Bacteriological diagnosis and molecular strain typing of Mycobacterium bovis and Mycobacterium caprae.

The primary isolation of a Mycobacterium sp. of the Mycobacterium tuberculosis complex from an infected animal provides a definitive diagnosis of tuberculosis. However, as Mycobacterium bovis and Mycobacterium caprae are difficult to isolate, particularly for animals in the early stages of disease, success is dependent on the optimal performance of all aspects of the bacteriological process, from the initial choice of tissue samples at post-mortem examination or clinical samples, to the type of media and conditions used to cultivate the microorganism. Each step has its own performance characteristics, which can contribute to sensitivity and specificity of the procedure, and may need to be optimized in order to achieve the gold standard diagnosis. Having isolated the slow-growing mycobacteria, species identification and fine resolution strain typing are keys to understanding the epidemiology of the disease and to devise strategies to limit transmission of infection. New technologies have emerged that can now even discriminate different isolates from the same animal. In this review we highlight the key factors that contribute to the accuracy of bacteriological diagnosis of M. bovis and M. caprae, and describe the development of advanced genotyping techniques that are increasingly used in diagnostic laboratories for the purpose of supporting detailed epidemiological investigations.

Control strategies for wildlife tuberculosis in Ireland.

The principal domestic maintenance host for Mycobacterium bovis is infected cattle. In countries where comprehensive surveillance schemes have been applied, tuberculosis rarely affects an animal to the extent that it presents with clinical disease. In the latter stages of an eradication campaign, the aim is to maintain the disease-free status of clear herds and eliminate foci of infection in herds as well as restricting movement of infected animals from these herds, other than to slaughter. However, the eradication of tuberculosis from cattle herds may be compromised if infected wildlife species, such as Eurasian badgers (Meles meles), share the same environment and contribute to transmission of infection. The options for dealing with tuberculosis in the wildlife reservoir hosts are limited to segregation of domestic animals from the wildlife, culling of the wildlife host or vaccination. Options are further limited by conservation and social reasons, particularly where culling is concerned. In Ireland and the UK, vaccination of badgers against M. bovis, if successfully employed, could directly facilitate the completion of bovine tuberculosis eradication. Programmes of research into vaccination of badgers are being undertaken in both countries, and there is clear evidence that vaccination induces protection. Vaccine trials in captive badgers have established that the M. bovis bacille Calmette-Guérin (BCG) vaccine can induce a protective response that limits the distribution and severity of tuberculosis disease following experimental challenge. In Ireland, a large-scale field trial of oral BCG vaccination is being conducted to measure the protection generated in wild badgers subjected to natural transmission of infection and to estimate vaccine efficacy. The results will provide a framework for the development and implementation of a national strategy to address the disease in badger populations and if successful will remove this major impediment to tuberculosis eradication from cattle.

Primary isolation of Mycobacterium bovis from bovine tissues: conditions for maximising the number of positive cultures.

In studies of Mycobacterium bovis infection in animals a definitive diagnosis requires the isolation of the organism. However, the optimum conditions for the primary isolation of M. bovis have not been determined. The aim of this study was to determine for primary isolation of M. bovis, (a) the incubation time required to achieve maximum sensitivity (i.e., the number of positive samples identified), (b) the effect of decontaminants on bacterial growth rates, and (c) the influence of media and the number of slopes of media on the number of positive samples detected. Two agar-based media, modified Middlebrook 7H11 (7H11) and tuberculosis blood agar (B83), and an egg-based medium, Stonebrink's (SB) were compared. Three decontaminants, 2% (w/v) sodium hydroxide (NaOH), 0.75% (w/v) and 0.075% (w/v) cetylpyridinium chloride (CPC, also called hexadecylpyridinium chloride, HPC) and 0.5% (w/v) benzalkonium chloride (BC) were evaluated against treatment with sterile distilled water. The inoculated media slopes were incubated for up to 15 weeks. Colonies first appeared after 2 weeks on all media types and 75% of positive slopes were identified by 8 weeks. An incubation time of 15 weeks was required to identify all positive samples. The slowest growth was associated with inocula that contained the fewest viable bacilli. The time to the appearance of colonies was influenced by medium type: the median time to detection of colonies was 28 days on 7H11 and B83, and 36 days on SB. However, SB returned the greatest number of positive samples. Decontamination procedures increased the minimum incubation time required to detect positive cultures, probably due to the toxic effect of the decontaminants. Increasing the number of inoculated slopes resulted in an increased number of positive samples and a decreased time to the detection of colonies. Overall, the detection of M. bovis was significantly influenced by the choice of media, the decontaminant and the duration of incubation of cultures.

