Serological test performance for bovine tuberculosis in cattle from herds with evidence of on-going infection in Northern Ireland.

The ability to accurately identify infected hosts is the cornerstone of effective disease control and eradication programs. In the case of bovine tuberculosis, accurately identifying infected individual animals has been challenging as all available tests exhibit limited discriminatory ability. Here we assess the utility of two serological tests (IDEXX Mycobacterium bovis Ab test and Enfer multiplex antibody assay) and assess their performance relative to skin test (Single Intradermal Comparative Cervical Tuberculin; SICCT), gamma-interferon (IFNγ) and post-mortem results in a Northern Ireland setting. Furthermore, we describe a case-study where one test was used in conjunction with statutory testing. Serological tests using samples taken prior to SICCT disclosed low proportions of animals as test positive (mean 3% positive), despite the cohort having high proportions with positive SICCT test under standard interpretation (121/921; 13%) or IFNγ (365/922; 40%) results. Furthermore, for animals with a post-mortem record (n = 286), there was a high proportion with TB visible lesions (27%) or with laboratory confirmed infection (25%). As a result, apparent sensitivities within this cohort was very low (≤15%), however the tests succeeded in achieving very high specificities (96-100%). During the case-study, 7/670 (1.04%) samples from SICCT negative animals from a large chronically infected herd were serology positive, with a further 17 animals being borderline positive (17/670; 2.54%). Nine of the borderline animals were voluntarily removed, none of which were found to be infected post-mortem (no lesions/bacteriology negative). One serology test negative animal was subsequently found to have lesions at slaughter with M. bovis confirmed in the laboratory.

Liver fluke (Fasciola hepatica) co-infection with bovine tuberculosis (bTB) in cattle: A retrospective animal-level assessment of bTB risk in dairy and beef cattle.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, remains a persistent problem for cattle industries in endemic countries. The frequency, quality, and performance of tests, and the presence of wildlife reservoirs, have been identified as impediments to eradication. Recently, exposure to helminth infection (Fasciola hepatica) has been associated negatively with the disclosure of bTB. Here, for the first time, we assess impact of concurrent infections of Fasciola hepatica and the disclosure of bTB at the animal-level using large surveillance datasets. We utilized a dataset of 138,566 animal records from an abattoir from Northern Ireland (2011-2013). The presence of F. hepatica infection was assessed from macroscopic tissue inspection at abattoir. Multivariable models were developed to assess co-infection associations with bTB status based on: Single Intradermal Comparative Tuberculin Test (SICTT), lesion, bacteriological confirmation, including either all animals, or only skin-test negative animals (lesions at routine slaughter; LRS; confirmed nonreactors at routine slaughter; cNRs) or positive (reactors) animals alone, respectively. The relationship between skin tuberculin reaction sizes and fluke status was also explored for a subset of animals with field recordings (n = 24,680). Controlling for known risk factors (e.g., climatic, herd, and individual level characteristics), we did not find significant associations between the SICTT (standard or severe interpretation), lesion, nor confirmation status of animals and their liver fluke status. The only exception was a negative association between liver fluke positivity, and LRS or cNRs, respectively; though effect-sizes were small (e.g., LRS Odds-Ratio: 0.87; 95% CI: 0.76-1.00). There was limited evidence of a relationship between tuberculin reaction sizes during SICTT testing and liver fluke infection status. These data do not support the contention that the detection of bTB using skin-tests or reactor postmortem follow-up may be compromised by co-infection at a population level, but the relationship with lesion formation (pathogenesis) may indicate an impact for postmortem surveillance.

Bovine tuberculosis visible lesions in cattle culled during herd breakdowns: the effects of individual characteristics, trade movement and co-infection.

