Effects of sustained control of brushtail possums on levels of Mycobacterium bovis infection in cattle and brushtail possum populations from Hohotaka, New Zealand

AIMS: To examine the effect of reducing the abundance of brushtail possums (Trichosurus vulpecula) on the distribution and prevalence of bovine tuberculosis (Mycobacterium bovis, Tb) in possums and the incidence of Tb in domestic cattle on a group of farms in the central North Island, New Zealand. METHODS: The cumulative yearly incidence of Tb infection from 12 cattle herds was estimated from annual tuberculin testing and abattoir inspection data over the period 1983-98. Intensive control of possum populations began for six of the herds in 1988, five herds in 1994 and the remaining herd in 1996. The prevalence and distribution of macroscopic M. bovis infection in possums and an index of possum abundance was estimated during yearly cross-sectional surveys from 1988 to 1998. This enabled formal testing of the link between the abundance of tuberculous possums and the incidence of Tb in cattle. RESULTS: Before possum control, infected possums were clustered in foci on or adjacent to the farms with the highest annual incidence of tuberculosis in cattle, and had an overall prevalence of macroscopic M. bovis infection of 2.3%. The prevalence of disease declined to zero with ongoing possum control, although infected possums continued to be found during the first 5 years of control. Maintaining the possum population at an average of 22.1% of its pre-control density significantly reduced the odds of the cumulative yearly incidence of Tb in cattle by 77% during the first 5 years of possum control and a further 65% in the second 5-year period. Nine of 11 tuberculous possums identified since the start of possum control were found within the areas where infected possums were clustered during the pre-control survey, suggesting that the persistence of infection within these clusters rather than infected immigrants was the source of ongoing disease. Annual estimates of the prevalence of tuberculous possums broadly followed the predictions of Barlow's possum-Tb model for a controlled possum population. CONCLUSION: The results support the hypothesis that tuberculous possums transmit bovine tuberculosis to domestic cattle, and therefore that reducing the abundance of tuberculous possums reduces the incidence of Tb in cattle. If the level of possum culling is sufficient, it appears that M. bovis infection may be eradicated from possum populations. Better information on population density, rate of increase and annual culling rates would have been needed for a truly independent examination of the Barlow possum-Tb model.     

Broad-scale possum and ferret correlates of macroscopic Mycobacterium bovis infection in feral ferret populations

AIMS: To examine the relationships between the prevalence of macroscopic Mycobacterium bovis infection (bovine tuberculosis) in feral ferrets (Mustela furo), the abundance of ferrets, and the abundance of brushtail possums (Trichosurus vulpecula) . METHODS: Data on the prevalence of macroscopic M. bovis infection in ferrets, abundance of ferrets, and abundance of possums were analysed from 12 comparable independent broad-scale surveys. RESULTS: The prevalence of macroscopic M. bovis infection in ferrets was positively correlated with possum abundance but unrelated to ferret abundance, suggesting that possums are an important source of M. bovis infection in ferrets. The lack of any positive relationship between the prevalence of M. bovis infection in ferrets and ferret abundance does not support the hypothesis that per capita transmission rates, and therefore disease prevalence, should be higher at higher ferret abundance. CONCLUSION: The results support the hypothesis that tuberculous possums are the major underlying source of M. bovis infection for feral ferrets in New Zealand.     

The relationship between prevalence of Mycobacterium bovis infection in feral ferrets and possum abundance

