An ELISA Using Synthetic Mycolic Acid-Based Antigens with DIVA Potential for Diagnosing Johne's Disease in Cattle.

THE PROBLEM: Ante-mortem diagnosis of Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is normally achieved through faecal culture, PCR, or serological tests, but agreement as to which samples are positive for Johne's disease is often poor and sensitivities are low, particularly in early-stage infections. The potential solution: Mycobacterial cells contain very complex characteristic mixtures of mycolic acid derivatives that elicit antibodies during infection; this has been used to detect infections in humans. Here, we explore its application in providing an assay differentiating infected from vaccinated animals (DIVA assay) for Johne's disease in cattle. METHOD: Antibody responses to different classes of mycolic acid derivatives were measured using ELISA for serum from cattle positive for MAP by both faecal PCR and commercial serum ELISA, or just by PCR, and from animals from herds with no history of Johne's disease, bovine tuberculosis reactors, BCG-vaccinated, BCG-vaccinated and M. bovis-infected, and Gudair-vaccinated animals. RESULTS: The best-performing antigens, ZAM295 and ST123-the latter a molecule present in the cells of MAP but not of Mycobacterium bovis-achieved a sensitivity of 75% and 62.5%, respectively, for serum from animals positive by both faecal PCR and a commercial MAP serum ELISA, at a specificity of 94% compared to 80 no-history negatives. Combining the results of separate assays with two antigens (ST123 and JRRR121) increased the sensitivity/specificity to 75/97.5%. At the same cut-offs, animals vaccinated with Gudair or BCG vaccines and bTB reactors showed a similar specificity. The specificity in BCG-vaccinated but M. bovis-infected animals dropped to 85%. Combining the results of two antigens gave a sensitivity/specificity of 37.5/97.5% for the full set of 80 PCR-positive samples, detecting 30 positives compared 16 for IDEXX. CONCLUSION: Serum ELISA using synthetic lipids distinguishes effectively between MAP-negative cattle samples and those positive by both PCR and a commercial MAP serodiagnostic, without interference by Gudair or BCG vaccination. It identified almost twice as many PCR positives as the commercial serodiagnostic, offering the possibility of earlier detection of infection.

Temporal dynamics of the early immune response following Mycobacterium bovis infection of cattle.

Bovine tuberculosis is an infectious disease of global significance that remains endemic in many countries. Mycobacterium bovis infection in cattle is characterized by a cell-mediated immune response (CMI) that precedes humoral responses, however the timing and trajectories of CMI and antibody responses determined by newer generation assays remain undefined. Here we used defined-antigen interferon-gamma release assays (IGRA) and an eleven-antigen multiplex ELISA (Enferplex TB test) alongside traditional tuberculin-based IGRA and IDEXX M. bovis antibody tests to assess immune trajectories following experimental M. bovis infection of cattle. The results show CMI responses developed as early as two-weeks post-infection, with all infected cattle testing positive three weeks post-infection. Interestingly, 6 of 8 infected animals were serologically positive with the Enferplex TB assay as early as 4 weeks post-infection. As expected, application of the tuberculin skin test enhanced subsequent serological reactivity. Infrequent M. bovis faecal shedding was observed but was uncorrelated with observed immune trajectories. Together, the results show that early antibody responses to M. bovis infection are detectable in some individuals and highlight an urgent need to identify biomarkers that better predict infection outcomes, particularly for application in low-and-middle income countries where test-and-slaughter based control methods are largely unfeasible.

Vaccination of calves with Bacillus Calmette-Guerin Danish strain 1331 results in a duration of immunity of at least 52 weeks.

