Cost-based optimization of the stopping threshold for local disease surveillance during progressive eradication of tuberculosis from New Zealand wildlife.

Bovine tuberculosis (TB) is managed in New Zealand largely via population reduction of the major wildlife disease reservoir of Mycobacterium bovis, the introduced brushtail possum Trichosurus vulpecula. New Zealand aims to eradicate M. bovis infection from its livestock and wildlife within 40 years, as the culmination of progressive regional eradication programmes. Declarations of regional eradication are decided after extensive possum population control and post-control surveillance; hence, we developed a modelling framework, based on eco-epidemiological simulation data, to provide cost-evaluated options for deciding when to make these declarations. A decision-support framework evaluated potential costs of wildlife surveillance (and recontrol, if required) with respect to the calculated probability of successful eradication of M. bovis from wildlife. This enabled expected costs to be predicted in terms of stopping thresholds, allowing selection of optimal stopping rules based on minimizing costs. We identified factors that could influence optimal stopping values applied during regional eradication. Where vector/disease surveillance was inexpensive (for example, using low-cost detection devices or sentinel wildlife hosts) optimization involved setting a higher rather than lower stopping value, as it would be cheaper to minimize the risk of making a false declaration of eradication than to remedy any such failure. In addition, any cost of recontrol would largely depend on the time to rediscovery of residual M. bovis infection in wildlife, which would in turn be linked to the level of ongoing passive surveillance (with more rapid detection of re-emergent infection among wildlife in farmland situations than in remote forested regions). These two scenarios would favour different optimal stopping rules, as would the consideration of stakeholder confidence and socio-political issues, which are discussed. The framework presented here provides guidance to assess the economics underlying eradication of bovine TB from New Zealand farming; this eliminates reliance upon a pre-determined and uniform stopping rule for ceasing active management.

Livestock as sentinels for an infectious disease in a sympatric or adjacent-living wildlife reservoir host.

A central question to address in managing wildlife diseases is how much effort and resources are required to reduce infection prevalence to below a requisite threshold? This requires surveillance for infection in at least one species involved in the infection cycle, a process that is often expensive and time-consuming but one which could be enhanced using additional sources of readily-obtainable surveillance data. We demonstrate how surveillance data from ruminant livestock monitored for bovine tuberculosis (bTB) in New Zealand can be employed in spatially-explicit modelling to help predict the probability of freedom from Mycobacterium bovis infection in a sympatric wildlife reservoir species, the brushtail possum (Trichosurus vulpecula). We apply the model to a case study and compare resulting probabilities of freedom when utilizing (1) livestock data only, (2) wildlife data only, and (3) combined livestock-plus-wildlife surveillance data. Results indicated that the greatest probability of M. bovis eradication was achieved using wildlife monitoring data supplemented with livestock surveillance data. This combined approach lessened the time required for a confident (95% probability) declaration of regional eradication. However, the combined model was sensitive to the precision of the input parameters, and we describe ways to account for this. In a broad sense, this modelling approach is flexible in that any spatial arrangement of wildlife habitat and farms can be analysed, provided infection is readily detectable in both the wild and domestic animal(s) of interest. It is applicable to monitoring any communicable wildlife disease that affects regularly-tested livestock. The potential benefits to wildlife disease management include reduced surveillance costs and more rapid achievement of targeted reductions in disease prevalence.

Valuing conservation benefits of disease control in wildlife: A choice experiment approach to bovine tuberculosis management in New Zealand's native forests.