BACKGROUND: Bovine tuberculosis (bTB), caused by Mycobacterium bovis, remains a significant problem for livestock industries in many countries worldwide including Northern Ireland, where a test and slaughter regime has utilised the Single Intradermal Comparative Cervical Tuberculin (SICCT) test since 1959. We investigated the variation in post-mortem confirmation based on bTB visible lesion (VL) presence during herd breakdowns using two model suites. We investigated animal-level characteristics, while controlling for herd-level factors and clustering. We were interested in potential impacts of concurrent infection, and therefore we assessed whether animals with evidence of liver fluke infection (Fasciola hepatica; post-mortem inspection), M. avium reactors (animals with negative M. bovis-avium (b-a) tuberculin reactions) or Bovine Viral Diarrhoea Virus (BVDV; RT-PCR tested) were associated with bTB confirmation. RESULTS: The dataset included 6242 animals removed during the 14 month study period (2013-2015). bTB-VL presence was significantly increased in animals with greater b-a reaction size at the disclosing SICCT test (e.g. b-a = 5-9 mm vs. b-a = 0 mm, adjusted Odds ratio (aOR): 14.57; p < 0.001). M. avium reactor animals (b-a < 0) were also significantly more likely to disclose VL than non-reactor animals (b-a = 0; aOR: 2.29; p = 0.023). Animals had a greater probability of exhibiting lesions with the increasing number of herds it had resided within (movement; log-herds: aOR: 2.27-2.42; p < 0.001), if it had an inconclusive penultimate test result (aOR: 2.84-3.89; p < 0.001), and with increasing time between tests (log-time; aOR: 1.23; p = 0.003). Animals were less likely to have VL if they were a dairy breed (aOR: 0.79; p = 0.015) or in an older age-class (e.g. age-quartile 2 vs. 4; aOR: 0.65; p < 0.001). Liver fluke or BVDV variables were not retained in either multivariable model as they were non-significantly associated with bTB-VL status (p > 0.1). CONCLUSIONS: Our results suggest that neither co-infection of liver fluke nor BVDV had a significant effect on the presence of VLs in this high-risk cohort. M. avium tuberculin reactors had a significantly increased risk of disclosing with a bTB lesion, which could be related to the impact of co-infection with M. avium subsp. paratuberculosis (MAP) affecting the performance of the SICCT however further research in this area is required. Movements, test history, breed and age were important factors influencing confirmation in high-risk animals.

Gamma interferon responses to proteome-determined specific recombinant proteins in cattle experimentally- and naturally-infected with paratuberculosis.

Johne's disease (JD), is a fatal enteritis of animals caused by infection with Mycobacterium avium subspecies paratuberculosis (Map). Diagnosis of subclinical JD is problematic as test sensitivity is limited. Th1 responses to Map are activated early, thus detection of a cell-mediated response, indicated by measuring interferon gamma (IFN-γ) stimulated by mycobacterial antigens, may give the first indication of sub-clinical infection. Crude extracts of Map (PPDJ) have been used to detect the cell-mediated response in infected cattle. More specific, quantifiable antigens may improve test specificity and reproducibility. Map-specific proteins, MAP_3651c and MAP_0268c, raised a cell-mediated immune response in sub-clinically infected sheep. Results presented in this manuscript demonstrate these proteins elicit a cell-mediated response in experimental and natural infections of cattle. Individual ranked IFN-γ responses of experimentally infected calves to PPDJ showed a high, statistically significant association with ranked responses of recombinant Map antigens. Responses of infected animals were higher than the control group. Threshold values determined using data from an experimental infection were applied to naturally infected animals. Some animals exhibited responses above these threshold values. Responses to MAP_3651c on a farm categorised as high-risk for JD showed strong evidence (P<0.001) that responses were significantly different to lower-risk farms. The IGRA test may prove to be an additional tool for the diagnosis of JD, and inclusion of specific antigens a refinement however, understanding and interpretation of IGRA results remain challenging and further investigation will be required to determine whether the IGRA test can detect exposure and hence predict clinical JD.

Liver fluke (Fasciola hepatica) infection in cattle in Northern Ireland: a large-scale epidemiological investigation utilising surveillance data.

BACKGROUND: Liver fluke (Fasciola hepatica) is a widespread parasite of ruminants which can have significant economic impact on cattle production. Fluke infection status at the animal-level is captured during meat inspection of all animals processed for human consumption within Northern Ireland. These national datasets have not been analysed to assess their utility in uncovering patterns in fluke infection at animal- and herd-levels in Northern Ireland. METHODS: We utilised a dataset of 1.2 million animal records from ~18,000 herds across 3 years (2011-2013) to assess animal- and herd-level apparent prevalence and risk-factors associated with fluke infection. Animal-level apparent prevalence was measured as the proportion of animals exhibiting evidence of fluke infection at slaughter; between herd-level infection prevalence was measured by binary categorisation of herds (infected or not). "Within herd" infection prevalence was measured using the proportion of animals within a herd that showed evidence of fluke infection per year (ranging from 0-100%). "Within herd" infection prevalence at the herd level was investigated using multivariable modelling. RESULTS: At the animal level, the proportion of animals slaughtered that exhibited evidence of infection was 21-25% amongst years. Across herds, the proportion of herds with at least one infected animal, varied between 61 and 65%. However, there was a significant sampling effect at the herd-level; all herds where at least 105 animals slaughtered over the study period exhibited evidence of fluke infection (100%). There was significant variation in terms of within-herd infection prevalence. Risk factors included herd type, long-term weather variation, geographic location (region) and the abattoir. CONCLUSIONS: Liver fluke apparent prevalence was high at the herd-level across years. However, there was lower prevalence at the animal level, which may indicate significant variation in the exposure to fluke infection within herds. The proportion infected within-herds varied significantly in time and space, and by abattoir, herd-type and some weather variables. These data are a useful source of information on a widespread endemic disease, despite known limitations in terms of test performance (low sensitivity). As well as informing on the distribution and severity of liver fluke infection, these analyses will be used to investigate the effect of co-infection on risk for bovine tuberculosis.