Tuberculosis in cattle herds are sentinels for Mycobacterium bovis infection in European badgers (Meles meles): the Irish Greenfield Study.

In Ireland badgers are removed in response to tuberculosis (TB) breakdowns in cattle herds (focal culling). Prevalence studies, conducted using a detailed post mortem and bacteriological examination, showed that 36-50% of badgers were infected with Mycobacterium bovis. Focal culling forms part of the medium term national strategy for the control of bovine TB in cattle and is based on the premise that badgers in areas with herd breakdowns have a higher prevalence of infection than the badger population at large. However, the hypothesis that cattle can be used as sentinels for infection in the badger population has never been formally tested. In this study we tested the hypothesis by determining the infection prevalence in badgers in areas where there had been historically, a consistently low prevalence of infection in cattle. Low cattle TB prevalence areas were defined as those herds with ≤ 2 standard reactors in the annual round of skin testing over the preceding 5 years (Greenfield sites). Using GIS, and adjusting for variation in land use, previous culling and cattle density, 198 Greenfield sites were identified and surveyed, and 138 areas with badger setts or signs of badger activity were identified. A single badger was removed from 87 sites and all were examined using detailed post mortem and bacteriological procedures. A prevalence of M. bovis infection of 14.9% was found in the Greenfield site badgers. This prevalence was significantly lower (P<0.001) than in badgers removed during focal culling (36.6%). The results validate the use of cattle as sentinels for TB in badgers and support the medium term national strategy for the control of bovine TB. The geographic variation in M. bovis infection prevalence in the Irish badger populations will be used when devising strategies for the incorporation of badger vaccination into the long term bovine TB control programme.

Mycobacterium bovis infection in the Eurasian badger (Meles meles): the disease, pathogenesis, epidemiology and control.

Eurasian badgers (Meles meles) are an important wildlife reservoir of tuberculosis (Mycobacterium bovis) infection in Ireland and the United Kingdom. As part of national programmes to control tuberculosis in livestock, considerable effort has been devoted to studying the disease in badgers and this has lead to a rapid increase in our knowledge of tuberculosis in this host. Tuberculosis in badgers is a chronic infection and in a naturally-infected population the severity of disease can vary widely, from latent infection (infection without clinical signs and no visible lesions) to severe disease with generalized pathology. The high prevalence of pulmonary infection strongly supports the lungs as the principal site of primary infection and that inhalation of infectious aerosol particles is the principal mode of transmission. However, other routes, including transmission via infected bite wounds, are known to occur. The ante-mortem diagnosis of infection is difficult to achieve, as clinical examination and immunological and bacteriological examination of clinical samples are insensitive diagnostic procedures. Because infection in the majority of badgers is latent, the gross post-mortem diagnosis is also insensitive. A definitive diagnosis can only be made by the isolation of M. bovis. However, to gain a high level of sensitivity in the bacteriological examination, a large number of tissues from each badger must be cultured and sensitive culture methods employed. The transmission and maintenance of M. bovis in badger populations are complex processes where many factors influence within-population prevalence and rates of transmission. Badger social structures and the longevity of infected animals make them an ideal maintenance host for M. bovis infection. Badgers are directly implicated in the transmission of infection to cattle and the inability to eradicate the disease from cattle is, in part, a consequence of the interactions between the two species. A detailed understanding and knowledge of the epidemiology and pathogenesis of the disease are recognized as fundamental for devising new strategies to control infection with a view to limiting interspecies transmission. Vaccination, in spite of formidable challenges, is seen as the best long-term strategy option and studies with captive badgers have shown that vaccination with M. bovis bacillus Calmette-Guérin (BCG) induces protection when delivered by a variety of routes. Continued research is required to develop effective technologies to control the disease both in badgers and cattle. A combination of strategies, which employ the optimal use and targeting of resources, is likely to make a significant contribution towards eradication of the disease.

