Understanding how bovine tuberculosis (TB) is maintained in wildlife reservoirs is critical for the management of this disease impacting cattle in many countries. For the primary wildlife reservoir of the disease in New Zealand, the brushtail possum (Trichosurus vulpecula), transmission of this contagious bacterial disease among possums is often assumed to be linked to home range overlap. Identifying drivers of possum home range, and home range overlap between individuals, is thus important for informing wildlife reservoir TB management in New Zealand. We monitored four sub-populations of free-living possums in New Zealand native forests during 10 consecutive months using live trapping, to give the first direct insight into how the frequency and area of overlaps alters with density, sex and age. A total of 832 individuals were captured (average 9.3, range from 1 to 40 captures per animal with a median value of 7) and 35,820 home range overlaps were recorded. Both the number and area of overlaps were significantly associated with age class, with 66.6% of overlaps occurring between adults, 30% between adults and juveniles, and only 3.4% between juveniles. Overall, adult males showed significantly higher numbers of overlaps than expected, while adult and juvenile females showed significantly lower numbers of overlaps than expected and no differences were observed in juvenile males. In addition, males exhibited more and larger overlaps than females. The number and size of overlaps per individual decreased with increasing local population density. Understanding shared areas of activity among individuals can provide insights into the interactions occurring and potential pathways for diseases transmitted by contact such as TB. These results can inform to develop effective strategies for the control of diseases carried and dispersed by possums.

