Jackie Whitford

USE OF RELEASED PIGS AS SENTINELS FOR MYCOBACTERIUM BOVIS

Identifying the presence of bovine tuberculosis (TB; Mycobacterium bovis) in wildlife is crucial in guiding management aimed at eradicating the disease from New Zealand. Unfortunately, surveys of the principal wildlife host, the introduced brushtail possum (Trichosurus vulpecula), require large samples (>95% of the population) before they can provide reasonable confidence that the disease is absent. In this study, we tested the feasibility of using a more wide-ranging species, feral pig (Sus scrofa), as an alternative sentinel capable of indicating TB presence. In January 2000, 17 pigs in four groups were released into a forested area with a low density of possums in which TB was known to be present. The pigs were radiotracked at 2 wk intervals from February to October 2000, and some of them were killed and necropsied at various intervals after release. Of the 15 pigs successfully recovered and necropsied, one killed 2 mo after release had no gross lesions typical of TB, and the only other pig killed at that time had greatly enlarged mandibular lymph nodes. The remainder were killed at longer intervals after release and all had gross lesions typical of TB. Mycobacterium bovis was isolated from all 15 pigs by mycobacterial culture. Home range sizes of pigs varied widely and increased with the length of time the pigs were in the forest, with minimum convex polygon range-size estimates averaging 10.7 km2 (range 4.7–20.3 km2) for the pigs killed after 6 mo. A 6 km radius around the kill site of each pig would have encompassed 95% of all of their previous locations at which they could have become infected. However, one pig shifted 35 km, highlighting the main limitation of using unmarked feral pigs as sentinels. This trial indicates use of resident and/or released free-ranging pigs is a feasible alternative to direct prevalence surveys of possums for detecting TB presence.

Reduced spillover transmission of Mycobacterium bovis to feral pigs (Sus scofa) following population control of brushtail possums (Trichosurus vulpecula).

In New Zealand, bovine tuberculosis (bTB) is present in domestic cattle and deer herds primarily as the result of on-going disease transmission from the primary wildlife host, the brushtail possum (Trichosurus vulpecula). However, bTB is also present in other introduced free-ranging mammalian species. Between 1996 and 2007, we conducted a series of studies to determine whether poison control of possum populations would have any effect on the prevalence of Mycobacterium bovis infection in sympatric feral pigs (Sus scrofa). We compared trends in the prevalence of bTB infection in feral pigs in six study areas: possum numbers were reduced in three areas, but not in the other three, effectively providing a thrice-replicated before-after-control-intervention design. Before possum control, the overall prevalence of culture-confirmed M. bovis infection in feral pigs was 16.7-94.4%, depending on area. Infection prevalence varied little between genders but did vary with age, increasing during the first 2-3 years of life but then declining in older pigs. In the areas in which possum control was applied, M. bovis prevalence in feral pigs fell to near zero within 2-3 years, provided control was applied successfully at the whole-landscape scale. In contrast, prevalence changed much less or not at all in the areas with no possum control. We conclude that feral pigs in New Zealand acquire M. bovis infection mainly by inter-species transmission from possums, but then rarely pass the disease on to other pigs and are end hosts. This is in contrast to the purported role of pigs as bTB maintenance hosts in other countries, and we suggest the difference in host status may reflect differences in the relative importance of the oral route of infection in different environments. Despite harbouring M. bovis infection for a number of years, pigs in New Zealand do not sustain bTB independently, but are good sentinels for disease prevalence in possum populations.

Sustained protection against tuberculosis conferred to a wildlife host by single dose oral vaccination

