Field application of immunoassays for the detection of Mycobacterium bovis infection in the African buffalo (Syncerus caffer)

Application of the Enfer chemiluminescent multiplex ELISA system for the detection of Mycobacterium bovis infection in goats

Parallel testing increases detection of Mycobacterium bovis-infected African buffaloes (Syncerus caffer)

Mycobacterium bovis causes bovine tuberculosis (bTB), a chronic infectious disease with significant veterinary, public health, and economic consequences. The interferon-gamma (IFN-γ) assay is increasingly used alongside the Single Intradermal Comparative Tuberculin Test (SICTT) in Ireland's national bTB eradication programme, but age-specific patterns associated with IFN-γ positivity or post-mortem visible lesion detection (VLD) have not been fully characterised. This retrospective cohort study includes 267,674 SICTT-negative cattle tested with IFN-γ between May 2019 and December 2023 in high-risk Irish herds. Mixed-effects logistic regression models quantify associations between age and (i) IFN-γ positivity and (ii) VLD at slaughter among IFN-γ-positive cattle. Models adjust for sex, herd type, prior inconclusive SICTTs, number of prior 'risky' SICTT tests, and herd-level breakdown size (% of animals positive). Overall, 9.6% of SICTT-negative cattle test positive to IFN-γ. Our findings show that IFN-γ positivity increases with age, peaks in cattle aged 4-6 years, plateaus until 8 years, and declines thereafter. Relative to beef breeding herds, dairy, mixed, and 'other' herd types are associated with higher IFN-γ positivity, as is a history of prior inconclusive SICTTs, and fewer prior 'risky' SICTT exposures. Among IFN-γ-positive cattle, 21.9% exhibit VLD at slaughter. VLD positivity shows a U-shaped relationship with age, highest in the youngest (0-2 years), reducing in cattle aged 2-4, then increasing linearly to oldest (>8 years) cattle. The VLD odds are approximately half in dairy herds compared with beef breeding herds and are elevated in herds in the largest quartile of breakdowns (>6.25% of animals positive). The interpretation of these results should consider that IFN-γ-positivity and VLD likely reflect different stages of bTB infection, with early immune responses detected ante-mortem and visible lesions at post-mortem representing later stage disease; the absence of visible lesions therefore does not exclude M. bovis infection. It appears that age-specific IFN-γ positivity and VLD in high-risk herds are likely shaped by production systems, prior risky SICTT exposures, and herd-level outbreak dynamics rather than simple cumulative risk. The IFN-γ testing helps to identify infected cattle missed by SICTT, particularly in the early infection or large herd breakdowns and serves to support targeted, risk-based deployment to optimize Ireland's bTB eradication programme.

The present study was carried out to investigate the diagnostic potential of gamma interferon (IFN-ã) assay and single intradermal comparative tuberculin test (SICTT), including species specification of bovine tuberculosis infection in different livestock farms of Assam and Meghalaya. A total of 199 animals (cattle and buffalo) were examined for bovine tuberculosis symptoms and swab samples were cultured. Biochemical tests and PCR were used for species specification of bovine tuberculosis. Out of 199 cases examined, 33 (16.58%) showed positive for SICTT, 39 (19.59%) for IFN-ã and 35(17.59%) for PCR. Based on PCR targeting pncA region, the confirmation was done for M. Bovis. IFN-ã thus ensures a sensitive and specific detection of early bovine tuberculosis infection together with SICTT and hence may be considered as a screening method of choice.

