Transposon libraries identify novel Mycobacterium bovis BCG genes involved in the dynamic interactions required for BCG to persist during in vivo passage in cattle.

BACKGROUND: BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens remains unlicensed. BCG's efficacy relates to its ability to persist in the host for weeks, months or even years after vaccination. It is unclear to what degree this ability to resist the host's immune system is maintained by a dynamic interaction between the vaccine strain and its host as is the case for pathogenic mycobacteria. RESULTS: To investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which a BCG vaccination program has been proposed. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutated genes were identified as affecting in vivo fitness, they included genes encoding known mycobacterial virulence functions such as mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism. Many other attenuating genes had not previously been recognised as having a virulence phenotype. To test these genes, we generated and characterised three knockout mutants that were predicted by transposon mutagenesis to be attenuating in vivo: pyruvate carboxylase, a hypothetical protein (BCG_1063), and a putative cyclopropane-fatty-acyl-phospholipid synthase. The knockout strains survived as well as wild type during in vitro culture and in bovine macrophages, yet demonstrated marked attenuation during passage in bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host. CONCLUSION: These data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host's innate immune system to allow it to persist, a property that is important for its protective efficacy.

Potential for rapid antibody detection to identify tuberculous cattle with non-reactive tuberculin skin test results.

BACKGROUND: Bovine tuberculosis (TB) control programs generally rely on the tuberculin skin test (TST) for ante-mortem detection of Mycobacterium bovis-infected cattle. RESULTS: Present findings demonstrate that a rapid antibody test based on Dual-Path Platform (DPP®) technology, when applied 1-3 weeks after TST, detected 9 of 11 and 34 of 52 TST non-reactive yet M. bovis-infected cattle from the US and GB, respectively. The specificity of the assay ranged from 98.9% (n = 92, US) to 96.0% (n = 50, GB) with samples from TB-free herds. Multi-antigen print immunoassay (MAPIA) revealed the presence of antibodies to multiple antigens of M. bovis in sera from TST non-reactors diagnosed with TB. CONCLUSIONS: Thus, use of serologic assays in series with TST can identify a significant number of TST non-reactive tuberculous cattle for more efficient removal from TB-affected herds.

Global gene transcriptome analysis in vaccinated cattle revealed a dominant role of IL-22 for protection against bovine tuberculosis.

Bovine tuberculosis (bTB) is a chronic disease of cattle caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex group of bacteria. Vaccination of cattle might offer a long-term solution for controlling the disease and priority has been given to the development of a cattle vaccine against bTB. Identification of biomarkers in tuberculosis research remains elusive and the goal is to identify host correlates of protection. We hypothesized that by studying global gene expression we could identify in vitro predictors of protection that could help to facilitate vaccine development. Calves were vaccinated with BCG or with a heterologous BCG prime adenovirally vectored subunit boosting protocol. Protective efficacy was determined after M. bovis challenge. RNA was prepared from PPD-stimulated PBMC prepared from vaccinated-protected, vaccinated-unprotected and unvaccinated control cattle prior to M. bovis challenge and global gene expression determined by RNA-seq. 668 genes were differentially expressed in vaccinated-protected cattle compared with vaccinated-unprotected and unvaccinated control cattle. Cytokine-cytokine receptor interaction was the most significant pathway related to this dataset with IL-22 expression identified as the dominant surrogate of protection besides INF-γ. Finally, the expression of these candidate genes identified by RNA-seq was evaluated by RT-qPCR in an independent set of PBMC samples from BCG vaccinated and unvaccinated calves. This experiment confirmed the importance of IL-22 as predictor of vaccine efficacy.

Bioinformatic and empirical analysis of novel hypoxia-inducible targets of the human antituberculosis T cell response.

We analyzed whole genome-based transcriptional profiles of Mycobacterium tuberculosis subjected to prolonged hypoxia to guide the discovery of novel potential Ags, by a combined bioinformatic and empirical approach. We analyzed the fold induction of the 100 most highly induced genes at 7 d of hypoxia, as well as transcript abundance, peptide-binding prediction (ProPred) adjusted for population-specific MHC class II allele frequency, and by literature search. Twenty-six candidate genes were selected by this bioinformatic approach and evaluated empirically using IFN-γ and IL-2 ELISPOT using immunodominant Ags (Acr-1, CFP-10, ESAT-6) as references. Twenty-three of twenty-six proteins induced an IFN-γ response in PBMCs of persons with active or latent tuberculosis. Five novel immunodominant proteins-Rv1957, Rv1954c, Rv1955, Rv2022c, and Rv1471-were identified that induced responses similar to CFP-10 and ESAT-6 in both magnitude and frequency. IL-2 responses were of lower magnitude than were those of IFN-γ. Only moderate evidence of infection stage-specific recognition of Ags was observed. Reconciliation of bioinformatic and empirical hierarchies of immunodominance revealed that Ags could be predicted, providing transcriptomic data were combined with peptide-binding prediction adjusted by population-specific MHC class II allele frequency.