The prevalence and distribution of Mycobacterium bovis infection in European badgers (Meles meles) as determined by enhanced post mortem examination and bacteriological culture.

The accurate diagnosis of Mycobacterium bovis infection in badgers is key to understanding the epidemiology of tuberculosis in this species and has significant implications for devising strategies to limit spread of the disease. In this study, badgers (n=215) in the Republic of Ireland were examined at post mortem and tissues were collected from a range of anatomical locations and pooled into groups for bacterial culture of M. bovis. By assessing confirmed gross visible lesions (VL) alone, infection was detected in 12.1% of badgers. However, by including the results of all culture positive pooled samples, the overall infection prevalence increased significantly to 36.3%. Two-thirds (66.7%) of infected animals had no visible lesions (NVL). While the thoracic cavity (lungs and pulmonary lymph nodes) was found to be the most common site of infection, in a proportion of animals infection was absent from the lungs and draining lymph nodes and was confined to the lymph nodes of the carcase or the head. This may indicate an early extrapulmonary dissemination of infection or alternatively, in the case of the head lymph nodes, a secondary pathogenic pathway involving the lymphoid tissues of the upper respiratory tract (URT).

Tuberculosis in European badgers (Meles meles) and the control of infection with bacille Calmette-Guérin vaccination.

The eradication of tuberculosis (Mycobacterium bovis infection) from cattle herds may be compromised if infected wildlife species, such as European badgers (Meles meles), share the same environment and contribute to transfer of infection. Options for dealing with tuberculosis in this wild reservoir host are limited by conservation and social concerns, despite a clear implication that infected badgers are involved with the initiation of tuberculosis in cattle herds. Vaccination of badgers against M. bovis, if successfully employed, would directly facilitate the completion of bovine tuberculosis eradication in affected areas. Vaccine trials in captive badgers have established that the M. bovis bacille Calmette-Guérin (BCG) vaccine can induce a protective response that limits the distribution and severity of tuberculosis disease following experimental challenge. The protective effect of the vaccine has been demonstrated when the vaccine was delivered by subcutaneous injection, deposited on mucous membranes, and given orally in a lipid formulation. A large-scale field trial of oral BCG vaccine has been designed to measure the protection generated in wild badgers subjected to natural transmission of infection and to estimate vaccine efficacy. These parameters will be estimated by comparing the prevalence of M. bovis infection in vaccinated and nonvaccinated badgers. The results will provide a framework for the development and implementation of a national strategy to eliminate the disease in badger populations and if successful will remove this major impediment to bovine tuberculosis eradication.

Experimental tuberculosis in the European badger (Meles meles) after endobronchial inoculation with Mycobacterium bovis: II. Progression of infection.

The aim of the study was to describe, over a period of 24 weeks, the pathological and bacteriological changes in badgers experimentally infected with Mycobacterium bovis. The badgers were infected by endobronchial instillation of 2.5 x10(4) colony forming units (cfu) M. bovis. After infection, the badgers were examined at 3 weekly intervals when blood and tracheal aspirates were collected. At 6, 12, 18 and 24 weeks post-infection (pi) three animals were euthanized and a detailed pathological and bacteriological examination was performed to assess the nature of the experimental disease. During the course of the study only one badger developed clinical signs of disease: a subcutaneous swelling on its head, first observed at 18 weeks pi. At post-mortem examination gross and histological lesions of tuberculosis were observed and M. bovis was recovered from all, except one badger. In the majority of badgers the endobronchial route of inoculation resulted in the establishment of infection that over 24 weeks was non-progressive with limited dissemination of infection from the thoracic cavity, mainly to the hepatic and mesenteric lymph nodes. However, in one of the badgers examined at 18 weeks pi and one at 24 weeks pi, infection was widely disseminated. The disease induced by the endobronchial inoculation displayed the characteristics of disease observed in naturally infected badgers.

The effect of varying levels of population control on the prevalence of tuberculosis in badgers in Ireland.