Ante-mortem bovine tuberculosis (bTB) tests for buffaloes include the single comparative intradermal tuberculin test (SCITT), interferon-gamma (IFN-γ) release assay (IGRA) and IFN-γ-inducible protein 10 release assay (IPRA). Although parallel test interpretation increases the detection of Mycobacterium bovis (M. bovis)-infected buffaloes, these algorithms may not be suitable for screening buffaloes in historically bTB-free herds. In this study, the specificities of three assays were determined using M. bovis-unexposed herds, historically negative, and a high-specificity diagnostic algorithm was developed. Serial test interpretation (positive on both) using the IGRA and IPRA showed significantly greater specificity (98.3%) than individual (90.4% and 80.9%, respectively) tests or parallel testing (73%). When the SCITT was added, the algorithm had 100% specificity. Since the cytokine assays had imperfect specificity, potential cross-reactivity with nontuberculous mycobacteria (NTM) was investigated. No association was found between NTM presence (in oronasal swab cultures) and positive cytokine assay results. As a proof-of-principle, serial testing was applied to buffaloes (n = 153) in a historically bTB-free herd. Buffaloes positive on a single test (n = 28) were regarded as test-negative. Four buffaloes were positive on IGRA and IPRA, and M. bovis infection was confirmed by culture. These results demonstrate the value of using IGRA and IPRA in series to screen buffalo herds with no previous history of M. bovis infection.

Background and Aim:Bovine tuberculosis (bTB) is an infectious disease of cattle, mainly caused by Mycobacterium bovis. This study aimed to evaluate the efficacy of the interferon-gamma (IFN-γ) assay and single-intradermal comparative tuberculin test (SICTT) in detecting bTB.Materials and Methods:In an earlier study, 150 positive, 83 inconclusive, and 480 negative animals from 24 cattle herds were screened using SICTT. From these groups, 125 positive, 17 inconclusive, and six negative animals were subsequently verified using the IFN-γ assay. Single-intradermal comparative tuberculin test outcomes were interpreted according to standard guidelines, whereas blood samples were collected and stimulated with purified protein derivatives. Sandwich enzyme-linked immunosorbent assay was used to measure secreted IFN-γ. Concordant and Bayesian latent class analyses were performed to evaluate test performance.Results:Results from the IFN-γ assay revealed that 83.2%, 64.7%, and 16.67% of the animals were positive in the SICTT-positive, inconclusive, and negative animal categories, respectively. Sensitivity (SE) and specificity (SP) of SICTT were 83.9% (95% confidence interval [CI]: 77.4–90.1) and 95.7% (95% CI: 86.9–99.7), respectively. Sensitivity and SP for the IFN-γ assay were 78.9% (95% CI: 71.9–85.4) and 83.9% (65.9–95.9), respectively. The use of both tests in parallel increases the SE of bTB detection (~94%), compared with SICTT alone.Conclusion:Use of the IFN-γ assay with SICTT in parallel, predominantly on cattle demonstrating an inconclusive SICTT outcome, boosts bTB detection rate in low resource settings.

Parallel measurement of IFN-γ and IP-10 in QuantiFERON®-TB Gold (QFT) plasma improves the detection of Mycobacterium bovis infection in African buffaloes (Syncerus caffer)