Buruli ulcer (BU) is a flesh-eating infection caused by Mycobacterium ulcerans that leads to significant community morbidity, with an estimated cost to Victoria in 2016 of more than $2.5 million.1 It was first recognised in Victoria, Australia, in 1948.2 Until the turn of the century, it was only reported in low numbers and a few localised regions, but the BU epidemic in Victoria has shown a consistent upward trend in recent years. In 2022, reported case numbers increased to 340, the equal highest annual case load recorded and more than five times the number reported 10 years previously. Alarmingly, the disease is also moving into new areas — in the past 5 years it has moved into a coastal region south of Geelong (Surf Coast), with urban outbreaks in the regional city of Geelong and in the inner north-western suburbs of Melbourne. The proportion of severe cases at diagnosis is also on the rise — since 2010, the proportion of people with infection presenting with World Health Organization category 2 or 3 lesions has increased from 20% to about 30%.3 Efforts to implement public health interventions to address this have been hindered by the lack of a clear understanding of the environmental reservoir and transmission mechanisms of M. ulcerans.1 An important discovery in the mid-2000s identified that possum faecal specimens (both common ringtail [Pseudocheirus peregrinus] and common brushtail [Trichosurus vulpecula] possums) were M. ulcerans DNA positive in the newly endemic area of Point Lonsdale, Victoria.4 On further assessment, 33% of captured possums either had evidence of clinical disease or were asymptomatic but had positive excreta. The rate of BU lesions or positive excreta was higher in ringtail (38%) than in brushtails (24%) possums. Interestingly, M. ulcerans isolates from possums were genetically indistinguishable from those from Victorian BU patients.5 More comprehensive One Health surveillance has found that the presence of M. ulcerans positive possum faecal samples correlates geographically with the occurrence of human BU cases, and the detection of positive samples in a new area can herald the onset of human cases in the next one to two years.6 Further evidence for the role of possums in BU transmission comes from a large comprehensive case–control study across Victoria's endemic regions from 2018 to 2020.7 This showed a greatly increased risk of BU among people reporting the presence of possums on their property, with a dose–response effect suggested by more possums reported correlating with a higher risk of BU. Additionally, when environmental samples were collected from case and control properties, a high proportion of possum faeces (mainly ringtail) was found to be M. ulcerans DNA positive, and a large percentage was shown to contain viable M. ulcerans that suggests transmissibility.8 Other environmental features associated with possums, such as known food sources, habitat and mobility, were also associated with detection of M. ulcerans at the property. Finally, to support possums carrying viable M. ulcerans organisms that can be transmitted to humans, a case was recently reported where a man bitten while helping a sick ringtail possum developed a BU lesion at the bite site within 6 months.9 The case for possums being a zoonotic reservoir and being involved in the transmission of M. ulcerans is now strong in Victorian endemic areas. Considering the urgent need to deal with the worsening Victorian BU epidemic, the challenge is how we can utilise this knowledge to reduce M. ulcerans transmission. Preventing contact between possums and humans via public education campaigns to increase awareness of potential risks, especially if possums are sick or unwell, may help. However, possums have adapted well to suburban settings, including gardens where their faeces can cover much of the ground, making limiting contact (direct or indirect) very difficult. Culling is not an appropriate solution as they are a protected species in Victoria and form an important part of the natural ecosystem, with unintended consequences likely to result if their social structure is disrupted. Culling would also likely result in significant public anger. We propose that research be undertaken to explore the effectiveness and feasibility of vaccinating ringtail possums with an oral bait bacille Calmette–Guérin (BCG) vaccine in the wild. If effective in reducing BU disease in possums, it should hopefully simultaneously reduce the occurrence in humans. Furthermore, it would benefit possums, as BU can lead to death, and this would be a safe intervention that could reduce both morbidity and mortality in possums. The aim would be to manage this zoonotic pathogen in its reservoir possum host with benefits for human and animal health, without damaging the local ecosystem. Oral bait vaccines have been used to control other diseases in wildlife such as rabies in Europe and North America and classical swine flu virus in Europe.10 Specifically, the use of oral bait BCG vaccine has been successfully employed to control bovine tuberculosis in wildlife such as badgers in Ireland and the United Kingdom, deer in North America, and wild boars in Spain.11 Most importantly, researchers in New Zealand, where bovine tuberculosis is a major problem in non-native Australian brushtail possums, have shown that they can successfully vaccinate possums with oral bait BCG and induce immunity to bovine tuberculosis lasting more than two years.10 This was effective both in laboratory studies and in wide-scale field trials, with up to a 95% reduction in the prevalence of bovine tuberculosis in vaccinated wild possum populations.12 It is reasonable to expect that the intervention would be equally effective in both ringtail and brushtail possums. Furthermore, BCG vaccine has been shown to be safe for numerous livestock and wildlife species. Whether BCG vaccination of ringtail possums could protect them against M. ulcerans infection has not been evaluated, but mice vaccinated with BCG are protected against BU.13 Two large randomised controlled trials of BCG vaccination in humans for the prevention of BU conducted in Uganda during the late 1960s and early 1970s found significant but short-lasting protection against BU.14, 15 In a recent case–control study in Victoria, a history of BCG vaccine was associated with a 40% reduction in the risk of BU (odds ratio, 0.6; 95% CI, 0.4–0.9).7 Although BCG vaccination of humans in endemic areas could also be considered, vaccinating possums would have the added benefits of protecting everyone in endemic areas regardless of vaccine status, protecting visitors living outside the area (Victorian endemic regions are high tourism areas), and providing health benefits for possums. Additionally, as possums likely play a role in introducing the disease into new areas, it may stop disease spread. Furthermore, the uptake of oral bait BCG by non-target species could be beneficial as M. ulcerans infection has been detected in a wide range of mammalian species in Australia.1 The accidental vaccination of these animals could therefore contribute to overall host immunity within the endemic ecosystem and subsequently reduce the risk to humans. The proposed research would involve several stages. The first would be to establish housing and husbandry conditions for ringtail possums in a research facility. Once established, this would be followed by attempts to develop a M. ulcerans infection model using both high dose (~ 103 colony-forming units) and low dose (~ 20–30 colony-forming units) M. ulcerans inocula, with monitoring of excreta and clinical signs of disease to determine infective dose and susceptibility to infection, incubation periods, immunological responses, and the routes and magnitude of M. ulcerans shedding. If successful, possums would then be vaccinated with BCG before M. ulcerans challenge, to assess the level and durability of BU protection by comparing vaccine with control groups. Blood samples would be collected to measure the immune responses to BCG vaccination, which would be correlated with immune protection. If shown to be effective, attempts would be made to optimise the palatability and feasibility of oral bait BCG delivery. Finally, testing the effectiveness of oral bait BCG vaccine against M. ulcerans in ringtail possums would be performed in laboratory and real-life field settings. Data obtained from these studies could be used to assess whether possum vaccination would be cost-effective in controlling BU, provide information on the epidemiology of infection in possums, and help develop disease transmission models. Studies could also be extended to brushtail possums. Although mosquitoes are considered to play a role in transmission,1 and mosquito prevention measures might have an impact on human case numbers, evidence for their effectiveness is lacking. It is therefore important to explore other interventions that may reduce transmission, such as possum vaccination.7 In south-eastern Australia, BU is a One Health challenge involving a complex interaction between the environment, insects, wildlife and humans, with many knowledge gaps remaining. However, there are currently no proven public health interventions to address the worsening epidemic of BU in this region. A proposal to vaccinate possums in the wild with oral bait BCG provides hope that an acceptable, safe, feasible and cost-effective intervention can be found that would benefit both human and possum populations by reducing the transmission of M. ulcerans and the incidence of BU in the region. Open access publishing facilitated by The University of Melbourne, as part of the Wiley – The University of Melbourne agreement via the Council of Australian University Librarians. No relevant disclosures. Not commissioned; externally peer reviewed.