We assess the non-monetary environmental benefits that accrue incidentally in New Zealand (NZ) from pest management conducted primarily to control an animal disease, bovine tuberculosis (TB). TB is an infectious disease that is one of the world's most serious animal health problems and, in many parts of the developing world, still a major mortality risk for humans. The incidence of TB in New Zealand (NZ) farmed livestock has been reduced progressively over the last 20 years, largely due to extensive and sustained population control of the main wildlife reservoir of disease, the introduced brushtail possum. Possums are also major pests that threaten indigenous forest biodiversity, and so extensive possum control for TB mitigation also incidental benefits conservation, but the extent and public value of this benefit has yet to be quantified. We conducted a choice experiment survey of the NZ public in an effort to value the native forest biodiversity benefits of TB-related possum control. We find strong public support for conservation outcomes consequent to TB-possum control in public native forests. The public place substantial value on the most observable biodiversity benefits of TB possum control, such as improved forest canopies and presence of native birds. The benefits, costs and values of TB-possum control are discussed in relation to the future directives of NZ's TB control programme, which is headed toward first regional and then national level disease eradication.

Field Trial of an Aerially-Distributed Tuberculosis Vaccine in a Low-Density Wildlife Population of Brushtail Possums (Trichosurus vulpecula).

Oral-delivery Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine in a lipid matrix has been shown to confer protection against M. bovis infection and reduce the severity of tuberculosis (TB) when fed to brushtail possums (Trichosurus vulpecula), the major wildlife vector of bovine TB in New Zealand. Here we demonstrate the feasibility of aerial delivery of this live vaccine in bait form to an M. bovis-infected wild possum population, and subsequently assess vaccine uptake and field efficacy. Pre-trial studies indicated a resident possum population at very low density (<0.6 possums/ha) at the field site, with a 5.1% prevalence of macroscopic TB lesions. Pilot studies indicated that flavoured lipid matrix baits in weather-proof sachets could be successfully sown aerially via helicopter and were palatable to, and likely to be consumed by, a majority of wild possums under free-choice conditions. Subsequently, sachet-held lipid baits containing live BCG vaccine were sown at 3 baits/ha over a 1360 ha area, equating to >5 baits available per possum. Blood sampling conducted two months later provided some evidence of vaccine uptake. A necropsy survey conducted one year later identified a lower prevalence of culture-confirmed M. bovis infection and/or gross TB lesions among adult possums in vaccinated areas (1.1% prevalence; 95% CI, 0-3.3%, n = 92) than in unvaccinated areas (5.6%; 0.7-10.5%, n = 89); P = 0.098. Although not statistically different, the 81% efficacy in protecting possums against natural infection calculated from these data is within the range of previous estimates of vaccine efficacy in trials where BCG vaccine was delivered manually. We conclude that, with further straightforward refinement to improve free-choice uptake, aerial delivery of oral BCG vaccine is likely to be effective in controlling TB in wild possums. We briefly discuss contexts in which this could potentially become an important complementary tool in achieving national eradication of TB from New Zealand wildlife.

Testing an Alternative Method for Estimating the Length of Fungal Hyphae Using Photomicrography and Image Processing.

This study aimed to develop and test an unbiased and rapid methodology to estimate the length of external arbuscular mycorrhizal fungal (AMF) hyphae in soil. The traditional visual gridline intersection (VGI) method, which consists in a direct visual examination of the intersections of hyphae with gridlines on a microscope eyepiece after aqueous extraction, membrane-filtration, and staining (e.g., with trypan blue), was refined. For this, (i) images of the stained hyphae were taken by using a digital photomicrography technique to avoid the use of the microscope and the method was referred to as "digital gridline intersection" (DGI) method; and (ii), the images taken in (i) were processed and the hyphal length was measured by using ImageJ software, referred to as the "photomicrography-ImageJ processing" (PIP) method. The DGI and PIP methods were tested using known grade lengths of possum fur. Then they were applied to measure the hyphal lengths in soils with contrasting phosphorus (P) fertility status. Linear regressions were obtained between the known lengths (Lknown) of possum fur and the values determined by using either the DGI (LDGI) (LDGI = 0.37 + 0.97 × Lknown, r2 = 0.86) or PIP (LPIP) methods (LPIP = 0.33 + 1.01 × Lknown, r2 = 0.98). There were no significant (P > 0.05) differences between the LDGI and LPIP values. While both methods provided accurate estimation (slope of regression being 1.0), the PIP method was more precise, as reflected by a higher value of r2 and lower coefficients of variation. The average hyphal lengths (6.5-19.4 m g-1) obtained by the use of these methods were in the range of those typically reported in the literature (3-30 m g-1). Roots growing in P-deficient soil developed 2.5 times as many hyphae as roots growing in P-rich soil (17.4 vs 7.2 m g-1). These tests confirmed that the use of digital photomicrography in conjunction with either the grid-line intersection principle or image processing is a suitable method for the measurement of AMF hyphal lengths in soils for comparative investigations.