AIM: To determine the relationship between the prevalence of macroscopic Mycobacterium bovis infection in feral ferrets (Mustela furo) and the abundance of brushtail possums (Trichosurus vulpecula). METHODS: The predictive power of a previously reported positive association between the prevalence of macroscopic M. bovis infection in ferrets and possum abundance was examined by undertaking surveys of M. bovis infection in ferrets at sites of low and high possum abundance. The association was then tested by a manipulative experiment that measured changes in the prevalence of macroscopic M. bovis infection in feral ferrets after reducing possum abundance. RESULTS: The positive relationship between the prevalence of macroscopic M. bovis infection in ferrets and possum abundance remained valid for new survey data, although the goodness of fit of the relationship was reduced. Experimentally reducing possum abundance reduced the odds of macroscopic M. bovis infection in ferrets by 80% in the years immediately following possum control (Odds Ratio=0.23, p=0.003). CONCLUSIONS: There is a causal link between possum abundance and the prevalence of macroscopic M. bovis infection in feral ferrets in areas in which M. bovis infection is known to occur in ferret populations. This suggests that possum-to-ferret transmission of disease occurs and accounts for most of the disease evident in ferret populations, though does not determine whether ferrets are spillover or maintenance hosts of M. bovis. Management to reduce the prevalence of M. bovis infection in ferrets should consider reducing possum abundance as a control tactic. KEY WORDS: Mustela furo, ferret, Trichosurus vulpecula, brushtail possum, Mycobacterium bovis, bovine tuberculosis.     

Resolution of Mycobacterium bovis infection in wild brushtail possums (Trichosurus vulpecula)

The brushtail possum (Trichosurus vulpecula) is the principal wildlife reservoir of Mycobacterium bovis in New Zealand (Morris and Pfeiffer 1995). Tuberculosis in possums is a progressive, fatal infection. The complete resolution of infection in possums has not been reported. The purpose of this communication is to present two cases of complete and spontaneous resolution of tuberculosis in wild, naturally infected possums. During an 11-year (1989-2000) longitudinal study of a possum population at Castlepoint in the Wairarapa, 90 bacteriologically-confirmed cases of M. bovis infection were observed, and 88 of the tuberculous possums were either found dead or were presumed to have died when they ceased to be trapped. As the home range of adult possums is stable (Efford et al 2000) it was assumed that if an adult possum could no longer be trapped, it had died. In many cases, the declining physical condition of a tuberculous possum prior to its disappearance supported the assumption. However, two cases survived and were euthanised, one 15 months and the other 30 months after they first showed clinical signs of infection. When examined post mortem, neither showed evidence of M. bovis infection. The case histories are described and the significance of the observations discussed.     

Habitat-related prevalence of macroscopic Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula), Hohonu Range, Westland, New Zealand

AIM: To identify broadscale habitat factors influencing the prevalence of macroscopic Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula) at a site in Westland, New Zealand. METHODS: During 1973/74, 1989/90 and 1997, we undertook repeated cross-sectional surveys of M. bovis infection in a possum population on the Hohonu Range , Westland. Data were analysed to determine the influence of site-specific habitat characteristics (land form, aspect, slope, altitude), distance from forest-pasture margin and time since infection on the spatial and temporal prevalence of macroscopic M. bovis infection. RESULTS: The prevalence of M. bovis infection was highest in 1973/74 (13.4%), compared with 1989/90 (3.1%) and 1997 (9.4%). The prevalence of macroscopic M. bovis infection was significantly influenced by habitat, as indexed by altitude and slope in this study site. Every 100 m increase in elevation was associated with a 29% decrease in the odds of infection, and every 10 degrees increase in slope was associated with a 20% decrease in the odds of infection. For possums caught in the lowland podocarp forest (altitude 100-200 m, average slope=5.7 degrees ), the odds of infection were nearly 30-fold higher than those for possums caught in high-altitude hardwood forest near the tree line (altitude 900-1000 m, average slope=28 degrees ). Whilst the prevalence of disease fluctuated markedly between surveys, its broadscale spatial distribution changed little over time. Proximity to the forest-pasture margin had no significant influence on the prevalence of disease, once the effect of habitat was taken into account. CONCLUSION: The prevalence of macroscopic M. bovis infection in possums was strongly influenced by habitat type, being highest in habitats that supported the highest density of possums, and lowest in habitats where population density was low. There was no evidence of progressive spread of M. bovis infection in possums into forest away from pasture-forest margins over the 24-year period of these surveys.     

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.     