Knowledge gained from veterinary immunology has played an important role in the control of microbial and parasitic diseases in New Zealand through the development and use of vaccines and diagnostic tests. In this article celebrating the 100th anniversary of the Journal, I follow the development of important discoveries in veterinary immunology which have led to major advances in the control of animal diseases.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhea, weight-loss, and eventual death in ruminants. Commercially available vaccine provides only partial protection against MAP infection and can interfere with the use of current diagnostic tests for bovine tuberculosis in cattle. Here, we characterized immune responses in calves to vaccines containing four truncated MAP antigens as a fusion (Ag85A 202-347 -SOD 1-72 -Ag85B 173-330 -74F 1-148+669-786 ), either displayed on protein particles, or expressed as a soluble recombinant MAP (rMAP) fusion protein as well as to commercially available Silirum ® vaccine. The rMAP fusion protein elicited the strongest antigen-specific antibody responses to both PPDA and recombinant antigen and strong and long-lasting T-cell immune responses to these antigens, as indicated by increased production of IFN-γ and IL-17A in antigen-stimulated whole blood cultures. The MAP fusion protein particle vaccine induced minimal antibody responses and weak IFN-γ responses but stimulated IL-17A responses to recombinant antigen. The immune response profile of Silirum ® vaccine was characterized by weak antibodies and strong IFN-γ and IL-17A responses to PPDA. Transcription analysis on antigen-stimulated leukocytes from cattle vaccinated with rMAP fusion protein showed differential expression of several immune response genes and genes involved in costimulatory signaling, TLR4 , TLR2 , PTX3 , PTGS2 , PD-L1 , IL1B , IL2 , IL6 , IL12B , IL17A , IL22 , IFNG , CD40 , and CD86 . Moreover, the expression of several genes of immune pathways correlated with cellular immune responses in the rMAP fusion protein vaccinated group. These genes have key roles in pathways of mycobacterial immunity, including autophagy, manipulation of macrophage-mediated killing, Th17- and regulatory T cells- (Treg) mediated responses. Calves vaccinated with either the rMAP fusion protein or MAP fusion protein particle vaccine did not induce reactivity to PPDA and PPDB in a comparative cervical skin test, whereas Silirum ® induced reactivity to these tuberculins in most of the vaccinated animals. Overall, our results suggest that a combination of recombinant MAP antigens in the form of a soluble fusion protein vaccine are capable of inducing strong antigen-specific humoral and a balanced Th1/Th17-cell immune response. These findings, together with the absence of reactivity to tuberculin, suggest this subunit vaccine could provide protective immunity against intracellular MAP infection in cattle without compromising the use of current bovine tuberculosis surveillance test.