Bacillus Calmette-Guérin (BCG) Danish strain 1331 (CattleBCG) is currently the lead vaccine candidate for the control of bovine tuberculosis (TB) in cattle in GB, where prior vaccination has shown to result in a significant reduction in bovine TB pathology induced by infection with Mycobacterium bovis (M. bovis). A critical knowledge gap in our understanding of CattleBCG is the duration of immunity post vaccination at the minimum intended vaccine dose. To this end, we performed an experiment where calves were vaccinated with a targeted dose of 106 CFU and, after a period of 52 weeks, experimentally infected with M. bovis. Post mortem examination performed 13 weeks after infection revealed a statistically significant reduction in the severity of TB pathology in the CattleBCG vaccinated group compared with the unvaccinated control group. Additionally, this study allowed us to further assess the diagnostic performance of a defined antigen DIVA reagent (DST-F) developed to detect infected amongst vaccinated animals. Our results demonstrate that when used in a skin test format, DST-F showed high specificity (100 %) in BCG-vaccinated animals when tested prior to infection, whilst detecting all infected animals when re-tested after infection. Furthermore, we also present results supporting the use of the DST-F reagent in an interferon-gamma release assay. In conclusion, the results of this study demonstrate a 52-week duration of immunity following administration of a minimum dose of CattleBCG. This evidence will be a fundamental component in our efforts to apply for UK marketing authorisation to enable vaccination of cattle as a significant additional control measure in the ongoing fight against bovine TB in GB.

Differences in skin test reactions to official and defined antigens in guinea pigs exposed to non-tuberculous and tuberculous bacteria.

The single and comparative intradermal tuberculin tests (SITT and CITT) are official in vivo tests for bovine tuberculosis (TB) diagnosis using bovine and avian purified protein derivatives (PPD-B and PPD-A). Infection with bacteria other than Mycobacterium tuberculosis complex (MTC) can result in nonspecific reactions to these tests. We evaluated the performance of the skin test with PPDs and new defined antigens in the guinea pig model. A standard dose (SD) of Rhodococcus equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis, M. avium subsp. avium, M. avium subsp. hominissuis, M. scrofulaceum, M. persicum, M. microti, M. caprae and M. bovis, and a higher dose (HD) of M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis were tested using PPD-B, PPD-A, P22, ESAT-6-CFP-10-Rv3615c peptide cocktail long (PCL) and fusion protein (FP). The SD of R. equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare and M. avium subsp. paratuberculosis did not cause any reactions. The HD of M. nonchromogenicum, M. monacense, M. intracellulare, and M. avium subsp. paratuberculosis and the SD of M. avium subsp. hominissuis, M. scrofulaceum and M. persicum, caused nonspecific reactions (SIT). A CITT interpretation would have considered M. avium complex and M. scrofulaceum groups negative, but not all individuals from M. nonchromogenicum HD, M. monacense HD and M. persicum SD groups. Only animals exposed to M. bovis and M. caprae reacted to PCL and FP. These results support the advantage of complementing or replacing PPD-B to improve specificity without losing sensitivity.

A digitally enhanced home-based physical activity intervention for high-risk middle school youth during COVID-19.

COVID-19 significantly impacted physical activity among high-risk youth. Camp from Home, a digitally enhanced home-based intervention, was developed to address physical activity disparities among middle school youth during COVID-19. Camp from Home enrolled 62 youth in 54 families from five schools in Philadelphia during the summer of 2020. The 6-week intervention comprised of (1) three home deliveries of "activity kits" including exercise equipment and activity booklets, (2) asynchronous sport and exercise videos posted to a private YouTube channel, and (3) supportive text-messages from health coaches. YouTube analytics and self-report surveys completed by parents and youth at baseline and at the end of programming were used to assess engagement, acceptability, and preliminary efficacy. Youth participants were 12.4 (1.2) years, 38.7% female and 90.3% Black/African American. At follow-up, 41 parents (75.9%) and 34 youth (54.8%) completed measures. Youth self-reported increases in self-efficacy (ΔM(sd) = 0.4(1.0), p = .03) and physical activity (ΔM(sd) = 4.2(7.9), p = .004), despite suboptimal engagement in digital program components. Overall, participants highly rated the program. Activity kits and text-messages from health coaches were rated as most helpful. Most parents (95.1%) and youth (83.8%) expressed interested in participating again in the future. A 6-week digitally enhanced, home-based physical activity intervention was acceptable and feasible among parents and youth during the summer of 2020, with youth reporting improvements in self-efficacy and physical activity. Summer programs are critical for reducing disparities in physical activity and hold potential for addressing key barriers for high-risk youth even outside the context of COVID-19.