Behavioural studies on the potential for direct transmission of tuberculosis from feral ferrets (Mustela furo) and possums (Trichosurus vulpecula) to farmed livestock

Studies were conducted to evaluate the response of cattle and deer to ferrets which were sedated so they behaved like terminally tuberculous animals, and to compare this with the response of cattle, deer and sheep to sedated possums. Six groups of deer and two groups of cattle were exposed to a sedated ferret and to a sedated possum. Both livestock species showed interest in the possum by sniffing and licking it, but they only briefly touched the ferret and no licking or extended investigation was observed. The proportion of available time spent in physical contact with the possum by cattle was 7.7 times as high as for the ferret, and for the deer was 5.7 times as high. The behavioural response of three groups of sheep to a sedated possum was investigated, and sheep showed limited interest beyond viewing the possum from a distance. The amount of time spent by sheep investigating the possum was very low and the intensity of exploration was also low. For possums, at least one deer was within 1.5 m (an estimate of the distance that tuberculosis can be transmitted by aerosol) for 50.9% of observation time, and in physical contact with the possum for 9.5% of time. The figures for cattle were 69.3% and 17.3%, while those for sheep were 6.9% and 0.3%. In interactions with ferrets, the equivalent figures were 29.8% within 1.5 m and 2.2% in physical contact for cattle, and 20.8% and 1.1% for deer. Tuberculous possums commonly and tuberculous ferrets less commonly have lung lesions and/or discharging sinuses, and may excrete Mycobacterium bovis intermittently or continuously in aerosols or discharges. The exploratory behaviour of deer and cattle in this study would provide opportunities for them to become infected with M. bovis if they had contact with infectious possums, and less probably with ferrets. The response of sheep to possums suggests that they would be much less likely to contract the disease.     

Interactions between beef cattle and simulated tuberculous possums on pasture

Brushtail possums (Trichosurus vulpecula) were sedated with ketamine and placed within a fenced observation area with 17 Hereford cross steers. The behaviour of the sedated possums simulated that of terminally ill possums. Behavioural analysis was carried out during eight observation periods in which sedated possums were successfully released and interactions occurred. Cattle were attracted from over 50 m away by the movements of the possums, and for 34% of observation time cattle investigated the possums. Inactive animals engendered far less interest, and cattle investigated them for 3% of observation time. An average of seven to eight cattle came in physical contact with the possum in each of the observation periods. Cattle actively investigated the possum while following it as it moved, commonly close enough to be within aerosol transmission distance, and some sniffed the possum, touched it with their noses, and in many cases licked it extensively. All of these activities would expose the cattle to high risk of receiving an infective dose of Mycobacterium bovis, especially via the respiratory route. The type and duration of contact between individual steers and the possum varied between animals and observation period. The cattle exposed themselves to greater risk, overall, during the first week of observations, and there was evidence of habituation with increased contact. Although all 17 steers came within 5 metres of the possum at some point during the observation periods, some individual cattle behaved in ways which would put them at greater risk of contracting tuberculosis than did others. Cattle showed little interest in possum carcasses placed on pasture. These were unlikely to be a significant source of infection for cattle, although they could be for scavenger species.     

Ranging behaviour and duration of survival of wild brushtail possums (Trichosurus vulpecula) infected with Mycobacterium bovis

AIM: To quantify the duration of survival of possums (Trichosurus vulpecula) infected with Mycobacterium bovis, and identify aspects of their behaviour which may influence the likelihood of disease transmission to domestic stock or wildlife. METHODS: Capture and den locations of 14 naturally infected tuberculous possums, eight possums experimentally infected with M. bovis and eight non-infected possums were recorded between May 1998 and February 2000 at a study site near Castlepoint on the Wairarapa coast of the North Island in New Zealand. Denning behaviour was observed weekly using radiotelemetry, and possums were captured, examined and released bi-monthly. Data were used to estimate survival period; create denning, activity, and total ranges; and to identify extended forays by possums as individuals and groups. RESULTS: Seventeen tuberculous possum carcasses were recovered, of which 14 (82%) were close to or within their activity range. Denning ranges were known for 10/17 possums that died. Four tuberculous possums were found dead within their denning range. Three possums made extended forays in the 3 weeks before death. Twelve possums were found dead in dense scrub, three in long grass in open woodland and two on pasture. Mean duration of survival of naturally infected possums following detection of clinical signs was 3.4 months (95% CI=2.1-5.4) and the instantaneous mortality rate was 0.293 per month (95% CI=0.184-0.470). Signs of disease were obvious for about 3 weeks prior to death. Tuberculous possums were commonly trapped on only part of the area where the total non-infected population was trapped. CONCLUSION: Most tuberculous possums died within their activity range and in scrub, representing a risk of transmission of M. bovis to wildlife and livestock that forage in scrub. Smaller proportions dying on pasture represent a less frequent, but highly visible risk. Tuberculous possums were clustered on the study site, and localised possum control operations would be more effective if focussed on such areas.     