Bacillus Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis (M. bovis), is the lead candidate vaccine for control of bovine tuberculosis (TB) in cattle. However, BCG vaccination sensitises cattle to bovine tuberculin, thus compromising the use of the current bovine TB surveillance tests. To address this, we have developed a diagnostic skin test that is not compromised by BCG vaccination and is able to detect BCG vaccinated animals that subsequently develop bovine TB following exposure to M. bovis. Building on previous work using 'in house' formulated protein cocktail reagents, we herein present test performance data for a single fusion protein (DST-F) containing the mycobacterial antigens ESAT-6, CFP-10 and Rv3615c formulated as a 'ready to use' reagent by a commercial manufacturer. Our results demonstrate that, unlike tuberculin reagents, a diagnostic skin test using DST-F maintained high specificity in BCG vaccinated animals. Furthermore, the DST-F skin test demonstrated a high relative sensitivity in identifying M. bovis infected animals, including those where BCG vaccination failed to prevent bovine TB pathology following experimental exposure to M. bovis. The DST-F is currently undergoing field trials in Great Britain to support its licensure and commercialisation.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhoea, weight loss, and eventual death in ruminants. Commercially available vaccines provide only partial protection against MAP infection and can compromise the use of bovine tuberculosis diagnostic tests. Here, we report the development of a protein-particle-based vaccine containing MAP antigens Ag85A 202–347 -SOD 1–72 -Ag85B 173–330 -74F 1–148+669–786 as a fusion (‘MAP fusion protein particle’). The fusion antigen displayed on protein particles was identified using mass spectrometry. Surface exposure and accessibility of the fusion antigen was confirmed by flow cytometry and ELISA. The MAP fusion protein particle vaccine induced strong antigen-specific T-cell immune responses in mice, as indicated by increased cytokine (IFN-γ and IL-17A) and costimulatory signals (CD40 and CD86) in these animals. Following MAP-challenge, a significant reduction in bacterial burden was observed in multiple organs of the mice vaccinated with the MAP fusion protein particle vaccine compared with the PBS group. The reduction in severity of MAP infection conferred by the MAP fusion protein particle vaccine was similar to that of Silirum and recombinant protein vaccines. Overall, the results provide evidence that MAP antigens can be engineered as a protein particulate vaccine capable of inducing immunity against MAP infection. This utility offers an attractive platform for production of low-cost particulate vaccines against other intracellular pathogens.

Bovine tuberculosis is an intractable problem where ‘test-and-cull’ policies are not affordable or socially acceptable, or where Mycobacterium bovis infection is sustained by wildlife reservoirs. Recent studies in domestic livestock and wildlife have demonstrated that vaccination with BCG could be a valuable control measure, particularly when integrated with other strategies.

The brushtail possum is the main reservoir of bovine tuberculosis in New Zealand. Disease prevalence is generally higher in males than in females. This has conventionally been assumed due to greater infection rates of males, but recent work has raised the hypothesis that it may instead be driven by survival differences. With bovine tuberculosis transmission among possums most likely occurring between individuals in close proximity, here we analyse social networks built on data from wild possums collared with contact loggers inhabiting a native New Zealand forest, to investigate whether there is mechanistic support for higher male infection rates. Our results revealed that adult female possums were generally just as connected with adult male possums as other adult males are, with male–female connection patterns not being significantly different. This result suggest that the new ‘survivorship’ hypothesis for the sex bias is more likely than the conventional ‘infection rate’ hypothesis.