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.

Use of the SpO2/FiO2 Diagram to Assess Gas Exchange in Horses Under General Anaesthesia.

The aim of this study was to evaluate the validity of the SpO2/FiO2 diagram in estimating gas exchange in horses under general anaesthesia. In this prospective, controlled clinical study were included 10 horses under general anaesthesia. FiO2 was progressively reduced with the following steps: 0.6, 0.4, 0.3 and 0.21; SpO2 was recorded at each step. An arterial blood sample was collected at the steps of 1.0 and 0.21, to calculate intrapulmonary shunt with the Fshunt formula. The Fshunt value calculated at 0.21 FiO2 was defined as "Fshunt 0.21", the one calculated at 1.0 FiO2 as "Fshunt 1.0". The FiO2 vs SpO2 data points were analyzed using a computer algorithm which uses the haemoglobin and a fixed value for arterial-venous oxygen difference of 3.5 mL/dL. The algorithm estimates a shunt value fitting the obtained data with an ideal SpO2/FiO2 curve. The value of shunt (Sshunt) was considered for the study. Correlation between "Fshunt 1.0", "Fshunt0.21" and SShunt was determined using the Spearman Rank Correlation Coefficient test, the analysis of the regression curve and the coefficient of determination (r2). Values of P < .05 were considered statistically significant. A significant and strong correlation (P = .0069; r = 0.839; r2=0.6194) and a significant and moderate correlation (P = .0443; r = 0.644; r2=0.2336) was found between Sshunt and "Fshunt 1.0" and between Sshunt and "Fshunt 0.21", respectively. The SpO2/FiO2 diagram proved to be a useful and non-invasive tool to characterize gas exchange in horses under general anaesthesia and mechanical ventilation.

Field evaluation of specific mycobacterial protein-based skin test for the differentiation of Mycobacterium bovis-infected and Bacillus Calmette Guerin-vaccinated crossbred cattle in Ethiopia.

Bovine tuberculosis (bTB) challenges intensive dairy production in Ethiopia and implementation of the test and slaughter control strategy is not economically acceptable in the country. Vaccination of cattle with Bacillus Calmette-Guerin (BCG) could be an important adjunct to control, which would require a diagnostic test to differentiate Mycobacterium bovis (M. bovis)-infected and BCG-vaccinated animals (DIVA role). This study describes an evaluation of a DIVA skin test (DST) that is based on a cocktail (DSTc) or fusion (DSTf) of specific (ESAT-6, CFP-10 and Rv3615c) M. bovis proteins in Zebu-Holstein-Friesians crossbred cattle in Ethiopia. The study animals used were 74 calves (35 BCG vaccinated and 39 unvaccinated) aged less than 3 weeks at the start of experiment and 68 naturally infected 'TB reactor' cows. Six weeks after vaccination, the 74 calves were tested with the DSTc and the single intradermal cervical comparative tuberculin (SICCT) test. The TB reactor cows were tested with the DSTc and the SICCT test. Reactions to the DSTc were not observed in BCG-vaccinated and unvaccinated calves, while SICCT test reactions were detected in vaccinated calves. DSTc reactions were detected in 95.6% of the TB reactor cows and single intradermal tuberculin positive reactions were found in 98.2% (95% confidence interval, CI, 92.1-100%). The sensitivity of the DSTc was 95.6% (95% CI, 87.6-99.1%), and significantly (p < .001) higher than the sensitivity (75%, 95% CI, 63.0-84.7%) of the SICCT test at 4 mm cut-off. DSTf and DSTc reactions were correlated (r = 0.75; 95% CI = 0.53-0.88). In conclusion, the DSTc could differentiate M. bovis-infected from BCG-vaccinated cattle in Ethiopia. DST had higher sensitivity than the SICCT test. Hence, the DSTc could be used as a diagnostic tool for bTB if BCG vaccination is implemented for the control of bTB in Ethiopia and other countries.