We examined the effect of varying levels of badger population control on the prevalence of Mycobacterium bovis infection in badgers in four counties of Ireland. In the 'Removal' and 'Buffer' areas, proactive culling was conducted to substantially reduce and subsequently maintain badger populations at a low level for five years. In the 'Reference' areas, localised reactive culling was conducted in association with herd breakdowns. The infection status of badgers was determined using bacteriology. A total of 2696 badgers were recruited into the study, and 19.0% were found to be infected with M. bovis. The two population control strategies had differing effects on the subsequent prevalence of tuberculosis in badger populations. Proactive culling led to a long term decrease in the prevalence of tuberculosis in the re-emergent populations. Although there was an overall decline in the disease prevalence, no consistent trend in disease prevalence as a result of reactive culling was observed.

Adverse reactions to Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination against tuberculosis in humans, veterinary animals and wildlife species.

The Mycobacterium bovis strain, bacille Calmette-Guérin (BCG) is one of the most widely used human vaccines and remains one of the safest vaccines available. It has been used in human populations for over 80 years and 100 million children receive the vaccine annually. It has also been employed extensively for vaccine studies in laboratory animal hosts and is currently being developed for use in a variety of livestock and wild animals. Despite the large number of doses delivered since is first usage in 1921, reports of adverse reactions arising from the use of the BCG vaccine are relatively uncommon and where serious reactions do occur they are often the result of vaccination of immuno-compromised individuals. Factors that may influence the development of adverse reactions to BCG include the potency and dose of the vaccine strain, the route of delivery, the age and immune status of the host, and the skill levels of the operator administering the vaccine. Circumstances affecting the notification of adverse reactions include the lack of clear case definitions of abnormal vaccine reactions, and a scarcity of systematic surveillance and functioning reporting systems. With continued use of the BCG and the development of a new generation of prophylactic and therapeutic vaccines against tuberculosis in different host species, the risk factors associated with adverse reactions may need to be reappraised.

Does reactive badger culling lead to an increase in tuberculosis in cattle?

The conclusion from the randomised badger culling trial was that localised badger culling not only fails to control but can actually increase the incidence of bovine tuberculosis in cattle. Professor Simon More and colleagues from University College Dublin question that conclusion, arguing that the data do not provide sufficient evidence to rule out alternative hypotheses.

Experimental tuberculosis in the European badger (Meles meles) after endobronchial inoculation of Mycobacterium bovis: I. Pathology and bacteriology.

The aim was to develop an endobronchial infection procedure for the study of Mycobacterium bovis infection in badgers. The badgers were anaesthetised and a cannula was passed per os to the tracheal bifurcation. When in place 1 ml of M. bovis suspension was inoculated. Three concentrations of M. bovis suspension were used; <10 colony forming units (cfu), approximately 10(2) cfu and approximately 3 x 10(3) cfu. The badgers were examined at three weekly intervals for clinical signs of disease and a tracheal aspirate was collected at each examination. The badgers were euthanased 17 weeks post infection (pi) and at the post mortem examination a wide range of tissues were examined for gross and histopathological lesions of tuberculosis and cultured for M. bovis. A sample of bronchial alveolar lavage (BAL) fluid was collected at post mortem for culture. At post mortem examination 17 weeks after infection, gross and histopathological lesions of tuberculosis were observed in all badgers inoculated with the high and medium dose and 1/3 inoculated with the low dose. M. bovis was recovered from all inoculated badgers. Infection in the high dose group was more widely disseminated than in the other groups. The number of sites with gross and histopathological lesions increased with increasing dose of M. bovis. All tracheal aspirates were negative on culture and only one BAL, collected from a badger of the high dose group, was positive on culture. No clinical signs due to the experimental infection were observed. The endobronchial route of inoculation is an effective route for establishing experimental infection, and could be used for studies of tuberculosis pathogenesis, immunology of M. bovis infection in badgers and for challenging badgers in vaccine protection studies. Badgers appeared to be very susceptible to infection by this procedure even with a dose of < 10 cfu but appear to control and limit the resulting infection.

The role of wild animal populations in the epidemiology of tuberculosis in domestic animals: how to assess the risk.