Bovine tuberculosis (TB) continues to be an intractable problem in many countries, particularly where "test and slaughter" policies cannot be implemented or where wildlife reservoirs of Mycobacterium bovis infection serve as a recurrent source of infection for domestic livestock. Alternative control measures are urgently required and vaccination is a promising option. Although the M. bovis bacille Calmette-Guérin (BCG) vaccine has been used in humans for nearly a century, its use in animals has been limited, principally as protection against TB has been incomplete and vaccination may result in animals reacting in the tuberculin skin test. Valuable insights have been gained over the past 25 years to optimise protection induced by BCG vaccine in animals and in the development of tests to differentiate infected from vaccinated animals (DIVA). This review examines factors affecting the efficacy of BCG vaccine in cattle, recent field trials, use of DIVA tests and the effectiveness of BCG vaccine in other domestic livestock as well as in wildlife. Oral delivery of BCG vaccine to wildlife reservoirs of infection such as European badgers, brushtail possums, wild boar, and deer has been shown to induce protection against TB and could prove to be a practical means to vaccinate these species at scale. Testing of BCG vaccine in a wide range of animal species has indicated that it is safe and vaccination has the potential to be a valuable tool to assist in the control of TB in both domestic livestock and wildlife.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, which is characterized by chronic progressive granulomatous enteritis. The infection leads to wasting and weight loss in the animals and eventually death, causing considerable production losses to the agricultural industry worldwide. Currently available ELISA- and PCR-based diagnostic tests have limited sensitivity and specificity during early MAP infection in cattle, suggesting that there is an urgent demand for alternative diagnostic tests. Circulating microRNA (miRNA) have recently gained attention as potential biomarkers for several diseases in humans. However, knowledge and use of miRNA as biomarkers in diseases of ruminants, including Johne's disease, are very limited. Here we used NanoString nCounter technology (NanoString, Seattle, WA), a digital platform for amplification-free and hybridization-based quantitative measurement of miRNA in the sera of noninfected and naturally MAP-infected cattle with different severity of infection. Using probes developed against human miRNA, 26 miRNA were detected in cattle serum; 13 of these miRNA were previously uncharacterized for cattle. Canonical discrimination analysis using 20 miRNA grouped animals into 4 distinct clusters based on their disease status, suggesting that the levels of these miRNA can reflect disease severity. A model was developed using a combination of 4 miRNA (miR-1976, miR-873-3p, miR-520f-3p, and miR-126-3p), which distinguished moderate and severely infected animals from noninfected animals. Our study demonstrated the ability of the NanoString nCounter technology to detect differential expression of circulating miRNA in cattle and contributes to widely growing evidence that miRNA can be used as biomarkers in infectious diseases in cattle.

The global burden of bovine tuberculosis (bTB) remains poorly characterized, with spill-over impacts on multiple species. The "One Health" concept is especially relevant given the bidirectional risk of cattle infecting humans with Mycobacterium bovis and humans infecting cattle with Mycobacterium tuberculosis. "Test and cull" is the traditional bTB control method, but the strategy may not be economically feasible or culturally acceptable where cattle are highly prized or their killing is a religious taboo; it is also less effective when there are wildlife reservoirs of infection. Vaccination with M. bovis bacille Calmette-Guerin (BCG) provides protection against bTB, but its use in animals has been limited. The Jerusalem One Health workshop considered key bTB knowledge gaps and innovative solutions. Knowledge gaps identified included (a) the poorly quantified prevalence of M. bovis infection and disease in cattle, domestic camelids and human populations in developing countries, (b) the absence of alternatives to a "test and cull" strategy in settings where the killing of infected animals is culturally or economically unacceptable, or where affected species are protected and (c) an understanding of the induction of mucosal immunity against bTB. We summarize discussions on the use of BCG vaccination in domestic animals and wildlife and list potential projects to address the knowledge gaps identified.

Vaccination of cattle with Mycobacterium bovis BCG has been shown to protect against infection with virulent strains of M. bovis, and against resultant bovine tuberculosis (TB). Here we report on a large-scale trial in New Zealand where free-ranging cattle were vaccinated with 3 x 10(5) BCG via injection, a lower dose than any previously trialed in cattle against exposure to a natural force of M. bovis infection. In a multi-year enrolment study involving >800 animals, three cohorts of 1-2 year old cattle were randomised to receive vaccine or to serve as non-vaccinated controls. Cattle were slaughtered and subject to standard abattoir post mortem examination for M. bovis culture-positive TB lesions after up to 3.7 years of in-field exposure; additionally, lymph node samples from approximately half of the cattle were examined further to identify infection in the absence of lesions. Overall TB prevalence, as identified by gross lesions detected at slaughter, was low among farmed cattle at the study site (<4% annually). There were two lesioned cases among 520 vaccinated trial cattle (0.38%) compared to eight among 297 non vaccinated trial cattle (2.69%). Trial vaccine efficacy was 85.7% against abattoir-detectable TB (statistically significant protection), and 86.7% when adjusted for duration of exposure. BCG vaccination did not significantly affect the response rates of cattle to ante mortem skin- or blood-tests in diagnostic tests conducted >7 months post-vaccination. Use of a reduced, yet effective, dose of BCG would increase the cost effectiveness of using this vaccine in a bovine TB control programme. (C) 2018 Elsevier Ltd. All rights reserved.