Bovine tuberculosis, a zoonotic disease caused primarily by Mycobacterium bovis, poses a significant threat to cattle health and farming livelihoods within the United Kingdom (UK). Disease control in cattle is complicated by the persistence of M. bovis in European badgers, the UK's principal wildlife reservoir. Accurate diagnostic tools for both species are essential for effective surveillance and disease control. Many existing badger serodiagnostic tests, which include MPB70, MPB83, and ESAT6-CFP10 antigens, have relatively modest sensitivities (~50%-60%), limiting their utility in surveillance. To address this issue, we used an unbiased and comprehensive antigen discovery approach to identify new diagnostic targets. This strategy identified Rv3616c as a novel antigen with promising diagnostic test potential for M. bovis infection in badgers. Overlapping peptides spanning the full Rv3616c amino acid sequence were screened to identify the most diagnostically informative epitopes. A pool of four Rv3616c peptides, used in an indirect enzyme-linked immunosorbent assay (ELISA), had a sensitivity of 85.71% (95% CI: 77.19-91.96), a specificity of 94.80% (95% CI: 90.35-97.59), and a diagnostic accuracy of 91.51% (95% CI: 87.54-94.54). The existing validated Badger M. bovis Ab Test, when used alone, had a sensitivity of 73.47% (95% CI: 63.59-81.88); however, parallel interpretation with the Rv3616c ELISA could increase overall sensitivity to 91.84% (95% CI: 84.55-96.41), with minimal loss of specificity. These findings support the use of Rv3616c-derived peptides in serodiagnostic tests to improve the detection of M. bovis infection in badgers and enhance tuberculosis surveillance in this wildlife reservoir.IMPORTANCEAccurate diagnosis of Mycobacterium bovis infection in wildlife reservoirs is essential for controlling bovine tuberculosis (bTB), a zoonotic disease that threatens human health, animal welfare, and farming livelihoods. In the United Kingdom, European badgers are the principal wildlife reservoir, complicating efforts to eradicate bTB in cattle. Existing serodiagnostic tests for badgers have moderate sensitivity, limiting effectiveness in surveillance. To address this, this study used an unbiased, comprehensive antigen discovery approach and identified several new diagnostic targets, including the Rv3616c protein. A test based on specific Rv3616c-derived peptides had a high diagnostic accuracy (91.51%) and, when used in parallel with a validated test, improved test sensitivity while maintaining specificity. These synthetic peptides are scalable, cost-effective, and adaptable to different diagnostic platforms. The findings reveal an antigen with diagnostic potential that could inform the development of new tests for bTB surveillance in wildlife, supporting One Health principles and global tuberculosis elimination strategies.

The effect of the tuberculin test and the consequences of a delay in blood culture on the sensitivity of a gamma-interferon assay for the detection of Mycobacterium bovis infection in cattle

Modification of the QuantiFERON-TB Gold (In-Tube) assay for the diagnosis of Mycobacterium bovis infection in African buffaloes ( Syncerus caffer)

The most widely used ante-mortem diagnostic tests for tuberculosis in cattle are the tuberculin skin test and the interferon-gamma (IFN-γ) release assay, both of which measure cell-mediated immune responses to Mycobacterium bovis infection. However, limitations in the performance of these tests results in a failure to identify all infected animals. In attempting to increase the range of diagnostic tests for tuberculosis, measurement of the cytokine IP-10 in antigen-stimulated blood has previously been shown to improve the detection of M. tuberculosis and M. bovis infection, in humans and African buffaloes (Syncerus caffer), respectively. In the present study, 60 cattle were identified by the single intradermal comparative tuberculin test as tuberculosis reactors (n = 24) or non-reactors (n = 36) and the release of IFN-γ and IP-10 in antigen-stimulated whole blood from these animals was measured using bovine specific ELISAs. There was a strong correlation between IP-10 and IFN-γ production in these samples. Moreover, measurement of the differential release of IP-10 in response to stimulation with M. bovis purified protein derivative (PPD) and M. avium PPD distinguished between reactor and non-reactor cattle with a sensitivity of 100% (95% CI, 86%–100%) and a specificity of 97% (95% CI, 85%–100%). These results suggest that IP-10 might prove valuable as a diagnostic biomarker of M. bovis infection in cattle.

