History of Veterinary Immunology in New Zealand.

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.

Test performance data demonstrates utility of a cattle DIVA skin test reagent (DST-F) compatible with BCG vaccination.

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 subsp. paratuberculosis antigens induce cellular immune responses in cattle without causing reactivity to tuberculin in the tuberculosis skin test.

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 (Ag85A202-347-SOD1-72-Ag85B173-330-74F1-148+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.

Self-assembled particulate vaccine elicits strong immune responses and reduces Mycobacterium avium subsp. paratuberculosis infection in mice.

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 Ag85A202-347-SOD1-72-Ag85B173-330-74F1-148+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.

BCG vaccination for bovine tuberculosis; conclusions from the Jerusalem One Health workshop.

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.

Efficacy and Safety of BCG Vaccine for Control of Tuberculosis in Domestic Livestock and Wildlife.

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.

Low-dose BCG vaccination protects free-ranging cattle against naturally-acquired bovine tuberculosis.

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 105 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.

Detection of microRNA in cattle serum and their potential use to diagnose severity of Johne's disease.

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.

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

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

Immunological properties and protective efficacy of a single mycobacterial antigen displayed on polyhydroxybutyrate beads.

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.

Tuberculin Skin Testing Boosts Interferon Gamma Responses to DIVA Reagents in Mycobacterium bovis-Infected Cattle.

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 ESAT-6-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.

Factors affecting the gamma interferon test in the detection of bovine tuberculosis in cattle.

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.

Engineering Mycobacteria for the Production of Self-Assembling Biopolyesters Displaying Mycobacterial Antigens for Use as a Tuberculosis Vaccine.

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, ß-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.

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.

Experimental infection of New Zealand Merino sheep with a suspension of Mycobacterium avium subspecies paratuberculosis (Map) strain Telford: Kinetics of the immune response, histopathology and Map culture.

A long-term study was undertaken to monitor immune responses, faecal cultures and clinical disease in sheep experimentally infected with Mycobacterium avium subspecies paratuberculosis (Map) strain Telford. New Zealand Merino lambs (N=56) were challenged with three oral doses of Map suspension. The lambs were weighed and faecal and blood samples obtained at different time-points. At 63 weeks post-challenge, surviving sheep were euthanised and samples of liver, ileo-caecal valve and mesenteric lymph node were collected for histopathology and Map culture. High IFN-γ and antibody responses were evident as early as 8 weeks post-C1 which persisted until the end of the trial. Approximately 92% of the sheep shed Map in faeces at 36 weeks post-challenge, with the prevalence decreasing to around 40% at the end of the trial. Thirteen sheep progressively lost weight and were euthanised between weeks 32 and 58 post-challenge. Nearly 58% of surviving sheep exhibited histo-pathological lesions in at least one of the three tissues sampled, while 42% showed acid-fast bacilli in at least one tissue. A positive Map culture in at least one tissue was obtained from approximately 85% of sheep. These results indicate that the three doses of Map challenge were highly effective in establishing Johne's disease in NZ Merino lambs.

Experimental Infection Models of Tuberculosis in Domestic Livestock.

In this article we present experimental Mycobacterium bovis infection models in domestic livestock species and how these models were applied to vaccine development, biomarker discovery, and the definition of specific antigens for the differential diagnosis of infected and vaccinated animals. In particular, we highlight synergies between human and bovine tuberculosis (TB) research approaches and data and propose that the application of bovine TB models could make a valuable contribution to human TB vaccine research and that close alignment of both research programs in a one health philosophy will lead to mutual and substantial benefits.

Vaccination of cattle with a high dose of BCG vaccine 3 weeks after experimental infection with Mycobacterium bovis increased the inflammatory response, but not tuberculous pathology.

A study was undertaken to determine whether BCG vaccination of cattle post-challenge could have an effect on a very early Mycobacterium bovis infection. Three groups of calves (n = 12/group) were challenged endobronchially with M. bovis and slaughtered 13 weeks later to examine for tuberculous lesions. One group had been vaccinated prophylactically with BCG Danish vaccine 21 weeks prior to challenge; a second group was vaccinated with a 4-fold higher dose of BCG Danish 3 weeks post-challenge and the third group, remained non-vaccinated. Vaccination prior to challenge induced only minimal protection with just a significant reduction in the lymph node lesion scores. Compared to the non-vaccinated group, BCG vaccination post-challenge produced no reduction in gross pathology and histopathology, but did result in significant increases in mRNA expression of pro-inflammatory mediators (IFN-γ, IL-12p40, IL-17A, IRF-5, CXCL9, CXCL10, iNOs, and TNF-α) in the pulmonary lymph nodes. Although there was no significant differences in the gross pathology and histopathology between the post-challenge BCG and non-vaccinated groups, the enhanced pro-inflammatory immune responses observed in the post-challenge BCG group suggest caution in the use of high doses of BCG where there is a possibility that cattle may be infected with M. bovis prior to vaccination.

Vaccination of Sheep with a Methanogen Protein Provides Insight into Levels of Antibody in Saliva Needed to Target Ruminal Methanogens.

Methane is produced in the rumen of ruminant livestock by methanogens and is a major contributor to agricultural greenhouse gases. Vaccination against ruminal methanogens could reduce methane emissions by inducing antibodies in saliva which enter the rumen and impair ability of methanogens to produce methane. Presently, it is not known if vaccination can induce sufficient amounts of antibody in the saliva to target methanogen populations in the rumen and little is known about how long antibody in the rumen remains active. In the current study, sheep were vaccinated twice at a 3-week interval with a model methanogen antigen, recombinant glycosyl transferase protein (rGT2) formulated with one of four adjuvants: saponin, Montanide ISA61, a chitosan thermogel, or a lipid nanoparticle/cationic liposome adjuvant (n = 6/formulation). A control group of sheep (n = 6) was not vaccinated. The highest antigen-specific IgA and IgG responses in both saliva and serum were observed with Montanide ISA61, which promoted levels of salivary antibodies that were five-fold higher than the second most potent adjuvant, saponin. A rGT2-specific IgG standard was used to determine the level of rGT2-specific IgG in serum and saliva. Vaccination with GT2/Montanide ISA61 produced a peak antibody concentration of 7 × 1016 molecules of antigen-specific IgG per litre of saliva, and it was estimated that in the rumen there would be more than 104 molecules of antigen-specific IgG for each methanogen cell. Both IgG and IgA in saliva were shown to be relatively stable in the rumen. Salivary antibody exposed for 1-2 hours to an in vitro simulated rumen environment retained approximately 50% of antigen-binding activity. Collectively, the results from measuring antibody levels and stablility suggest a vaccination-based mitigation strategy for livestock generated methane is in theory feasible.