Experimental infection with BCG as a model of tuberculosis in the brushtail possum (Trichosurus vulpecula)

AIMS: To study the development and progression of lesions produced following experimental inoculations of possums with Bacille Calmette-Guérin (BCG) Pasteur Strain 1173P2 and to compare these with lesions that occurred following natural Mycobacterium bovis infection. METHODS: Possums were inoculated with 5 x 106 colony forming units (cfu) of BCG via the intra-dermal (I/D) route into the dorsum of the neck (n=38) or the left brachium (n=7),orally (n=10), via the endobronchial (E/B) route (n=12), or intravenously (I/V) (n=10, half of which received 5 x 106 cfu and half of which received 5 x 107 cfu of BCG). The possums were humanely killed between 1-4 weeks post inoculation (p.i.), and the nature and distribution of lesions examined grossly and histopathologically. RESULTS: The distribution of lesions following I/D inoculation via either route was similar to that of the natural disease, but there were few lesions in the lung. Endobronchial inoculation resulted in pulmonary disease but produced few lesions outside the respiratory tract. Lesions produced by I/V inoculation were similar in distribution to those seen in terminally ill tuberculous possums. No lesions were produced following oral inoculation. Regression of lesions commenced after 3 weeks p.i. CONCLUSIONS: Although the phenomenon of lesion resolution restricts the use of BCG to the study of early lesion development, it avoids the overwhelming disease induced using M. bovis and thus allows the early phases of the development and progression of tuberculosis in this species to be observed. Intradermal inoculation produced evidence that infection through the skin is associated with lesions in superficial lymph nodes, whereas pulmonary disease was associated with E/B inoculation. The results are consistent with the hypothesis that both percutaneous and respiratory routes are important in natural infection of possums with M. bovis.     

The effect of fertility control on the transmission of bovine tuberculosis in wild brushtail possums

AIM: To determine the effect of fertility control on the rate of transmission of bovine tuberculosis (Tb), caused by Mycobacterium bovis, in brushtail possums (Trichosurus vulpecula). METHODS: At two study sites with a history of Tb infection in the resident possum population, a sample of adult male and female possums (n=50), determined by palpation to be Tb-free, was surgically sterilised by gonadectomy. A sample of untreated Tb-free male and female possums (n=54) served as controls. Each study site was trapped every 2 months over a 3-year period, and the Tb status of the individuals in the trial assessed. At the conclusion of the trial, all remaining experimental individuals were killed, necropsied and examined for characteristic lesions typical of Tb. The rate of transmission of Tb infection was estimated using the incidence of tuberculous cases in the experimental animals and comparing it between sites, sex and sterilisation treatment. RESULTS: Sterilisation by gonadectomy resulted in a reduction in the rate of transmission of Tb in male possums by 53%, but a corresponding increase in sterilised females for reasons that are still unclear. However, this interaction was statistically weak (p=0.10). When the sexes were combined, there was no overall effect of sterilisation on the rate of transmission of Tb (p=0.43). Sterility treatment notwithstanding, there was a highly significant difference in the rate of transmission between the sexes (p=0.01), being almost one order of magnitude higher in untreated males compared with untreated females. CONCLUSIONS: Although lacking strong statistical support, these results suggest that fertility control that targets endocrine control of fertility may provide some additional benefits for disease control over that achieved by reductions in population density, by reducing the rate of transmission of M. bovis in male possums. However, additional studies are needed to confirm the validity of these results. The large difference in the rate of transmission of M. bovis in untreated males compared with untreated females suggests that transmission of Tb among males may be an important driver of the dynamics of Tb infection in possums.     

