected tissues were collected from possums from four different populations known to be infected with M. bovis. Tissue pools from individual animals, and combined pools from multiple animals, were cultured for M. bovis. RESULTS: In the four populations investigated, the prevalence of possums with macroscopic lesions confirmed by culture to be infected with M. bovis ranged from 1 to 19 (mean 31/283; 10.9)%. The prevalence of possums with non-visible lesions that were culture positive for M. bovis in the same populations ranged from 4 to 10 (mean 24/283; 8.5)%. The mean of the log10 cfu of M. bovis of the macroscopic lesions and of the culture- positive samples that did not have visible lesions was 3.85 (SE 0.26) and 1.46 (SE 0.26) log10 cfu, respectively (p<0.01). Mycobacterium bovis was cultured from pools of 30-50 animals in the four populations studied. CONCLUSIONS: The finding of M. bovis infection in possums with non-visible lesions identified a potential deficiency of declaring possum populations free of M. bovis on the basis of absence of macroscopic lesions. The culturing of pools of selected tissues from multiple animals without visible lesions can be used to reduce laboratory costs of possum surveys without a major reduction in the ability to detect M. bovis infection.

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.

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.

Protection of free-living and captive possums against pulmonary challenge with Mycobacterium bovis following oral BCG vaccination.

In New Zealand, possums (Trichosurus vulpecula) are the main wildlife reservoir for bovine tuberculosis (Tb), which they transmit to livestock. This study investigated oral vaccination with lipid-formulated Mycobacterium bovis BCG and subsequent protection against virulent M. bovis challenge in wild-caught possums. Possums were trapped from the field and either hand-vaccinated and released back into the wild, or acclimatised to captive conditions prior to voluntary uptake of flavoured vaccine. Possums were subsequently exposed to pulmonary challenge with virulent M. bovis, administered either by instillation of a liquid suspension as an intra-tracheal challenge (field animals) or in micro-droplets as an aerosol (captive animals). Field studies indicated that the relative risk of death in wild possums due to Tb was 2.4 times greater in control compared with orally-vaccinated possums, with the vaccine conferring protection to possums in both good and poor body condition. Laboratory studies indicated that oral vaccination conferred protection in cage-acclimatised possums, with >3log(10) reduction in lung bacterial burdens among vaccinated animals. This study provides evidence that lipid-formulated BCG oral vaccine can provide significant protection to possums in field as well as laboratory conditions, which may favour the use of this formulation as a delivery method for controlling wildlife Tb.

Efficacy, immune responses and side-effects of vaccines against Johne's disease in young red deer (Cervus elaphus) experimentally challenged with Mycobacterium avium subsp paratuberculosis.

AIMS: To test the efficacy of a commercially available and an experimental vaccine against Johne's disease in young red deer (Cervus elaphus), using experimental challenge with live virulent Mycobacterium avium subsp paratuberculosis (M. ptb), measure injection-site reactions, and assess the effects of vaccination and challenge on results of subsequent skin tests and ancillary blood tests for bovine tuberculosis (Tb). METHODS: Ninety 6-8-week-old red deer fawns were randomly allocated to three equal groups of 30, and received either a 1-ml S/C injection of either a commercially available whole-cell killed vaccine with a mineral-oil adjuvant (COM), or a live attenuated M. ptb experimental vaccine with a lipid adjuvant (EXP), or were unvaccinated controls. Ten weeks later (Week 10), all 90 fawns received an oral challenge with approximately 10(8) cfu of a bovine strain of M. ptb daily for 4 days. The fawns were regularly weighed and monitored for clinical signs of Johne's disease, and regularly blood-sampled and tested for antibodies to M. ptb, using the Paralisa test, an IgG1 ELISA, and for antibodies to Mycobacterium bovis, using a similar test. A mid-cervical tuberculin skin test (MCT) was administered at Week 23, and comparative cervical skin tests (CCTs) were administered at Weeks 37 and 57. All animals were electively killed at Week 59, injection sites inspected, gastrointestinal tracts examined for gross lesions, and samples taken for culture and histopathology. RESULTS: There were no clinical cases of Johne's disease but, at slaughter, more gross lesions in intestinal lymph nodes were observed in Control (20%) than COM animals (0%; p<0.05). This latter group also had less severe histopathological lesions in samples of intestines and lymph nodes compared with the Control group (p<0.05), but not deer in the EXP group. Over 89% of deer in all three groups were shown by culture to be infected with M. ptb, while only 21-33% of faecal samples were culture-positive. Time to positive culture was longer for COM vs EXP and Control groups (p<0.01), reflecting fewer M. ptb organisms in samples from the ileocaecal valve (ICV) in that group. Almost all (>or=90%) deer reacted to the MCT at Week 23, and there were no significant differences between groups. One or two deer in each group were classified as Tb reactors to the CCT at Week 37, and none were classified as Tb reactors to the CCT at Week 57. At the time of challenge, over 50% of deer in the COM group were classified as positive (9/28) or suspicious (7/28) for M. ptb antibodies in the Paralisa test, one animal in the EXP group was classified as suspicious, and all the Controls were negative. From Week 23 to the end of the trial, 25/28 (89%) deer in the COM group were Paralisa-positive or -suspicious. The proportion of animals in the EXP and Control groups that were Paralisa-positive peaked at Week 39 (60% and 55%, respectively). The majority of deer in the COM group had significant levels of antibody to M. bovis 10 weeks after vaccination, while the proportion of M. bovis-antibody positive Control deer rose gradually throughout the trial, reaching 23/30 (77%) at slaughter. Injection-site lesions in COM deer ranged from 10-38 mm in diameter 4 weeks after vaccination, and then resolved. Minimal injection-site lesions were observed in EXP deer. At slaughter, 14 months after vaccination, 19/28 deer in the COM group had 5-15-mm nodules that were easily trimmed from the carcass. CONCLUSIONS: The experimental challenge with M. ptb produced subclinical Johne's disease in the majority of deer, but did not cause any clinical disease. The number and severity of gross and microscopic lesions was significantly reduced in the COM compared with Control and EXP groups; vaccination of the EXP group did not appear to give significant protection. Deer vaccinated with the commercial vaccine are likely to give a false-positive reaction to the MCT but should have an avian reaction to the CCT, if it is carried out >12 months after vaccination. Most of the deer vaccinated with the commercial vaccine produced significant levels of antibodies against both M. ptb and M. bovis, which interfered with ancillary Tb tests. If this vaccine or similar oil-based vaccines are used on deer farms in the future, it may be advisable to only vaccinate animals destined for slaughter, that would not need to be Tb-tested, but would be 'works-monitored' for evidence of Tb instead.