A molecularly defined skin test reagent for the diagnosis of bovine tuberculosis compatible with vaccination against Johne's Disease.

Tuberculin Purified Protein Derivatives (PPDs) exhibit multiple limitations: they are crude extracts from mycobacterial cultures with largely unknown active components; their production depends on culture of mycobacteria requiring expensive BCL3 production facilities; and their potency depends on the technically demanding guinea pig assay. To overcome these limitations, we developed a molecularly defined tuberculin (MDT) by adding further antigens to our prototype reagent composed of ESAT-6, CFP-10 and Rv3615c (DIVA skin test, DST). In vitro screening using PBMC from infected and uninfected cattle shortlisted four antigens from a literature-based list of 18 to formulate the MDT. These four antigens plus the previously identified Rv3020c protein, produced as recombinant proteins or overlapping synthetic peptides, were formulated together with the three DST antigens into the MDT to test cattle experimentally and naturally infected with M. bovis, uninfected cattle and MAP vaccinated calves. We demonstrated significant increases in MDT-induced skin responses compared to DST in infected animals, whilst maintaining high specificity in unvaccinated or MAP vaccinated calves. Further, MDT can also be applied in in vitro blood-based interferon-gamma release assays. Thus, MDT promises to be a robust diagnostic skin and blood test reagent overcoming some of the limitations of PPDs and warrants full validation.

Genetic screening for the protective antigenic targets of BCG vaccination.

Bovine tuberculosis is an important animal health problem and the predominant cause of zoonotic tuberculosis worldwide. It results in serious economic burden due to losses in productivity and the cost of control programmes. Control could be greatly improved by the introduction of an efficacious cattle vaccine but the most likely candidate, BCG, has several limitations including variable efficacy. Augmentation of BCG with a subunit vaccine booster has been shown to increase protection but the selection of antigens has hitherto been left largely to serendipity. In the present study, we take a rational approach to identify the protective antigens of BCG, selecting a BCG transposon mutant library in naïve and BCG-vaccinated cattle. Ten mutants had increased relative survival in vaccinated compared to naïve cattle, consistent with loss of protective antigen targets making the mutants less visible to the BCG immune response. The immunogenicity of three putative protective antigens, BCG_0116, BCG_0205 (YrbE1B) and BCG_1448 (PPE20) was investigated using peptide pools and PBMCs from BCG vaccinated cattle. BCG vaccination induced PBMC to release elevated levels of IP10, IL-17a and IL-10 in response to all three antigens. Taken together, the data supports the further study of these antigens for use in subunit vaccines.

Biomarkers of cell-mediated immunity to bovine tuberculosis.

Whole blood based assays, particularly interferon gamma (IFN-γ) release assays (IGRAs), are used for the diagnosis of both bovine and human tuberculosis (TB). The aim of the current study was to evaluate a panel of cytokines and chemokines for potential use as diagnostic readouts indicative of Mycobacterium bovis (M. bovis) infection in cattle. A gene expression assay was used to determine the kinetics of the response to M. bovis purified protein derivative and a fusion protein consisting of ESAT-6, CFP10, and Rv3615c upon aerosol infection with ∼104 cfu of M. bovis. The panel of biomarkers included: IFN-γ, CXCL9, CXCL10, CCL2, CCL3, TNF-α, IL-1α, IL-1ß, IL-1Ra, IL-22, IL-21 and IL-13. Protein levels of IFN-γ, CXCL9, and CXCL10 were determined by ELISA. Findings suggest that CXCL9, CXCL10, IL-21, IL-13, and several acute phase cytokines may be worth pursuing as diagnostic biomarkers of M. bovis infection in cattle.

Potential of the dual IFN-γ/IL-2 fluorescence-immunospot assay to distinguish different stages in bovine tuberculosis.