A neurological syndrome in a free-living population of possums (Trichosurus vulpecula)

AIMS: To determine the clinical and pathological features of a neurological disease syndrome in a free-living possum population in New Zealand and to compare this syndrome with wobbly possum disease. METHODS: An outbreak of a neurological disease in possums in the Rotorua district was investigated in 1994. A variety of tissues was collected and investigated microbiologically and histopathologically. Tissues stored from clinically affected possums were homogenised, clarified and inoculated into healthy possums by the intra-peritoneal route. The clinical signs and histopathological lesions in naturally-infected and in experimentally-inoculated possums were assessed and compared with those of possums affected with wobbly possum disease. RESULTS: Histopathological investigation of three of the naturally-affected possums revealed non-suppurative encephalitis with perivascular cuffing, diffuse non-suppurative meningitis and focal non-suppurative myocarditis. These lesions were suggestive of a viral infection. No pathogenic bacteria were recovered and no viruses were isolated in tissue culture. A neurological disease, indistinguishable from wobbly possum disease, was reproduced in five out of the eight experimentally inoculated possums. In two experimental cases the clinical signs were very mild and, in most cases of the natural and experimental disease, histopathological lesions in the central nervous system were mild in comparison with wobbly possum disease. Possums which did not develop clinical signs of neurological disease or have lesions in the central nervous system did have infiltrations of mononuclear inflammatory cells in the liver and kidney. CONCLUSIONS: This neurological disease, reported for the first time in a free-living population, closely resembles and may be the same as wobbly possum disease. The milder nature of this disease could suggest there may be more than one strain of the aetiological agent.     

Mycobacterium bovis infection in the brush-tailed possum (Trichosurus vulpecula): II. Comparison of experimental infections with an Australian cattle strain and a New Zealand possum strain

Experimentally induced M. bovis infection in brush-tailed possums (Trichosurus vulpecula) with a possum-adapted strain from New Zealand, produced a more rapidly progressive, severe disease than did that induced by an Australian cattle strain. Possums infected with either strain excreted large numbers of mycobacteria in urine, faeces and discharges from abscesses. Aerosol transmission of infection occurred only with the New Zealand possum strain. The infectious aerosol most probably was of respiratory origin but urine may have been the source.All possums had focal interstitial nephritis consistent with Leptospira infection. Histopathological evidence of severe stress was observed in only one possum; the remainder had either mild or no stress-associated lesions.     

Natural transmission of Mycobacterium bovis infection in captive brushtail possums (Trichosurus vulpecula)

AIMS: To examine natural transmission of bovine tuberculosis (Mycobacterium bovis infection) in captive brushtail possums (Trichosurus vulpecula) and to determine if this mode of transmission could be employed to challenge possums in vaccination studies. METHODS: Three experiments were conducted. In Experiment 1, 11 pairs of possums were housed together in cages, one of the pair having been experimentally infected with M. bovis. Of the in-contact possums 5/11 had been vaccinated with bacille Calmette-Guerin (BCG). In Experiment 2, three susceptible possums were placed in a colony of 19 possums that had been experimentally infected with M. bovis. In Experiment 3, the four most socially active possums in each of two colonies (24 possums in one colony and 23 in the other) were experimentally infected with M. bovis, and 10 of the remaining possums in each colony were vaccinated with BCG. RESULTS: In Experiment 1, transmission of M. bovis infection occurred in only 1/11 pairs. In Experiment 2, none of the three in-contact possums became infected. In Experiment 3, infection was transmitted to 5/20 in-contact possums in one colony and 12/19 in-contact possums in the other. The possums that became infected by natural transmission were significantly more socially interactive than those that remained free of infection (p<0.05). CONCLUSIONS: When susceptible and infected possums were randomly mixed, the rate of transmission of M. bovis was low, but when highly sociable possums were infected the rate of transmission increased markedly. The risk of transmission was dependent on the close proximity of infected and susceptible possums and the frequency and duration of their social interactions. Natural transmission from experimentally infected to incontact possums in a colony would be a useful way of studying the pathogenesis of tuberculosis in this species, and the social behaviour of the possums studied should be taken into account. The high degree of variation in the rate of natural transmission of M. bovis infection between possums makes this mode of transmission unreliable for assessing vaccine efficacy.     