Multisystemic Listeriosis in a Common Brushtail Possum (Trichosurus vulpecula) and Two Common Ringtail Possums (Pseudocheirus peregrinus).

A single free-ranging common brushtail possum (Trichosurus vulpecula) and 2 captive sibling common ringtail possums (Pseudocheirus peregrinus)from a zoological facility in Sydney, Australia, were diagnosed with multisystemic listeriosis. The brushtail was found dead in an animal enclosure while the ringtails presented with signs of cardiovascular collapse and died shortly thereafter. All 3 animals were culture positive forListeria monocytogenesand demonstrated focal suppurative lesions within the brainstem in addition to fulminant disease in other areas of the thorax and/or abdomen. Listeriosis in phalangeriformes species has rarely been reported, and brainstem lesions have not previously been described. It is speculated that access to the brainstem by the organism may have occurred hematogenously or via retrograde migration along cranial nerves. Sources of infection and the possibility of transmission between animals are also discussed.

Assessing Movements of Brushtail Possums (Trichosurus vulpecula) in Relation to Depopulated Buffer Zones for the Management of Wildlife Tuberculosis in New Zealand.

In New Zealand, managing the threat of bovine tuberculosis (TB) to livestock includes population reduction of potentially infectious wildlife, primarily the brushtail possum (Trichosurus vulpecula). Population control is often targeted on forested buffer zones adjacent to farmland, in order to limit movements of possums across the buffer and reduce the risk of disease transmission to livestock. To assess the effectiveness of buffers in protecting livestock we analysed GPS telemetry data from possums located in untreated forest adjacent to buffers, and used these data to characterise patterns of movement that could lead to possums reaching farmland during the season when most dispersal occurs. Analyses of movement data showed that the direction of dispersal by sub-adult and adult possums and the extent of long exploratory movements were not biased toward forest buffers, even though these provided vacant habitat as suitable for possums as untreated forest. Instead, dispersal and exploratory movements were uncommon even for sub-adult possums and such events typically lasted <10 days. Dispersing possums settled predominantly in river valleys. A simulation model was developed for the 3-6-month dispersal season; it demonstrated a probability of <0.001 that an infected possum, originating from a low-density population with low disease prevalence in untreated forest, would move across 3 km of recently controlled forest buffer to reach farmland. Our results indicate short-term reduction in the risk of TB transmission from possums to livestock in New Zealand by the use of depopulated buffer zones, while acknowledging that the threat of disease spread from untreated forest is likely to increase over time as possum population density and, potentially, TB prevalence among those possums, increase in the buffer zone.

Managing and eradicating wildlife tuberculosis in New Zealand.