Human studies have identified the potential of measuring Mycobacterium tuberculosis specific IFN-γ and/or IL-2 secreting T cell subsets to distinguish different clinical stages of human tuberculosis (TB). To assess these functional T cell subsets in different states of bovine TB we have established a bovine dual IFN-γ/IL-2 fluorescence-immunospot (FluoroSpot) assay and analysed the frequencies of Mycobacterium bovis (M. bovis) specific IL-2 and/or IFN-γ producing cells in PBMC from 30 cattle naturally infected with M. bovis. Depending on their post mortem results the animals were grouped in 22 cattle with visible lesions (VL) and 8 cattle without visible lesions (NVL). In response to bovine tuberculin purified protein derivative (PPD-B) the frequencies of cytokine producing cells and proportions of IL-2 single producers were significantly higher in VL compared to NVL while PWM-induced cytokine responses were similar between the two groups. Dual IL-2+IFN-γ+ T cells could be identified as the largest PPD-B responsive T cell subset in both cattle groups. In conclusion, our FluoroSpot is a valid method to enumerate individual antigen-specific IFN-γ+ and IL-2+ T cell subsets ex vivo. The greater levels of single IL-2 producing T cells associated with the presence of pathology could be a potential biomarker for active TB in cattle.

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.

Tuberculosis Detection in Paratuberculosis Vaccinated Calves: New Alternatives against Interference.

Paratuberculosis vaccination in cattle has been restricted due to its possible interference with the official diagnostic methods used in tuberculosis eradication programs. To overcome this drawback, new possibilities to detect Mycobacterium bovis infected cattle in paratuberculosis vaccinated animals were studied under experimental conditions. Three groups of 5 calves each were included in the experiment: one paratuberculosis vaccinated group, one paratuberculosis vaccinated and M. bovis infected group and one M. bovis infected group. The performance of the IFN-gamma release assay (IGRA) and the skin test using conventional avian and bovine tuberculins (A- and B-PPD) but also other more specific antigens (ESAT-6/CFP10 and Rv3615c) was studied under official and new diagnostic criteria. Regarding the IGRA of vaccinated groups, when A- and B-PPD were used the sensitivity reached 100% at the first post-challenge sampling, dropping down to 40-80% in subsequent samplings. The sensitivity for the specific antigens was 80-100% and the specificity was also improved. After adapting the diagnostic criteria for the conventional antigens in the skin test, the ability to differentiate between M. bovis infected and non-infected animals included in paratuberculosis vaccinated groups was enhanced. Taking for positive a relative skin thickness increase of at least 100%, the single intradermal test specificity and sensitivity yielded 100%. The comparative intradermal test was equally accurate considering a B-PPD relative skin increase of at least 100% and greater than or equal to that produced by A-PPD. Using the specific antigens as a proteic cocktail, the specificity and sensitivity reached 100% considering the new relative and absolute cut-offs in all experimental groups (Δ≥30% and Δmm ≥ 2, respectively). Results suggest that the interference caused by paratuberculosis vaccination in cattle could be completely overcome by applying new approaches to the official tuberculosis diagnostic tests.

Oral vaccination of cattle with heat inactivated Mycobacterium bovis does not compromise bovine TB diagnostic tests.

In this study we investigated whether oral uptake of a heat inactivated M. bovis wildlife vaccine by domestic cattle induced systemic immune responses that compromised the use of tuberculin or defined antigens in diagnostic tests for bovine TB. Positive skin test and blood-based IFN-γ release assay (IGRA) results were observed in all calves vaccinated via the parenteral route (i.e. intramuscular). In contrast, no positive responses to tuberculin or defined antigens were observed in either the skin test or IGRA test when performed in calves vaccinated via the oral route. In conclusion, our results suggest that the heat inactivated M. bovis vaccine could be used to vaccinate wildlife in a baited form in conjunction with the following in cattle: (i) continuation of existing tuberculin skin testing or novel skin test formats based on defined antigens; and (ii) the use of IGRA tests utilizing tuberculin or defined antigens.

Protection associated with a TB vaccine is linked to increased frequency of Ag85A-specific CD4(+) T cells but no increase in avidity for Ag85A.