Experimental infections in young red deer (Cervus elaphus) with a bovine and an ovine strain of Mycobacterium avium subsp paratuberculosis.

AIMS: To compare the virulence of a 'bovine' and an 'ovine' strain of Mycobacterium avium subsp paratuberculosis (M. ptb) in red deer (Cervus elaphus) after experimental inoculation orally, and to examine the relationship between the dose of the bovine strain given and immunological, clinical and histopathological outcomes in young red deer. METHODS: Newly-weaned 4-month-old male red deer (n=81) were randomly assigned to one of five groups. Three groups (n=16) received high (10(9) colony forming units (cfu); HB), medium (10(7) cfu; MB) or low (10(3) cfu; LB) oral doses of a bovine strain of M. ptb, one group (n=16) received medium (10(7) cfu; MO) doses of an ovine strain of M. ptb, and a Control group (n=17) was not dosed. The HB and Control groups were grazed together, the MB and LB groups were grazed together, and the MO group was grazed alone, in separate small paddocks on a quarantined area of the farm for 45 weeks. Liveweight, clinical signs and immunoglobulin G1 (IgG1) antibody levels were monitored for up to 45 weeks. Deer affected with Johne's disease were euthanised when they showed obvious clinical signs. Unaffected deer were slaughtered at the end of the trial (Week 45), and all deer were necropsied. Faeces and tissue samples were cultured for M. ptb, and fixed tissues were examined for histopathology. RESULTS: Between 21 and 38 weeks post-challenge (pc), 5/16 animals in the HB group developed early signs of Johne's disease and were euthanised. The remaining deer in the five groups were all apparently healthy and reached good liveweights (approximately 100 kg average), and were euthanised and examined 45 weeks pc. Three deer (two HB and one MB) had small caseous lesions in their jejunal lymph nodes (JJLNs) and one HB animal had a small caseous lesion in a retropharyngeal lymph node. The remaining animals had no grossly-visible lesions. Mycobacterium avium subsp paratuberculosis was cultured from samples from 100% of the HB and MB animals, 50% of the LB group, 69% of the MO group and all Control animals. Thus all Control deer were infected by natural transmission from the HB group but none developed signs of clinical disease. Examination of histological sections of jejunum, ileocaecal valve (ICV) and associated lymph nodes showed a gradation of severity of lesions that was positively correlated (p<0.001) with dose of the bovine strain administered; mean lesion severity scores were 4.8, 2.9 and 0.9 for HB, MB and LB groups, and 2.2 and 0.9 for the Control and MO groups, respectively. IgG1 antibody levels at the time of euthanasia were also correlated with lesion severity scores at slaughter (p<0.001). CONCLUSIONS: The ovine strain of M. ptb used in this study was less virulent for red deer than the bovine strain. The correlation between dose of the bovine strain and the severity of lesions suggests that clinical Johne's disease in yearling red deer likely results from high oral challenge with a bovine strain whilst they are young. The minimum oral infective dose may be close to 10(3) cfu for this bovine strain.