Should they stay, or should they go? Relative future risk of bovine tuberculosis for interferon-gamma test-positive cattle left on farms.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a serious infectious disease that remains an ongoing concern for cattle farming worldwide. Tuberculin skin-tests are often used to identify infected animals (reactors) during test-and-cull programs, however, due to relatively poor sensitivity, additional tests can be implemented in parallel. For example, in Northern Ireland interferon-gamma (IFN-g) testing is used in high-risk herds. However, skin-test negative animals which are positive to the IFN-g test are not required by law to be slaughtered - therefore the final choice for these animals' fate is left with the owner. During this study we investigated whether these animals represented a greater risk of becoming a skin reactor, relative to IFN-g test negative animals from the same herds. Our study population included 1107 IFN-g positive animals from 239 herds. A Cox-proportional hazard model indicated that animals which tested IFN-g positive were 2.31 times (95% CI: 1.92-2.79; P < 0.001) more likely to become a reactor compared with IFN-g negative animals. Animals from dairy herds, and from herds in the south-east, were of higher risk than animals from beef herds and other regions, respectively. Our findings suggest that IFN-g positive animals represent a higher risk of failing a skin-test in the future, indicating the value of IFN-g testing for identifying early-stage infected animals. These IFN-g positive animals are not under any disease restriction, and may move freely (trade), which may put recipient herds at increased risk. Our findings provide important evidence for stakeholders engaged in bTB eradication programs.

Immunity, safety and protection of an Adenovirus 5 prime--Modified Vaccinia virus Ankara boost subunit vaccine against Mycobacterium avium subspecies paratuberculosis infection in calves.

Vaccination is the most cost effective control measure for Johne's disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) but currently available whole cell killed formulations have limited efficacy and are incompatible with the diagnosis of bovine tuberculosis by tuberculin skin test. We have evaluated the utility of a viral delivery regimen of non-replicative human Adenovirus 5 and Modified Vaccinia virus Ankara recombinant for early entry MAP specific antigens (HAV) to show protection against challenge in a calf model and extensively screened for differential immunological markers associated with protection. We have shown that HAV vaccination was well tolerated, could be detected using a differentiation of infected and vaccinated animals (DIVA) test, showed no cross-reactivity with tuberculin and provided a degree of protection against challenge evidenced by a lack of faecal shedding in vaccinated animals that persisted throughout the 7 month infection period. Calves given HAV vaccination had significant priming and boosting of MAP derived antigen (PPD-J) specific CD4+, CD8+ IFN-γ producing T-cell populations and, upon challenge, developed early specific Th17 related immune responses, enhanced IFN-γ responses and retained a high MAP killing capacity in blood. During later phases post MAP challenge, PPD-J antigen specific IFN-γ and Th17 responses in HAV vaccinated animals corresponded with improvements in peripheral bacteraemia. By contrast a lack of IFN-γ, induction of FoxP3+ T cells and increased IL-1ß and IL-10 secretion were indicative of progressive infection in Sham vaccinated animals. We conclude that HAV vaccination shows excellent promise as a new tool for improving control of MAP infection in cattle.

Ante-mortem diagnosis of caprine tuberculosis in persistently infected herds: influence of lesion type on the sensitivity of diagnostic tests.