There is a need to improve the efficacy of Bacille Calmette-Guérin (BCG) vaccination against tuberculosis in humans and cattle. Previously, we found boosting BCG-primed cows with recombinant human type 5 adenovirus expressing antigen 85A (Ad5-85A) increased protection against Mycobacterium bovis infection compared to BCG vaccination alone. The aim of this study was to decipher aspects of the immune response associated with this enhanced protection. We compared BCG-primed Ad5-85A-boosted cattle with BCG-vaccinated cattle. Polyclonal CD4(+) T cell libraries were generated from pre-boost and post-boost peripheral blood mononuclear cells - using a method adapted from Geiger et al. (2009) - and screened for antigen 85A (Ag85A) specificity. Ag85A-specific CD4(+) T cell lines were analysed for their avidity for Ag85A and their Ag85A epitope specificity was defined. Boosting BCG with Ad5-85A increased the frequencies of post-boost Ag85A-specific CD4(+) T cells which correlated with protection (reduced pathology). Boosting Ag85A-specific CD4(+) T cell responses did not increase their avidity. The epitope specificity was variable between animals and we found no clear evidence for a post-boost epitope spreading. In conclusion, the protection associated with boosting BCG with Ad5-85A is linked with increased frequencies of Ag85A-specific CD4(+) T cells without increasing avidity or widening of the Ag85A-specific CD4(+) T cell repertoire.

CD4+ and γδ T Cells are the main Producers of IL-22 and IL-17A in Lymphocytes from Mycobacterium bovis-infected Cattle.

Gene transcription studies have identified dual roles for the cytokines IL-17A and IL-22 in bovine tuberculosis, where they show potential as both predictors of vaccine success and correlates of infection. To allow for a detailed investigation of the cell populations responsible for production of these cytokines, we have utilised a novel bovine IL-22 specific recombinant antibody for flow cytometry. Bovine tuberculin (PPDB) induced greater IL-22 and IL-17A production in Mycobacterium bovis (M. bovis)-infected cattle compared to non-infected controls, while PWM-induced cytokine levels were similar between the two groups. In M. bovis-infected animals, PPDB specific IL-22 and IL-17A responses were observed in both CD4+ T cell and γδ T cell populations. Although both cytokines were detected in both cell types, IL-22/IL-17A double producers were rare and confined mainly to the γδ T cell population. These results support previous gene transcription studies and extend the observation of increased IL-22 and IL-17A responses in M. bovis-infected animals to the level of protein production. We were also able to characterise the cell populations responsible for these disease-related cytokine responses. The data generated can be used to further our understanding of the immunopathology of bovine tuberculosis and to produce more sensitive and specific immune-diagnostic reagents.

Bovine Tuberculosis in Cattle: Vaccines, DIVA Tests, and Host Biomarker Discovery.

Bovine tuberculosis remains a major economic and animal welfare concern worldwide. Cattle vaccination is being considered as part of control strategies. This approach, used alongside conventional control policies, also requires the development of vaccine-compatible diagnostic assays to distinguish vaccinated from infected animals (DIVA). We discuss progress made on optimizing the only potentially available vaccine, bacille Calmette Guérin (BCG), and on strategies to improve BCG efficacy. We also describe recent advances in DIVA development based on the detection of host cellular immune responses by blood-testing or skin-testing approaches. Finally, to accelerate vaccine development, definition of host biomarkers that provide meaningful stage-gating criteria to select vaccine candidates for further testing is highly desirable. Some progress has also been made in this area of research, and we summarize studies that defined either markers predicting vaccine success or markers that correlate with disease stage or severity.

Development of immune-diagnostic reagents to diagnose bovine tuberculosis in cattle.

Bovine tuberculosis remains a major economic and animal welfare concern worldwide. As part of control strategies, cattle vaccination is being considered. This approach, used alongside conventional control policies, also requires the development of vaccine compatible diagnostic assays to distinguish infected from vaccinated animals (DIVA). In this review we discuss recent advances in DIVA development based on the detection of host cellular immune responses by blood testing or skin testing approaches.