Tuberculosis (TB) due to Mycobacterium bovis infection was first identified in brushtail possums (Trichosurus vulpecula) in New Zealand in the late 1960s. Since the early 1970s, possums in New Zealand have been controlled as part of an ongoing strategy to manage the disease in livestock. The TB management authority (TBfree New Zealand) currently implements three strategic choices for disease-related possum control: firstly TB eradication in areas selected for eradication of the disease from livestock and wildlife, secondly Free Area Protection in areas in which possums are maintained at low densities, normally along a Vector Risk Area (VRA) boundary, and thirdly Infected Herd Suppression, which includes the remaining parts of VRA where possums are targeted to minimise the infection risk to livestock. Management is primarily through a range of lethal control options. The frequency and intensity of control is driven by a requirement to reduce populations to very low levels (usually to a trap-catch index below 2%), then to hold them at or below this level for 5-10 years to ensure disease eradication.Lethal possum control is implemented using aerial- and ground-based applications, under various regulatory and operational constraints. Extensive research has been undertaken aimed at improving the efficacy and efficiency of control. Aerial applications use sodium fluoroacetate (1080) bait for controlling possums over extensive and rugged areas of forest that are difficult to access by foot. Ground-based control uses a range of toxins (primarily, a potassium cyanide-based product) and traps. In the last 5 years there has been a shift from simple possum population control to the collection of spatial data on possum presence/absence and relative density, using simple possum detection devices using global positioning system-supported data collection tools, with recovery of possum carcasses for diagnostic necropsy. Such data provide information subsequently used in predictive epidemiological models to generate a probability of TB freedom.The strategies for managing TB in New Zealand wildlife now operate on four major principles: firstly a target threshold for possum population reduction is defined and set, secondly an objective methodology is applied for assessing whether target reductions have been achieved, thirdly effective control tools for achieving possum population reductions are used, and fourthly the necessary legislative support is in place to ensure compliance. TBfree New Zealand's possum control programme meets these requirements, providing an excellent example of an effective pest and disease control programme.

Epidemiology and control of Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula), the primary wildlife host of bovine tuberculosis in New Zealand.

The introduced Australian brushtail possum (Trichosurus vulpecula) is a maintenance host for bovine tuberculosis (TB) in New Zealand and plays a central role in the TB problem in this country. The TB-possum problem emerged in the late 1960s, and intensive lethal control of possums is now used to reduce densities to low levels over 8 million ha of the country. This review summarises what is currently known about the pathogenesis and epidemiology of TB in possums, and how the disease responds to possum control. TB in possums is a highly lethal disease, with most possums likely to die within 6 months of becoming infected. The mechanisms of transmission between possums remain unclear, but appear to require some form of close contact or proximity. At large geographic scales, TB prevalence in possum populations is usually low (1-5%), but local prevalence can sometimes reach 60%. Intensive, systematic and uniform population control has been highly effective in breaking the TB cycle in possum populations, and where that control has been sustained for many years the prevalence of TB is now zero or near zero. Although some uncertainties remain, local eradication of TB from possums appears to be straightforward, given that TB managers now have the ability to reduce possum numbers to near zero levels and to maintain them at those levels for extended periods where required. We conclude that, although far from complete, the current understanding of TB-possum epidemiology, and the current management strategies and tactics, are sufficient to achieve local, regional, and even national disease eradication from possums in New Zealand.

Not all types of host contacts are equal when it comes to E. coli transmission.

The specific processes that facilitate pathogen transmission are poorly understood, particularly for wild animal populations. A major impediment for investigating transmission pathways is the need for simultaneous information on host contacts and pathogen transfer. In this study, we used commensal Escherichia coli strains as a model system for gastrointestinal pathogens. We combined strain-sharing information with detailed host contact data to investigate transmission routes in mountain brushtail possums. Despite E. coli being transmitted via the faecal-oral route, we revealed that, strain-sharing among possums was better explained by host contacts than spatial proximity. Furthermore, and unexpectedly, strain-sharing was more strongly associated with the duration of brief nocturnal associations than day-long den-sharing. Thus, the most cryptic and difficult associations to measure were the most relevant connections for the transmission of this symbiont. We predict that future studies that employ similar approaches will reveal the importance of previously overlooked associations as key transmission pathways.

Clinical, microbiological and pathological findings of Mycobacterium ulcerans infection in three Australian Possum species.