Tuberculosis is present in wild animal populations in North America, Europe, Africa and New Zealand. Some wild animal populations are a source of infection for domestic livestock and humans. An understanding of the potential of each wild animal population as a reservoir of infection for domestic animals is reached by determining the nature of the disease in each wild animal species, the routes of infection for domestic species and the risk of domestic animals encountering an infectious dose. The mere presence of infection in a wild animal population does not of itself provide evidence of a significant wildlife reservoir. Although at times counterintuitive, wildlife populations with high disease prevalence may not necessarily have a role in the epidemiology of disease in domestic livestock. The key concepts used in deciding whether an infected wild animal population is involved in the epidemiology of tuberculosis in domestic livestock is illustrated by reference to six well-researched cases: the feral pig (Suis scrofa) and feral Asian water buffalo (Bubalus bubalis) in Australia, white tailed deer (Odocoileus virginianus) in Michigan, and the brushtail possum (Trichosurus vulpecula) and other species, such as the ferret (Mustela furo), in New Zealand. A detailed analysis of Mycobacterium bovis infection in the Eurasian badger (Meles meles) in Ireland and their role as a reservoir of infection for cattle is also presented.

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.

Conjunctival vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin.

AIM: To determine the efficacy of conjunctival vaccination of captive brushtail possums (Trichosurus vulpecula) with bacille Calmette-Guérin (BCG), as measured by immunological responses to vaccination and response to intratracheal challenge with Mycobacterium bovis. METHODS: Nine adult male brushtail possums were vaccinated by the instillation of a suspension of BCG strain Pasteur 1173P2 into the conjunctival sac of each eye. Each drop contained approximately 2.5 x 105 colony forming units (cfu). At 8 weeks post-vaccination (pv) the vaccinated possums and 10 unvaccinated possums were challenged by intratracheal instillation of approximately 100 cfu of M. bovis. Cellular immune responses to bovine purified protein derivative (PPD) antigen were measured using the lymphocyte proliferation assay (LPA). Possums surviving to 50-51 days after challenge were euthanised and subjected to detailed post-mortem examination, including histopathology, to assess protection against tuberculosis. Sections of lung and spleen were cultured for M. bovis. RESULTS: No conjunctival inflammation or other adverse reactions to the administration of the vaccine were evident macroscopically. The vaccinated group showed a systemic cellular immune response to bovine PPD antigen at 4 and 8 weeks pv, and the response at 8 weeks was significantly greater than at 4 weeks (p<0.05). Conjunctival vaccination induced significant levels of protective immunity, measured as less mass of tuberculous lesions in lung (p<0.05) and less dissemination of disease in vaccinated compared with unvaccinated possums (p<0.05). CONCLUSIONS: Conjunctival vaccination with BCG induced a significant level of protective immunity against M. bovis infection in possums. This route of vaccination, together with intranasal aerosol vaccination, could be utilised in the delivery of an aerosolised vaccine using a device that sprays the vaccine suspension into the eyes and nose of possums.

Use of an electronic nose to diagnose Mycobacterium bovis infection in badgers and cattle.

It is estimated that more than 50 million cattle are infected with Mycobacterium bovis worldwide, resulting in severe economic losses. Current diagnosis of tuberculosis (TB) in cattle relies on tuberculin skin testing, and when combined with the slaughter of test-positive animals, it has significantly reduced the incidence of bovine TB. The failure to eradicate bovine TB in Great Britain has been attributed in part to a reservoir of the infection in badgers (Meles meles). Accurate and reliable diagnosis of infection is the cornerstone of TB control. Bacteriological diagnosis has these characteristics, but only with samples collected postmortem. Unlike significant wild animal reservoirs of M. bovis that are considered pests in other countries, such as the brushtail possum (Trichosurus vulpecula) in New Zealand, the badger and its sett are protected under United Kingdom legislation (The Protection of Badgers Act 1992). Therefore, an accurate in vitro test for badgers is needed urgently to determine the extent of the reservoir of infection cheaply and without destroying badgers. For cattle, a rapid on-farm test to complement the existing tests (the skin test and gamma interferon assay) would be highly desirable. To this end, we have investigated the potential of an electronic nose (EN) to diagnose infection of cattle or badgers with M. bovis, using a serum sample. Samples were obtained from both experimentally infected badgers and cattle, as well as naturally infected badgers. Without exception, the EN was able to discriminate infected animals from controls as early as 3 weeks after infection with M. bovis, the earliest time point examined postchallenge. The EN approach described here is a straightforward alternative to conventional methods of TB diagnosis, and it offers considerable potential as a sensitive, rapid, and cost-effective means of diagnosing M. bovis infection in cattle and badgers.