Immunological approaches to the control of tuberculosis in wildlife reservoirs

Attempts to eradicate tuberculosis from cattle and farmed deer in some countries have been frustrated by the existence of wildlife reservoirs of Mycobacterium bovis infection. Possum control programmes in New Zealand using poisons have shown clearly that the brushtail possum is an important source of infection for cattle and farmed deer, and the sum of evidence strongly suggests that badgers serve as a source of infection for cattle in the UK. Bovine tuberculosis can only be eradicated from these countries by controlling M. bovis infection in both wildlife and domestic animals. The most promising options for control of M. bovis infection in wildlife in the longer term include the development of a tuberculosis vaccine for wildlife and a strategy for biological control of possums. The aim of this review is to address the problems and approaches involved in the control of wildlife tuberculosis from an immunological perspective.     

Aerosol vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin: the duration of protection

Bovine tuberculosis is endemic in wild brushtail possums (Trichosurus vulpecula) in New Zealand. The disease is controlled by reducing or eliminating infected possum populations, but control methods do not kill all possums in the targeted area, leaving some tuberculous possums to maintain the disease. Vaccination with bacille Calmette-Guérin (BCG) has been shown to provide significant levels of protection. Vaccination is a potential alternative or complementary control strategy if protection is long lasting. Captive possums were vaccinated with a single dose of BCG by intranasal aerosol and challenged by intratracheal instillation of Mycobacterium bovis 2, 6 or 12 months after vaccination. Vaccination produced significant immunity as measured by the lymphocyte proliferative response to bovine PPD and protection in response to challenge. The protective response was seen as a decrease in the mass of pulmonary lesions and decreased dissemination to the abdominal organs and body lymph nodes. The protective effect was strongest at 2 months after vaccination but was still present at a lower level at 12 months. Delivery of an aerosol vaccine to possums in the wild using a self-delivery system could contribute substantially to wildlife tuberculosis control.     

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.     

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.     

Natural infection of red deer with bovine tuberculosis

Six bovine tuberculosis-free red deer hinds were introduced in October 1993 to a 1.8 ha enclosure, within a larger field study site known to contain tuberculous possums, and kept there for 9 months. A Mycobacterium bovis-infected possum was found in the vicinity of the deer enclosure 3 weeks after the introduction. Subsequently, a further eleven infected possums were found in the area. The deer were monitored by repeated composite antibody detection ELISA and lymphocyte transformation assays for tuberculosis, interpreted in parallel, by skin testing and by routine culturing of samples collected from potential excretion sites. Lymphocyte transformation assay evidence of M. bovis infection in four hinds was first observed 4 months after introduction. One other hind became bovine tuberculin lymphocyte transformation assay positive in the 5th month. Positive or equivocal bovine reactivity remained evident at most test episodes. A comparative cervical skin test performed in July 1994, shortly before slaughter, was positive in these five hinds. Mycobacterium bovis was recovered off swabs from the oropharyngeal tonsils of two hinds during routine sampling. Detailed necropsy of the six deer revealed a single typical tuberculous lesion in only one, but culturing of various tissue specimens ascertained that the five blood test and comparative cervical skin test-positive animals were all infected. Mycobacterium bovis was cultured from the oropharyngeal tonsils of four and medial retropharyngeal lymph nodes of two of the deer with no typical gross lesions. Six additional tuberculosis-free hinds were introduced to the enclosure in April 1994 and kept there for 12 months. Four of these animals showed a positive lymphocyte transformation assay response to M. bovis after 9 weeks, but no significant reactivity thereafter. Concurrent observational studies suggest that five of the first six deer probably became infected through close inspection and investigation of the tuberculous possums, although the possibility of deer-to-deer transmission cannot be totally excluded. The likely deer-possum contact, and thus exposure to M. bovis, was related to the curiosity and social ranking of the hinds. The second group appear to have had transient exposure to M. bovis, possibly caused by direct contact with the infected hinds introduced earlier. This group never showed any curiosity toward, or interaction with, possums during the periods of observation.