High-Specificity Test Algorithm for Bovine Tuberculosis Diagnosis in African Buffalo (Syncerus caffer) Herds

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

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

Bovine tuberculosis (bTB) is a worldwide zoonosis that affects many species of domestic and wild animals. Mycobaterium bovis is the main cause of infection in water buffalo (Bubalus bubalis) and bovines and is of great concern for human health and for buffalo producers in Italy. The bTB eradication programme is based on slaughterhouse surveillance and intradermal skin tests. Other in vivo diagnostic methods such as the interferon-gamma (IFN-γ) assay have been developed and are widely used in cattle to accelerate the elimination of bTB positive animals. The present study is the first to assess the use and performance of IFN-γ assays, which is used as an ancillary test for bTB diagnosis in water buffalo, and presents the results of a field-evaluation of the assay from 2012 to 2019 during the buffalo bTB eradication programme in Italy. The study involved 489 buffaloes with a positive result to the single intradermal tuberculin test (SITT). The IFN-γ assays and single intradermal comparative tuberculin test were used as confirmation tests. Then, a total of 458 buffaloes, reared on officially tuberculosis-free (OTF) herds, that were confirmed bTB-free for at least the last 6 years were subjected to IFN-γ testing. Furthermore, to evaluate the IFN-γ test in an OTF herd with Paratuberculosis (PTB) infection, 103 buffaloes were subjected to SITT and IFN-γ test simultaneously. Four interpretative criteria were used, and the IFN-γ test showed high levels of accuracy, with sensitivity levels between 75.3% (CI 95% 71.2–79.0%) and 98.4% (CI 95% 96.7–99.4%) and specificity levels between 94.3% (CI 95% 91.2–96.50%) and 98.5% (CI 95% 96.9–99.4%), depending on the criterion used. Finally, in the OTF herd with PTB infection, in buffalo, the IFN-γ test displayed high specificity values according to all 4 interpretative criteria, with specificity levels between 96.7% (CI 95% 88.4–99.5%) and 100% (CI 95% 96.2–100%), while SITT specificity proved unsatisfactory, with a level of 45.3% (CI 95% 35.0–55.7%). Our results showed that the IFN-γ test in the buffalo species could reach high Sensitivity and Specificity values, and that the level of Sensitivity and Specificity could be chosen based on the interpretative criterion and the antigens used depending on the health status of the herd and the epidemiological context of the territory. The IFN-γ test and the use of different interpretative criteria proved to be useful to implement bTB diagnostic strategies in buffalo herds, with the possibility of a flexible use of the assay.

Bovine tuberculosis (bTB) is prevalent in intensive dairy farms in Ethiopia. Vaccination could be an alternative control approach given the socio-economic challenges of a test-and-slaughter control strategy. The efficacy of the BCG was evaluated on 40 Holstein-Friesian (HF) and zebu crossbred calves recruited from single intradermal cervical comparative tuberculin (SICCT) test negative herds and randomly allocated into two groups. Twenty-two calves were vaccinated within 2 weeks of age, and 18 were kept as a control. Six weeks post-vaccination, the two groups were exposed and kept mixed with known SICCT test positive cows for 1 year. Immune responses were monitored by interferon gamma (IFN-γ) release assay (IGRA), SICCT test, and antibody assay. Vaccinated calves developed strong responses to the SICCT test at the sixth week post-vaccination, but did not respond to ESAT-6/CFP-10 peptide antigen-based IGRA. During the exposure, IFN-γ response to the specific peptide cocktail [F(2.44, 92.67) = 26.96; p < 0.001] and skin reaction to the specific proteins cocktail [F(1.7, 64.3); p < 0.001] increased progressively in both groups while their antibody responses were low. The prevalence of bTB was 88.9% (95% CI: 65.3–98.6) and 63.6% (95% CI: 40.7–83.8) in the control and vaccinated calves, respectively, based on Mycobacterium bovis isolation, giving a direct protective efficacy estimate of 28.4% (95% CI: −2.7 to 50.1). The proportion of vaccinated calves with lesion was 7.0% (34/484) against 11.4% (45/396) in control calves, representing a 38% (95% CI: 5.8–59.4) reduction of lesion prevalence. Besides, the severity of pathology was significantly lower (Mann–Whitney U-test, p < 0.05) in vaccinated (median score = 2.0, IQR = 0–4.75) than in control (median score = 5, IQR = 3.0–6.25) calves. Moreover, survival from M. bovis infection in vaccinated calves was significantly (log-rank test: χ2 = 6.749, p < 0.01) higher than that of the control calves. In conclusion, the efficacy of BCG was low, but the reduced frequency and severity of lesion in vaccinated calves could suggest its potential role in containing onward transmission.