Caprine tuberculosis is a major health problem for goats, and an important zoonosis. Eradication programmes using the comparative skin test are being developed to aid in decreasing the prevalence of infection. However, persistent tuberculosis infections are found in herds subjected to eradication programmes. In the present study a commercial IFN-γ release assay and an experimental ELISA based on MPB70, were evaluated as potential ancillary tests to detect infection. The relationship between the three techniques (skin test, IFN-γ release and ELISA) and histopathological lesions was analyzed in 162 goats from herds with persistent tuberculosis infection. The presence of related pathogens (paratuberculosis and pseudotuberculosis) was also studied. The IFN-γ release assay and the ELISA had a higher sensitivity than the comparative skin test (65.3% and 66.3% vs 44.5%) using as a gold standard a combination of histopathological analysis and isolation. Used in combination, ELISA and the skin test detected 89.1% of goats with tuberculosis while a combination of IFN-γ assay and skin test detected 78.2%. The types of macroscopic and microscopic lesions reflected the results of diagnostic tests.

Fasciola hepatica is associated with the failure to detect bovine tuberculosis in dairy cattle.

Bovine tuberculosis (BTB) is a significant and intractable disease of cattle caused by Mycobacterium bovis. In the United Kingdom, despite an aggressive eradication programme, the prevalence of BTB is increasing with an unexplained, exponential rise in cases year on year. Here we show in a study involving 3,026 dairy herds in England and Wales that there is a significant negative association between exposure to the common, ubiquitous helminth parasite, Fasciola hepatica and diagnosis of BTB. The magnitude of the single intradermal comparative cervical tuberculin test used to diagnose BTB is reduced in cattle experimentally co-infected with M. bovis and F. hepatica. We estimate an under-ascertainment rate of about one-third (95% confidence interval 27-38%) among our study farms, in the hypothetical situation of no exposure to F. hepatica. This finding may in part explain the continuing spread of BTB and the failure of the current eradication programme in the United Kingdom.

BCG vaccination against tuberculosis in European badgers (Meles meles): a review.

Tuberculosis (TB) is a significant animal health problem in many parts of the world, and reservoirs of infection in wild animals complicate disease control efforts in farmed livestock, particularly cattle. Badgers (Meles meles) are a significant wildlife reservoir of Mycobacterium bovis infection for cattle in the United Kingdom (UK) and Republic of Ireland (ROI). Vaccination of badgers using an M. bovis strain bacille Calmette-Guérin (BCG) vaccine could potentially be an option in the national TB eradication strategy. Wildlife vaccination has been used successfully for other diseases in wildlife species, and may have a role to play in reducing M. bovis transmission at the wildlife-livestock interface. Research to date has provided evidence that BCG is protective in badgers, and a parenteral badger BCG vaccine has been licensed in the UK. Further research is required to develop effective strategies for vaccine deployment and to determine the effect of badger vaccination on cattle TB incidence.

The Effect of Mycobacterium avium Complex Infections on Routine Mycobacterium bovis Diagnostic Tests.

Bovine tuberculosis (bTB) is diagnosed in naturally infected populations exposed to a wide variety of other pathogens. This study describes the cell-mediated immune responses of cattle exposed to Mycobacterium avium subspecies paratuberculosis (Map) and Mycobacterium avium subspecies avium with particular reference to routine antefmortem Mycobacterium bovis diagnostic tests. The IFN-γ released in response to stimulated blood was found to peak later in the Map-exposed group and was more sustained when compared to the Maa-exposed group. There was a very close correlation between the responses to the purified protein derivatives (PPD) used for stimulation (PPDa, PPDb, and PPDj) with PPDa and PPDj most closely correlated. On occasion, in the Map-infected cattle, PPDb-biased responses were seen compared to PPDa suggesting that some Map-infected cattle could be misclassified as M. bovis infected using this test with these reagents. This bias was not seen when PPDj was used. SICCT results were consistent with the respective infections and all calves would have been classed skin test negative.

A New Experimental Infection Model in Ferrets Based on Aerosolised Mycobacterium bovis.

There is significant interest in developing vaccines to control bovine tuberculosis, especially in wildlife species where this disease continues to persist in reservoir species such as the European Badger (Meles meles). However, gaining access to populations of badgers (protected under UK law) is problematic and not always possible. In this study, a new infection model has been developed in ferrets (Mustela furo), a species which is closely related to the badger. Groups of ferrets were infected using a Madison infection chamber and were examined postmortem for the presence of tuberculous lesions and to provide tissue samples for confirmation of Mycobacterium bovis by culture. An infectious dose was defined, that establishes infection within the lungs and associated lymph nodes with subsequent spread to the mesentery lymph nodes. This model, which emphasises respiratory tract infection, will be used to evaluate vaccines for the control of bovine tuberculosis in wildlife species.

Bovine tuberculosis: effect of the tuberculin skin test on in vitro interferon gamma responses.