In Ireland, the interferon-gamma (IFN-γ) assay is routinely used as an ancillary test interpreted in parallel with the single intradermal comparative tuberculin test (SICTT) to maximize the detection of bovine tuberculosis (bTB) infected animals. Up until 2018, a positive test result was recorded in the IFN-γ ELISA assay following whole blood stimulation with purified protein derivative (PPD)-bovine (B), PPD-avian (A) and nil sample (N), using the interpretation criteria, B-N > 50 optical density units (OD), B > 100 and B-A > 0. Following a review of available data, the threshold of the B-A component changed to B-A > 80. As predicting the impact of changing the cut-off thresholds for the IFN-γ test de novo is challenging, the aims of this study were to follow animals that initially tested negative using the new IFN-γ assay interpretation criteria and investigate their future risk of disclosure with bTB, with a focus on animals that otherwise would have been removed when using the older interpretation criteria (0 < B-A ≤ 80). Enrolled animals (n = 28,669 cattle from 527 herds) were followed up for two years (2019–2021), or to point of bTB detection or death. At the end of follow-up, 1151 (4.0%) of enrolled animals were bTB cases. The majority of these cases were diagnosed using SICTT (80.5%). The cumulative number of positive animals that would have been removed if the old cut-off (0 < B-A ≤ 80) was used amounted to 1680 cattle (5.9% of the enrolled cohort). Of these, 127 (7.5%) were diagnosed with bTB during follow-up. In contrast, 1024 of the 1151 cattle which subsequently tested positive during the study period following a negative IFN-γ test would not have been identified with the old or new IFN-γ cut-off criteria. Survival analysis showed that animals that would have been removed under the old interpretation criteria were at increased risk of a positive diagnosis with bTB during follow-up compared to other test negative animals. A newly developed risk prediction model (using a Cox proportional hazard model) showed that age, animal number of SICTT tests, number of inconclusive SICTT tests, B-A (IFN-γ assay), B-N (IFN-γ assay), animals from store herds and the percentage of the rest of the herd that were positive during the breakdown were statistically significantly associated with bTB detection. However, inclusion of the IFN-γ OD variables did not show added value in terms of prediction performance of the model.

BackgroundBovine tuberculosis (bTB) is prevalent in dairy cattle in Ethiopia. Currently used diagnostic tools such as the single intradermal comparative tuberculin test (SICTT) are time consuming and labor intensive. A rapid, easy-to-use and cost-effective diagnostic test would greatly contribute to the control of bTB in developing countries like Ethiopia. In the present study, two point-of-care diagnostic tests were evaluated for the detection of bTB: LIONEX® Animal TB Rapid test, a membrane-based test for the detection of antibodies to Mycobacterium bovis in blood and ALERE® Determine TB Lipoarabinomannan (LAM) Ag, an immunoassay for the detection of lipoarabinomannan (LAM) antigen (Ag) of mycobacteria in urine. A combination of the SICTT and gamma interferon (IFN-γ) test was used as the gold standard for the validation of these point-of-care tests, as it was not feasible to slaughter the study animals to carry out the historical gold standard of mycobacterial culture. A total of 175 heads of cattle having three different bTB infection categories (positive SICTT, negative SICTT, and unknown SICTT status) were used for this study.ResultThe sensitivity and specificity of TB LAM Ag were 72.2% (95% CI = 62.2, 80.4) and 98.8% (95% CI = 93.6, 99.7), respectively, while the sensitivity and specificity of the LIONEX Animal TB rapid test assay were 54% (95% CI = 44.1 64.3) and 98.8% (95% CI = 93.6, 99.7) respectively. The agreement between TB LAM Ag and SICTT was higher (κ = 0.85; 95% CI = 0.65–0.94) than between TB LAM Ag and IFN-γ (κ = 0.67; 95% CI = 0.52–0.81). The agreement between LIONEX Animals TB Rapid blood test and SICTT was substantial, (κ = 0.63; 95% CI = 0.49–0.77) while the agreement between LIONEX Animal TB rapid blood test and IFN-γ test was moderate (κ = 0.53; 95% CI = 0.40–0.67). Analysis of receiver operating curve (ROC) indicated that the area under the ROC curve (AUC) for TB LAM Ag was 0.85 (95% CI = 0.79–0.91) while it was 0.76 (95% CI; =0.69–0.83) for LIONEX Animal TB rapid test assay.ConclusionThis study showed that TB LAM Ag had a better diagnostic performance and could potentially be used as ancillary either to SICTT or IFN-γ test for diagnosis of bTB.

Shorter-term risk of Mycobacterium bovis in Irish cattle following an inconclusive diagnosis to the single intradermal comparative tuberculin test

The comparative performance of the single intradermal test and the single intradermal comparative tuberculin test in Irish cattle, using tuberculin PPD combinations of differing potencies