BACKGROUND: Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans, with endemicity predominantly in sub-Saharan Africa and south-eastern Australia. The mode of transmission and the environmental reservoir(s) of the bacterium and remain elusive. Real-time PCR investigations have detected M. ulcerans DNA in a variety of Australian environmental samples, including the faeces of native possums with and without clinical evidence of infection. This report seeks to expand on previously published findings by the authors' investigative group with regards to clinical and subclinical disease in selected wild possum species in BU-endemic areas of Victoria, Australia. METHODOLOGY/PRINCIPAL FINDINGS: Twenty-seven clinical cases of M. ulcerans infection in free-ranging possums from southeastern Australia were identified retrospectively and prospectively between 1998-2011. Common ringtail possums (Pseudocheirus peregrinus), a common brushtail possum (Trichosurus vulpecula) and a mountain brushtail possum (Trichosurus cunninghami) were included in the clinically affected cohort. Most clinically apparent cases were adults with solitary or multiple ulcerative cutaneous lesions, generally confined to the face, limbs and/or tail. The disease was minor and self-limiting in the case of both Trichosurus spp. possums. In contrast, many of the common ringtail possums had cutaneous disease involving disparate anatomical sites, and in four cases there was evidence of systemic disease at post mortem examination. Where tested using real-time PCR targeted at IS2404, animals typically had significant levels of M. ulcerans DNA throughout the gut and/or faeces. A further 12 possums without cutaneous lesions were found to have PCR-positive gut contents and/or faeces (subclinical cases), and in one of these the organism was cultured from liver tissue. Comparisons were made between clinically and subclinically affected possums, and 61 PCR-negative, non-affected individuals, with regards to disease category and the categorical variables of species (common ringtail possums v others) and sex. Animals with clinical lesions were significantly more likely to be male common ringtail possums. CONCLUSIONS/SIGNIFICANCE: There is significant disease burden in common ringtail possums (especially males) in some areas of Victoria endemic for M. ulcerans disease. The natural history of the disease generally remains unknown, however it appears that some mildly affected common brushtail and mountain brushtail possums can spontaneously overcome the infection, whereas some severely affected animals, especially common ringtail possums, may become systemically, and potentially fatally affected. Subclinical gut carriage of M. ulcerans DNA in possums is quite common and in some common brushtail and mountain brushtail possums this is transient. Further work is required to determine whether M. ulcerans infection poses a potential threat to possum populations, and whether these animals are acting as environmental reservoirs in certain geographical areas.

Potential wildlife sentinels for monitoring the endemic spread of human buruli ulcer in South-East australia.

The last 20 years has seen a significant series of outbreaks of Buruli/Bairnsdale Ulcer (BU), caused by Mycobacterium ulcerans, in temperate south-eastern Australia (state of Victoria). Here, the prevailing view of M. ulcerans as an aquatic pathogen has been questioned by recent research identifying native wildlife as potential terrestrial reservoirs of infection; specifically, tree-dwelling common ringtail and brushtail possums. In that previous work, sampling of environmental possum faeces detected a high prevalence of M. ulcerans DNA in established endemic areas for human BU on the Bellarine Peninsula, compared with non-endemic areas. Here, we report research from an emergent BU focus recently identified on the Mornington Peninsula, confirming associations between human BU and the presence of the aetiological agent in possum faeces, detected by real-time PCR targeting M. ulcerans IS2404, IS2606 and KR. Mycobacterium ulcerans DNA was detected in 20/216 (9.3%) ground collected ringtail possum faecal samples and 4/6 (66.6%) brushtail possum faecal samples. The distribution of the PCR positive possum faecal samples and human BU cases was highly focal: there was a significant non-random cluster of 16 M. ulcerans positive possum faecal sample points detected by spatial scan statistics (P<0.0001) within a circle of radius 0.42 km, within which were located the addresses of 6/12 human cases reported from the area to date; moreover, the highest sample PCR signal strength (equivalent to ≥10(6) organisms per gram of faeces) was found in a sample point located within this cluster radius. Corresponding faecal samples collected from closely adjacent BU-free areas were predominantly negative. Possums may be useful sentinels to predict endemic spread of human BU in Victoria, for public health planning. Further research is needed to establish whether spatial associations represent evidence of direct or indirect transmission between possums and humans, and the mechanism by which this may occur.

Mycobacterium bovis: characteristics of wildlife reservoir hosts.