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.

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.

BCG vaccination reduces bovine tuberculosis transmission, improving prospects for elimination.

Bacillus Calmette-Guérin (BCG) is a routinely used vaccine for protecting children against Mycobacterium tuberculosis that comprises attenuated Mycobacterium bovis. BCG can also be used to protect livestock against M. bovis; however, its effectiveness has not been quantified for this use. We performed a natural transmission experiment to directly estimate the rate of transmission to and from vaccinated and unvaccinated calves over a 1-year exposure period. The results show a higher indirect efficacy of BCG to reduce transmission from vaccinated animals that subsequently become infected [74%; 95% credible interval (CrI): 46 to 98%] compared with direct protection against infection (58%; 95% CrI: 34 to 73%) and an estimated total efficacy of 89% (95% CrI: 74 to 96%). A mechanistic transmission model of bovine tuberculosis (bTB) spread within the Ethiopian dairy sector was developed and showed how the prospects for elimination may be enabled by routine BCG vaccination of cattle.

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.

Differential effects of Mycobacterium bovis--derived polar and apolar lipid fractions on bovine innate immune cells.

Mycobacterial lipids have long been known to modulate the function of a variety of cells of the innate immune system. Here, we report the extraction and characterisation of polar and apolar free lipids from Mycobacterium bovis AF 2122/97 and identify the major lipids present in these fractions. Lipids found included trehalose dimycolate (TDM) and trehalose monomycolate (TMM), the apolar phthiocerol dimycocersates (PDIMs), triacyl glycerol (TAG), pentacyl trehalose (PAT), phenolic glycolipid (PGL), and mono-mycolyl glycerol (MMG). Polar lipids identified included glucose monomycolate (GMM), diphosphatidyl glycerol (DPG), phenylethanolamine (PE) and a range of mono- and di-acylated phosphatidyl inositol mannosides (PIMs). These lipid fractions are capable of altering the cytokine profile produced by fresh and cultured bovine monocytes as well as monocyte derived dendritic cells. Significant increases in the production of IL-10, IL-12, MIP-1ß, TNFα and IL-6 were seen after exposure of antigen presenting cells to the polar lipid fraction. Phenotypic characterisation of the cells was performed by flow cytometry and significant decreases in the expression of MHCII, CD86 and CD1b were found after exposure to the polar lipid fraction. Polar lipids also significantly increased the levels of CD40 expressed by monocytes and cultured monocytes but no effect was seen on the constitutively high expression of CD40 on MDDC or on the levels of CD80 expressed by any of the cells. Finally, the capacity of polar fraction treated cells to stimulate alloreactive lymphocytes was assessed. Significant reduction in proliferative activity was seen after stimulation of PBMC by polar fraction treated cultured monocytes whilst no effect was seen after lipid treatment of MDDC. These data demonstrate that pathogenic mycobacterial polar lipids may significantly hamper the ability of the host APCs to induce an appropriate immune response to an invading pathogen.

Effects of vaccination against paratuberculosis on tuberculosis in goats: diagnostic interferences and cross-protection.

BACKGROUND: Most countries carrying out campaigns of bovine tuberculosis (TB) eradication impose a ban on the use of mycobacterial vaccines in cattle. However, vaccination against paratuberculosis (PTB) in goats is often allowed even when its effect on TB diagnosis has not been fully evaluated. To address this issue, goat kids previously vaccinated against PTB were experimentally infected with TB. RESULTS: Evaluation of interferon-γ (IFN-γ) secretion induced by avian and bovine tuberculins (PPD) showed a predominant avian PPD-biased response in the vaccinated group from week 4 post-vaccination onward. Although 60% of the animals were bovine reactors at week 14, avian PPD-biased responses returned at week 16. After challenge with M. caprae, the IFN-γ responses radically changed to show predominant bovine PPD-biased responses from week 18 onward. In addition, cross-reactions with bovine PPD that had been observed in the vaccinated group at week 14 were reduced when using the M. tuberculosis complex-specific antigens ESAT-6/CFP-10 and Rv3615c as new DIVA (differentiation of infected and vaccinated animals) reagents, which further maintained sensitivity post-challenge. Ninety percent of the animals reacted positively to the tuberculin cervical comparative intradermal test performed at 12 weeks post-infection. Furthermore, post-mortem analysis showed reductions in tuberculous lesions and bacterial burden in some vaccinated animals, particularly expressed in terms of the degree of extrapulmonary dissemination of TB infection. CONCLUSIONS: Our results suggest a degree of interference of PTB vaccination with current TB diagnostics that can be fully mitigated when using new DIVA reagents. A partial protective effect associated with vaccination was also observed in some vaccinated animals.