Bovine tuberculosis (bTB) is a disease of significant economic and zoonotic importance, therefore, optimising tests for the identification of Mycobacterium bovis infected cattle is essential. The Interferon Gamma (IFN-γ) Release Assay (IGRA) can diagnose M. bovis infected cattle at an early stage, is easy to perform and can be used alongside skin tests for confirmatory purposes or to increase diagnostic sensitivity. It is known that IGRA performance is sensitive to environmental conditions under which samples are taken and transported. In this study, the association between the ambient temperature on the day of bleeding and the subsequent IGRA result for bTB was quantified using field samples from Northern Ireland (NI). Results of 106,434 IGRA results (2013–2018) were associated with temperature data extracted from weather stations near tested cattle herds. Model dependent variables were the levels of IFN-γ triggered by avian purified protein derivative (PPDa), M. bovis PPD (PPDb), their difference (PPD(b-a)) as well as the final binary outcome (positive or negative for M. bovis infection). IFN-γ levels after both PPDa and PPDb stimulation were lowest at the extremes of the temperature distribution for NI. The highest IGRA positive probability (above 6%) was found on days with moderate maximum temperatures (6–16 °C) or moderate minimum temperatures (4–7 °C). Adjustment for covariates did not lead to major changes in the model estimates. These data suggest that IGRA performance can be affected when samples are taken at high or low temperatures. Whilst it is difficult to exclude physiological factors, the data nonetheless supports the temperature control of samples from bleeding through to laboratory to help mitigate post-collection confounders.

In South Africa, the largest proportion of the African wild dog (Lycaon pictus) population resides in regions where buffaloes have a high prevalence of Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB). Recent reports of deaths of wild dogs associated with bTB have raised concerns regarding the threat this disease might pose for this species. In order to understand the potential impact of the disease in wild dogs, diagnostic tools are required to identify infected individuals. The interferon gamma (IFN-γ) release assay (IGRA) is commonly used for tuberculosis (TB) screening of humans, cattle, and other species, and the aim of this study was to develop an IGRA for wild dogs to detect immune sensitization. Blood was collected from immobilized wild dogs from the Ann van Dyk Cheetah Centre (AvDCC; n=9) and Kruger National Park (KNP; n=31). Heparinized whole blood was incubated overnight in QuantiFERON®-TB Gold (QFT) blood collection tubes and with selected mitogens, after which the plasma fraction was harvested. Three canine IFN-γ enzymelinked immunosorbent assays (ELISAs) were compared for detection of wild dog IFN-γ in plasma and the R&D Quantikine canine IFN-γ ELISA was selected for measurement of M. bovis-specific IFN-γ release in plasma samples. An IGRA result was calculated as the concentration in plasma derived from the QFT TB Antigen tubes minus that in the QFT Nil tube. An IGRA cut-off value was calculated using the IGRA results of M. bovis-unexposed individuals from AvDCC. Using this cut-off value, 74% (23/31) of M. bovis-exposed KNP wild dogs were IGRA positive, indicating immune sensitization to TB antigens in these animals. Three M. bovis culture-positive wild dogs from KNP had IFN-γ concentrations between 758 and 1,445 pg/mL, supporting this interpretation. This warrants further investigation into the prevalence of M. bovis infection in the KNP population.

Field evaluation of the tuberculin skin test for the detection of Mycobacterium tuberculosis complex infection in communal goats (Capra hircus) in KwaZulu-Natal, South Africa

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)

Mycobacterium bovis (M. bovis), which causes bovine tuberculosis, is endemic in Canada's Wood Buffalo National Park and threatens the conservation of free-ranging bison. It also poses spillover risks to humans and livestock. Accurate diagnostic tools are critical for screening this threatened and culturally significant animal. Interferon gamma (IFNγ) release assays (IGRA) are commonly used for tuberculosis diagnosis in humans and other wildlife species. Hence, this study aimed to develop an IGRA-based approach for detecting M. bovis infection in bison. Animals from a tuberculosis-free captive bison farm (n = 8) were experimentally infected with M. bovis. The whole blood was collected prior to and following M. bovis challenge and incubated overnight in QuantiFERON®-TB Gold (QFT) blood collection tubes and pokeweed mitogen tubes. Plasma was harvested after centrifugation. Concentrations of IFNγ in plasma were quantified using the Mabtech bovine IFNγ ELISA Flex kit. IGRA responses were calculated as the difference in IFNγ concentrations between TB antigen and the nil tubes. A diagnostic cut-off value of 59 pg/ml (Se = 81 %, 95 % CI 57-93 %; Sp =100 %, 95 % CI 89-100 %; AUC = 0.92) was determined using known infection status to distinguish infected bison. Additionally, cut-off values for IFNγ concentrations for plasma from QFT-nil and pokeweed mitogen tubes were calculated to increase confidence in IGRA validity interpretation. The combination of the QFT stimulation platform and Mabtech bovine IFNγ ELISA shows promise as a diagnostic test to distinguish between M. bovis-infected and uninfected bison. These findings support the use of this tool for surveillance in free-ranging and captive Canadian bison populations and warrant further field validation.

An interferon-gamma release assay for the diagnosis of the Mycobacterium bovis infection in white rhinoceros (Ceratotherium simum)

Biomarkers of cell-mediated immunity to bovine tuberculosis

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 prevalence of bovine tuberculosis (BTB) infection in cattle was investigated in extensive and intensive production systems in three districts of northwestern Ethiopia. Single comparative intradermal tuberculin test (SCIDTT) was used in the study. The prevalence of BTB infection as determined by SCIDTT was 9.7% whereas the non-specific infection prevalence was 10.8%. In the extensive system the prevalence was 8.2% and 11.3%; under intensive system the prevalence was 22.1% and 6.3% for BTB and non-specific infections respectively. The prevalence of BTB was significantly higher in the intensive than extensive production systems. Of the 75 herds tested 41 (54.7%) had BTB infections, 68.9% of the BTB positive herds were in the extensive system and 40% of herds in the intensive systems (small dairy farms) had BTB infections.

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

Interference of paratuberculosis with the diagnosis of tuberculosis in a goat flock with a natural mixed infection