Mycobacterium bovis is the cause of tuberculosis in animals and sometimes humans. Many developed nations have long-standing programmes to eradicate tuberculosis in livestock, principally cattle. As disease prevalence in cattle decreases these efforts are sometimes impeded by passage of M. bovis from wildlife to cattle. In epidemiological terms, disease can persist in some wildlife species, creating disease reservoirs, if the basic reproduction rate (R0) and critical community size (CCS) thresholds are achieved. Recognized wildlife reservoir hosts of M. bovis include the brushtail possum (Trichosurus vulpecula) in New Zealand, European badger (Meles meles) in Great Britain and Ireland, African buffalo (Syncerus caffer) in South Africa, wild boar (Sus scrofa) in the Iberian Peninsula and white-tailed deer (Odocoileus virginianus) in Michigan, USA. The epidemiological concepts of R0 and CCS are related to more tangible disease/pathogen characteristics such as prevalence, pathogen-induced pathology, host behaviour and ecology. An understanding of both epidemiological and disease/pathogen characteristics is necessary to identify wildlife reservoirs of M. bovis. In some cases, there is a single wildlife reservoir host involved in transmission of M. bovis to cattle. Complexity increases, however, in multihost systems where multiple potential reservoir hosts exist. Bovine tuberculosis eradication efforts require elimination of M. bovis transmission between wildlife reservoirs and cattle. For successful eradication identification of true wildlife reservoirs is critical, as disease control efforts are most effective when directed towards true reservoirs.

A strategic approach to eradication of bovine TB from wildlife in New Zealand.

A review and amendment of New Zealand's National Pest Management Strategy for bovine tuberculosis (TB) has led to adoption of new strategy objectives for localized eradication of disease from the principal wildlife maintenance host and infecting vector for farmed cattle and deer, the brushtail possum Trichosurus vulpecula. Historic programmes have been based on management of disease within herds and control of wildlife directed towards reducing infected herd prevalence. From July 2011, the TB strategy has been redirected towards eradication of TB from possums and other wildlife over a total area of at least 2.5 million hectares over a 15-year period. The amended strategy is intended to provide large-scale proof of concept, using two extensive bush areas, that TB can be eradicated from wildlife in New Zealand in the longer term, leading to eventual savings in control programmes needed to protect cattle and deer herds from infection. Achievement of strategy objectives will be supported by major research together with technical and managerial improvements in wildlife TB control and surveillance, and these are reviewed.

Functional genotypes are associated with commensal Escherichia coli strain abundance within-host individuals and populations.

The selective pressures that determine genotype abundance and distribution frequently vary between ecological levels. Thus, it is often unclear whether the same functional genotypes will become abundant at different levels and how selection acting at these different scales is linked. In this study, we examined whether particular functional genotypes, defined by the presence or absence of 34 genes, of commensal Escherichia coli strains were associated with within-host abundance and/or host population abundance in a wild population of 54 adult mountain brushtail possums (Trichosurus cunninghami). Our results revealed that there was a positive correlation between a strain's relative abundance within individuals and the strain's abundance in the host population. We also found that strain abundance at both ecological levels was predicted by the same group of functional genes (agn43, focH, micH47, iroN, ygiL, ompT, kspmT2 and K1) that had associated patterns of occurrence. We propose that direct selection on the same functional genes at both levels may in part be responsible for the observed correlation between the ecological levels. However, a potential link between abundance within the host and excretion rate may also contribute.

High temporal variability in commensal Escherichia coli strain communities of a herbivorous marsupial.

Although Escherichia coli is an important model organism for bacterial research, few studies have explored the nature of temporal variation in E. coli strains within the intestinal tracts of host individuals. In this study the E. coli strains of 54 mountain brushtail possums were sampled on four occasions during a year. This allowed temporal changes to be quantified both at the host population level and within individuals. Escherichia coli strains were identified using a combination of rep-PCR profiles from two primers (CGG and ERIC) and phylogenetic group assigned by quadruplex PCR. The study revealed considerable changes in community structure within individuals among all time periods. In fact, temporal variation within individuals accounted for more of the variation in E. coli community structure than differences between animals. In contrast to the within-host dynamics, there were no significant differences among the time periods at the host population level. It was also found that there was no effect of host age or sex on strain community structure within host individuals. These findings highlight the importance of temporal variation in the ecology of E. coli, while the methods applied in this study may serve as a foundation for further work in this area.