Abstract The focus of this chapter is to provide an update on the progress in vaccination of tuberculosis (TB) in domestic (goats and cattle) and wild animals (badgers, ferrets, white-tailed deer, Sus scrofa, African buffaloes, and common brushtail possum).

The gamma interferon (IFN-γ) test has been used for many years as an ancillary test in the detection of bovine tuberculosis. We investigated the effect of skin testing and the length of time between blood collection and processing on the performance of the IFN-γ test. A series of blood samples were taken from groups of experimentally infected cattle ( n = 10), naturally infected ( n = 11), and uninfected animals ( n = 12) that were examined with a caudal fold skin test. Blood was taken on the day of tuberculin injection, 3 d later when the skin tests were read, and 11-19 d post-tuberculin injection, and was processed for the IFN-γ test at 8, 30, and 36 h postcollection. There were significant decreases in the IFN-γ responses with increasing time between blood collection and sample processing. Significantly greater responses were observed in both the purified protein derivative (PPD) and early secretory antigenic target protein 6/culture filtrate protein 10 IFN-γ tests for samples processed at 8 h postcollection compared with the same samples at 30 and 36 h postcollection, and greater responses for samples processed at 30 h compared with 36 h on 2 different days for the experimentally infected animals. There were no significant effects on IFN-γ responses that could be attributed to skin testing. The recommendation for IFN-γ testing in New Zealand is that samples should not be processed if in transit for >30 h, but blood samples can be collected for IFN-γ testing regardless of the timing of the skin test.

Mycobacterium bovis BCG vaccination sensitizes cattle to bovine tuberculin, which compromises the use of the current bovine tuberculosis (TB) surveillance tests. Although the performance of a blood test (that utilizes antigens expressed by Mycobacterium bovis but not by BCG) capable of discriminating infected from vaccinated animals (DIVA interferon gamma test [DIT]) has been evaluated in naturally infected TB field reactors, there is a need to perform similar analysis in a BCG-vaccinated M. bovis-infected population. Furthermore, we explored different scenarios under which a DIT may be implemented alongside BCG vaccination: (i) serial testing to resolve potential false-positive skin test results or (ii) a standalone test to replace the single intradermal comparative cervical tuberculin (SICCT) skin test. Our results demonstrated significantly better relative test sensitivity when the DIT was evaluated in a serial test scenario. Direct comparison of pre- and post-skin test blood samples revealed that the SICCT test induced significant boosting of the gamma interferon response in M. bovis-infected animals to both the ESAT-6-CFP-10 and Rv3615c peptide cocktails that comprise the DIT, which persisted for the ESAT6-CFP-10 reagent for at least 14 days. Importantly, no similar boosting effects were observed in noninfected BCG vaccinates, suggesting that DIVA blood testing after a recent skin test would have minimal impact on test specificity.

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

Summary In 2015, there were an estimated 10.4 million new tuberculosis (TB) cases and 1.4 million deaths worldwide. Bacille Calmette–Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the vaccine available against TB, but it is insufficient for global TB control. This study evaluated the immunogenicity of the Mycobacterium tuberculosis antigen Rv1626 in mice while assessing the effect of co‐delivering either Cpe30 (immunostimulatory peptide), CS.T3378–395 (promiscuous T helper epitope) or flagellin (TLR5 agonist) or a combination of all three immunostimulatory agents. Rv1626 and the respective immunostimulatory proteins/peptides were co‐displayed on polyhydroxybutyrate beads assembled inside an engineered endotoxin‐free mutant of Escherichia coli. Mice vaccinated with these beads produced immune responses biased towards Th1‐/Th17‐type responses, but inclusion of Cpe30, CS.T3378–395 and flagellin did not enhance immunogenicity of the Rv1626 protein. This was confirmed in a M. bovis challenge experiment in mice, where Rv1626 beads reduced bacterial cell counts in the lungs by 0.48 log10 compared with the adjuvant alone control group. Co‐delivery of immunostimulatory peptides did not further enhance protective immunity.