BACKGROUND Vaccination of wildlife against bovine tuberculosis (TB) is being considered by several countries to reduce the transmission of Mycobacterium bovis infection to livestock. In New Zealand, where introduced brushtail possums (Trichosurus vulpecula) are the major wildlife hosts, we have previously shown that repeat applications of a lipid-encapsulated oral bacille Calmette-Guerin (BCG) vaccine reduce the incidence of naturally acquired TB in wild possums. Here we extend this conceptual demonstration to an operational level, assessing long-term protection against TB conferred to free-living possums by a single oral immunisation. METHODS Possums in a non-TB area were randomly allocated to receive lipid-formulated BCG vaccine or remained unvaccinated. After initial trials to assess vaccine immunogenicity and establishment of protection within the first year post-vaccination, 13 individuals of each treatment group were relocated to a biosecurity facility and challenged (at 28 months post-vaccination) by subcutaneous injection of virulent M. bovis. RESULTS Vaccine immunogenicity and short-term protection were confirmed at 2 months and 12 months post-vaccination, respectively. In the long-term assessment, vaccinated possums had significantly reduced bacterial counts in peripheral lymph nodes compared to controls, with 0.6-2.3 log(10)-fold reductions in M. bovis burdens. DISCUSSION The magnitude of protective response by possums to experimental challenge at 28 months post-vaccination is known to equate to a high degree of protection against natural infection in this species. With techniques for oral bait delivery well advanced, the longevity of protection demonstrated here shows that an operable wildlife vaccine against TB is feasible.

Mortality rate and gross pathology due to tuberculosis in wild brushtail possums (Trichosurus vulpecula) following low dose subcutaneous injection of Mycobacterium bovis.

Gross pathology due to tuberculosis can be established experimentally in brushtail possums (Trichosurus vulpecula) within 7 weeks of injection of virulent Mycobacterium bovis into subcutaneous connective tissues of the peripheral limbs. This pathology involves lymphadenomegaly and development of gross lesions in peripheral lymph nodes, with subsequent gross lesions in the lungs and reticuloendothelial organs. Using this artificial infection model, we here assessed the mortality rate for possums in the wild, to provide new information on the likely survival period for New Zealands major wildlife host. Possums were trapped and inoculated with <50 CFU of M. bovis, then fitted with mortality signal emitting radio tracking collars, released and re-tracked for 6 months. Possum survival probability was 89% up to 12 weeks post-injection (p.i.), but cumulative mortality was rapid from then on. The median survival period, based on study of 38 possums, was 18 weeks p.i.; this corresponds with a predicted time interval of 11 weeks between first presentation of TB as palpable lymphadenomegaly and death for an average possum, shorter than period values currently used in possum TB epidemiological modelling. We also examined gross pathology in 11 possums by post mortem necropsy, and confirmed lymphadenomegaly and tuberculous lesions at 7 and 12 weeks p.i. Extra-peripheral gross lesions were more frequent among possums at 12 weeks p.i. than at 7 weeks, while the occurrence of lung lesions (the most likely cause of disease-induced mortality) was apparent in animals at 12 weeks but not at 7 weeks p.i. Our results suggest that the time course of TB from development of gross lesions to mortality may be shorter than previously estimated from field studies of naturally tuberculous possums.

Efficacy of oral BCG vaccination in protecting free-ranging cattle from natural infection by Mycobacterium bovis

Vaccination of cattle against bovine tuberculosis could be a valuable control strategy, particularly in countries faced with intractable ongoing infection from a disease reservoir in wildlife. A field vaccination trial was undertaken in New Zealand. The trial included 1286 effectively free-ranging cattle stocked at low densities in a remote 7600ha area, with 55% of them vaccinated using Mycobacterium bovis BCG (Danish strain 1311). Vaccine was administered orally in all but 34 cases (where it was injected). After inclusion, cattle were exposed to natural sources of M. bovis infection in cattle and wildlife, most notably the brushtail possum (Trichosurus vulpecula). Cattle were slaughtered at 3-5 years of age and were inspected for tuberculous lesions, with mycobacteriological culture of key tissues from almost all animals. The prevalence of M. bovis infection was 4.8% among oral BCG vaccinates, significantly lower than the 11.9% in non-vaccinates. Vaccination appeared to both reduce the incidence of detectable infection, and to slow disease progression. Based on apparent annual incidence, the protective efficacy of oral BCG vaccine was 67.4% for preventing infection, and was higher in cattle slaughtered soon after vaccination. Skin-test reactivity to tuberculin was high in vaccinates re-tested 70days after vaccination but not in non-vaccinates, although reactor animals had minimal response in gamma-interferon blood tests. In re- tests conducted more than 12 months after vaccination, skin-test reactivity among vaccinates was much lower. These results indicate that oral BCG vaccination could be an effective tool for greatly reducing detectable infection in cattle.