Percutaneous interdigital injection of Mycobacterium bovis as a model for tuberculous lesion development in wild brushtail possums (Trichosurus vulpecula).

Brushtail possums (Trichosurus vulpecula) are the major wildlife reservoir of Mycobacterium bovis, the causative agent of bovine tuberculosis (BTB), in New Zealand. Primary diagnosis of BTB in wild possums is by palpation to detect peripheral lymphadenomegaly followed by necropsy examination, which frequently identifies gross tuberculous lesions in the peripheral lymph nodes and lungs. Experimental infection studies were conducted with wild possums in an attempt to emulate field BTB, focussing on percutaneous administration of virulent M. bovis in the paws. In a preliminary study, viable M. bovis bacilli were recovered from lymph nodes draining fore- or hindlimbs 12 days after percutaneous injection. Subsequently, 21 wild possums were injected interdigitally with 500 colony forming units (cfu) of M. bovis, radio-collared and released; 17/18 possums recaptured 8 weeks later had an established M. bovis lymphatic infection, with 16 having culture-positive gross lesions in the superficial and/or deep axillary lymph nodes. A dual-site infection model was established, involving simultaneous interdigital injection of 100 cfu of M. bovis into front and rear paws of 19 wild possums; this identified that the average degree of lymphadenitis involved 30-fold enlargement of the draining lymph node by 7-8 weeks post injection (wpi). A time-course study demonstrated establishment of M. bovis infection in peripheral lymph nodes of 9/11 possums at 3-5 wpi of doses ranging from 60 to 190 cfu, but with no development of gross lesions; by 7 weeks, 8/8 animals injected similarly had both an established infection and gross lesions of peripheral lymph nodes. The incidence and progression of peripheral lesion development, together with indications of sequential infection of the lungs, liver and mesenteric lymph nodes(MLNs), indicates that a low-dose percutaneous M. bovis infection model is likely to emulate natural disease in possums.

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.

Contact patterns as a risk factor for bovine tuberculosis infection in a free-living adult brushtail possum Trichosurus vulpecula population.

The aim of this study was to identify risk factors for bovine tuberculosis (TB) in a free-roaming, capture-mark-recapture monitored possum Trichosurus vulpecula population in a 22-ha study site at Castlepoint, New Zealand from 1 April 1989 to 31 March 1994. A matched case-control design was used to evaluate the influence of sex, habitat and contact opportunities on TB risk. Cases comprised possums identified as TB-positive throughout the study period. Controls were selected from the group of possums that were captured and showed no clinical signs of TB throughout the study period. Measures derived from a social network analysis of possum capture locations such as degree, clustering coefficient (CC) and betweenness were used to represent potential contact opportunities among possums. Network analysis measures recorded for individual possums in the 12-month period before a diagnosis of TB were evaluated in a conditional logistic regression model. We found no evidence of an association between case status and the total number of possums with which there was potential contact (degree) (P=0.5). The odds of cases being exposed to unit increases in the number of TB-positive contacts was 2.50 (95% CI 1.24-5.05; P<0.01) times that of controls. This effect was conditional on the total number of potential contacts made, with a negative interaction with increasing degree. These findings indicate that potential contact with TB-positive possums increases the odds of disease whereas potential contact with large numbers of possums does not. This suggests that multiple contacts with TB-positive possum(s) are necessary for transmission of TB and this is more likely to occur in networks that are smaller. We challenge the hypothesis that contact with large numbers of individuals increases the probability of becoming TB infected and argue that individual contact behaviour is a determinant of the creation of TB foci within free-living possum populations.