Bovine tuberculosis (bTB) is a disease of zoonotic and economic importance. In many countries, control is based on test and slaughter policies and/or abattoir surveillance. For testing, cell mediated immune- (CMI-) based assays (i.e., tuberculin skin test (TST) supplemented by the interferon gamma (IFN-gamma) assay) are the primary surveillance and disease control tests for bTB. The combined use of the in vivo and in vitro CMI assays to increase overall sensitivity has raised the question of whether the IFN-gamma response is influenced by injection of purified protein derivatives (PPDs) for TST. Published data on the influence of the TST, applied as the caudal fold test (CFT) or the comparative cervical test (CCT), on the IFN-gamma assay are contradictory. Reviewing published data and including additional data, the following conclusions can be drawn: (1) in naturally infected cattle, PPD administration for the single or repeated short-interval CCT neither boosts nor depresses PPD-specific IFN-gamma production. Disparate results have been concluded from some studies using experimental infections, emphasizing the importance of confirming initial experimental-based findings with studies using cattle naturally infected with Mycobacterium bovis. (2) In cattle experimentally infected with M. bovis, PPD administration for CFT boosts PPD-specific IFN-gamma production for up to 7 days without any effect on test interpretation. Importantly, in naturally infected cattle, CFT-related boosting selectively increases the in vitroM. bovis PPD (PPD-B) response 3 days after CFT, resulting in an increased PPD-B response relative to the response to Mycobacterium avium PPD (PPD-A). In non-infected cattle, it cannot be excluded that the CFT induces a mild boost of the PPD-specific response, particularly in animals sensitized to environmental, non-tuberculous mycobacteria, thus decreasing the specificity of the IFN-gamma assay. (3) In general, there is a lack of data clearly characterizing the effect of TSTs on the IFN-gamma assay. Further studies are required to clearly describe the effects of both CFT and CCT in non-infected animals and in naturally infected cattle, especially in low reacting infected cattle.

The use of MPB70 and MPB83 to distinguish between bovine tuberculosis and paratuberculosis.

In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p<0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases.

Evaluation of blood assays for detection of Mycobacterium bovis in white-tailed deer (Odocoileus virginianus) in Michigan.

Surveillance and control activities related to bovine tuberculosis (TB) in free-ranging, Michigan white-tailed deer (Odocoileus virginianus) have been underway for over a decade, with significant progress. However, foci of higher TB prevalence on private lands and limited agency ability to eliminate them using broad control strategies have led to development and trial of new control strategies, such as live trapping, testing, and culling or release. Such strategies require a prompt, accurate live animal test, which has thus far been lacking. We report here the ability of seven candidate blood assays to determine the TB infection status of Michigan deer. Our aims were twofold: to characterize the accuracy of the tests using field-collected samples and to evaluate the feasibility of the tests for use in a test-and-cull strategy. Samples were collected from 760 deer obtained via five different surveys conducted between 2004 and 2007. Blood samples were subjected to one or more of the candidate blood assays and evaluated against the results of mycobacterial culture of the cranial lymph nodes. Sensitivities of the tests ranged from 46% to 68%, whereas specificities and negative predictive values were all >92%. Positive predictive values were highly variable. An exploratory analysis of associations among several host and sampling-related factors and the agreement between blood assay and culture results suggested these assays were minimally affected. This study demonstrated the capabilities and limitations of several available blood tests for Mycobacterium bovis on specimens obtained through a variety of field surveillance methods. Although these blood assays cannot replace mass culling, information on their performance may prove useful as wildlife disease managers develop innovative methods of detecting infected animals where mass culling is publicly unacceptable and cannot be used as a control strategy.

Experimental challenge models for Johne's disease: a review and proposed international guidelines.

An international committee of Johne's disease (JD) researchers was convened to develop guidelines for JD challenge studies in multiple animal species. The intent was to develop and propose international standard guidelines for models based on animal species that would gain acceptance worldwide. Parameters essential for the development of long-term and short-term infection models were outlined and harmonized to provide a "best fit" JD challenge model for cattle, goats, sheep, cervids, and mice. These models will be useful to study host-pathogen interactions, host immunity at the local and systemic level, and for evaluating vaccine candidates and therapeutics. The consensus guidelines herein list by animal species strains of Mycobacterium avium subsp. paratuberculosis used, challenge dose, dose frequency, age of challenge, route of challenge, preparation of inoculum, experimental animal selection, quality control, minimal experimental endpoints and other parameters.