Revaccination of neonatal calves with Mycobacterium bovis BCG reduces the level of protection against bovine tuberculosis induced by a single vaccination.

Cattle may provide a suitable model for testing ways of improving tuberculosis vaccine efficacy in human infants. A vaccination and challenge study was undertaken in calves to determine the optimal time to vaccinate neonatal animals with Mycobacterium bovis bacillus Calmette-Guérin (BCG) for protection against tuberculosis and to determine whether revaccination with BCG was beneficial. Calves (10 per group) were vaccinated with BCG within 8 h of birth or at 6 weeks of age, when immune responses to antigens of environmental mycobacteria were detectable, or vaccinated at birth and revaccinated at 6 weeks. A control group was not vaccinated. BCG vaccination at birth induced strong antigen-specific gamma interferon (IFN-gamma) and interleukin-2 (IL-2) responses and antigen-specific activation in CD4(+), CD8(+), and WC1(+) gammadelta T-cell subsets from blood. The proportions of animals per group with macroscopic tuberculous lesions after challenge were 0/10 for BCG at birth, 1/9 for BCG at 6 weeks, 4/10 for the revaccinated group, and 10/10 for the nonvaccinated group. There was no significant difference in the levels of protection between groups vaccinated at birth or at 6 weeks, while animals vaccinated both at birth and at 6 weeks had significantly less protection than those vaccinated only at birth. The revaccinated calves that subsequently developed tuberculous lesions had significantly stronger IFN-gamma and IL-2 responses to bovine purified protein derivative after the BCG booster than those in the same group that did not develop lesions. The results indicated that BCG vaccination at birth induced a high level of immunity and that the sensitization of very young animals to antigens of environmental mycobacteria by 6 weeks of age did not affect the effectiveness of BCG. However, BCG revaccination of these young animals was contraindicated.

Experimental infection of brushtail possums (Trichosurus vulpecula) with Mycobacterium bovis by conjunctival instillation.

In New Zealand, the brushtail possum (Trichosurus vulpecula) is the major wildlife reservoir of Mycobacterium bovis. Procedures for experimentally infecting possums are required to study the pathogenesis of the disease and to challenge possums in vaccine efficacy studies. Conjunctival instillation of a suspension of M. bovis was effective in producing bovine tuberculosis in captive possums. The experimental disease progressed slowly with the development of palpable lesions in superficial lymph node lesions, both characteristics of the disease in wild, naturally infected possums. At necropsy there was widespread distribution of macroscopic and microscopic lesions. The proportion of possums that became diseased, the rate of development and severity of lesions, the severity of clinical signs, all increased when the dose of M. bovis was increased. Of the three doses used, the medium dose (1000-2000 colony forming units) produced the disease with the most desired characteristics. As a procedure for exposing possums to infection with M. bovis the conjunctival route has advantages in that it is simple and safe to perform, and possums need only to be sedated for infection.

Social-network analysis of Mycobacterium bovis transmission among captive brushtail possums (Trichosurus vulpecula).

Wild brushtail possums (Trichosurus vulpecula) are the main source of Mycobacterium bovis infection for New Zealand livestock. The disease is spread principally by infectious aerosol; therefore, social interactions determine disease transmission. In captive possums, den-sharing behaviour provided the greatest risk of tuberculosis transmission between animals. Den sharing between individual possums was used as the basic measurement for quantifying close proximity between animals over extended periods. Social-network analysis (SNA) was used to model patterns of social behaviour and to predict tuberculosis transmission. There was great diversity between groups in their social behaviour-but there were consistent trends in the SNA measures (closeness and flow-betweenness). With time, the social distance between possums in the same group increased, the social network became more homogeneous and the possums less differentiated from each other. Alteration of the physical environment of the pens (such as changing the number of dens or relocating the group to a new pen) had an inconsistent effect on social structure when comparing different groups. During the infection-transmission study, the possums that became infected had greater closeness and flow-betweenness scores than those that remained free of infection. Although standard statistical descriptive measures (such as the number of partners and the frequency of den-sharing events) were greater for the infected than the infection-free possums, the SNA-specific measures were more precise and could be compared across time and between groups.