ABSTRACT Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. Recently, engineered polyhydroxyalkanoate (PHA) biobeads that were produced in both Escherichia coli and Lactococcus lactis and displayed mycobacterial antigens were found to induce significant cell-mediated immune responses in mice. We observed that such PHA beads contained host cell proteins as impurities, which we hypothesized to have the potential to induce immunity. In this study, we aimed to develop PHA beads produced in mycobacteria (mycobacterial PHA biobeads [MBB]) and test their potential as a TB vaccine in a mouse model. As a model organism, nonpathogenic Mycobacterium smegmatis was engineered to produce MBB or MBB with immobilized mycobacterial antigens Ag85A and ESAT-6 on their surface (A:E-MBB). Three key enzymes involved in the poly(3-hydroxybutyric acid) pathway, namely, β-ketothiolase (PhaA), acetoacetyl-coenzyme A reductase (PhaB), and PHA synthase (PhaC), were engineered into E. coli - Mycobacterium shuttle plasmids and expressed in trans . Immobilization of specific antigens to the surface of the MBB was achieved by creating a fusion with the PHA synthase which remains covalently attached to the polyester core, resulting in PHA biobeads displaying covalently immobilized antigens. MBB, A:E-MBB, and an M. smegmatis vector control (MVC) were used in a mouse immunology trial, with comparison to phosphate-buffered saline (PBS)-vaccinated and Mycobacterium bovis BCG-vaccinated groups. We successfully produced MBB and A:E-MBB and used them as vaccines to induce a cellular immune response to mycobacterial antigens. IMPORTANCE Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world's biggest global health burdens. In this study, we produced polyhydroxyalkanoate (PHA) biobeads in mycobacteria and used them as vaccines to induce a cellular immune response to mycobacterial antigens.

Context The Australian brushtail possums (Trichosurus vulpecula) introduction to New Zealand has exacted a heavy toll on native biodiversity and presented the country with its greatest wildlife reservoir host for bovine tuberculosis (TB). Management efforts to control both possums and TB have been ongoing for decades, and the biology of possums has been studied extensively in Australia and New Zealand over the past 50 years; however, we still do not have a clear understanding of its home-range dynamics. Aims To investigate determinants of home range size by using a uniquely large dataset in the Orongorongo Valley, a highly monitored research area in New Zealand and compare our findings with those of other studies. Methods Possum density was estimated, for subpopulations on four 13-ha cage-trap grids, by the spatially explicit capture–mark–recapture analysis of trapping data from 10 consecutive months. Home ranges were estimated from trap locations using a 100% minimum convex polygon (MCP) method for 348 individuals and analysed with respect to grid, age and sex. Key results Mean (standard error) possum density, estimated as 4.87 (0.19), 6.92 (0.29), 4.08 (0.21) and 4.20 (0.19) ha–1 for the four grids, was significantly negatively correlated with mean MCP home-range size. Grid, age, and the interaction of age and sex were significantly related to home-range size. Older possums had larger home ranges than did younger possums. When ‘juvenile cohort’ and ‘adult cohort’ data were analysed separately, to investigate the significant interaction, males in the ‘adult cohort’ had significantly larger home ranges than did females, with the grid effect still being apparent, whereas neither sex nor grid effects were significant for the ‘juvenile cohort’. Conclusions Our findings indicate that, in addition to density, age and sex are likely to be consistent determinants of possum home-range size, but their influences may be masked in some studies by the complexity of wild-population dynamics. Implications Our findings have strong implications regarding both disease transmission among possums and possum management. The fact that adult males occupy larger home ranges and the understanding that possum home range increases as population density decreases are an indication that males may be the primary drivers of disease transmission in possum populations. The understanding that possum home range increases as population density decreases could be a direct reflection of the ability of TB to persist in the wild that counteracts current management procedures. If individuals, and particularly males, infected with TB can withstand control measures, their ensuing home-range expansion will result in possible bacteria spread in both the expanded area of habitation and new individuals becoming subjected to infection (both immigrant possums and other control survivors). Therefore, managers should consider potential approaches for luring possum males in control operations.

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.