The safety of BCG vaccination in cattle: results from good laboratory practice safety studies in calves and lactating cows.

Bovine tuberculosis (bTB) is a global disease of livestock that has damaging economic, animal health and public health consequences. Conventional bTB disease control strategies, based around the testing and slaughter of cattle infected with bTB, are typically used to help limit or reduce the transmission of this disease but in many low- and middle-income countries such strategies may often be economically unviable, culturally unacceptable or logistically impracticable. The use of vaccination to protect cattle against bTB could provide a potentially more affordable, ethically acceptable and practical additional disease control measure. The protective efficacy of the commercially produced and readily available human vaccine against tuberculosis (Mycobacterium bovis Bacille Calmette-Guérin; BCG) in cattle has been demonstrated in many experimental laboratory and field studies. However, Good Laboratory Practice (GLP) studies assessing the safety of BCG vaccination in cattle have not previously been reported. We describe here the results of two GLP safety studies in which calves and lactating cows were vaccinated with BCG (Danish 1331 strain). From an animal health and welfare perspective, the results of these studies indicate that BCG vaccine is well tolerated in these categories of cattle with only transient and minor local or systemic reactions. Furthermore, there was no evidence that BCG was shed in raw milk, saliva or faeces collected from vaccinates and vaccination did not have a detrimental effect on milk yields in lactating cattle. These data, underpinned by GLP principles, further support the existing data on the safety of BCG vaccine in cattle and complement the abundant available cattle efficacy data for this potential cattle bTB vaccine.

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.

High-resolution transcriptomics of bovine purified protein derivative-stimulated peripheral blood from cattle infected with Mycobacterium bovis across an experimental time course.

OBJECTIVES: Improved bovine tuberculosis (bTB) diagnostics with higher sensitivity and specificity are urgently required. A better understanding of the peripheral blood transcriptional response of Mycobacterium bovis-infected animals after bovine purified protein derivative (PPD-b) stimulation of whole blood-an important component of current bTB diagnostics-will provide new information for development of better diagnostics. METHODS: RNA sequencing (RNA-seq) was used to study the peripheral blood transcriptome after stimulation with PPD-b across four time points (-1 wk pre-infection, and +1 wk, +2 wk, and +10 wk post-infection) from a 14-week M. bovis infection time course experiment with ten age-matched Holstein-Friesian cattle. RESULTS: In vitro PPD-b stimulation of peripheral blood from M. bovis-infected and non-infected cattle elicited a strong transcriptional response. Comparison of PPD-b stimulated, and unstimulated samples revealed higher expression of genes encoding cytokine receptors, transcription factors, and interferon-inducible proteins. Lower expression was seen for genes encoding proteins involved in antimicrobial activity, C-type lectin receptors, inhibition of signal transduction, and genes encoding metal ion transporters. CONCLUSIONS: A transcriptional signature associated with the peripheral blood response to PPD-b stimulation consisting of 170 genes was identified exclusively in the post-infection time points. Therefore, this represents a panel of potential biomarkers of M. bovis infection.

Differential detection of IgM and IgG antibodies to chimeric antigens in bovine tuberculosis.

Recent studies have suggested the potential of innovative serologic tests for accurate and rapid detection of bovine tuberculosis (bTB). Dual Path Platform (DPP) technology has been used to develop rapid animal-side antibody tests for Mycobacterium bovis infection in a range of livestock and wildlife host species. The present study evaluated diagnostic performance of DPP BovidTB IgM/IgG assay designed for differential detection of bovine IgM and IgG antibodies against two chimeric antigens, DID38 and TBf2, respectively, using 662 well-characterized serum samples from M. bovis-infected and bTB-free cattle collected in the United States, Great Britain, France, and South Africa. Test sensitivity and specificity ranged from 71% to 100% and from 95% to 100%, respectively, depending on the country, with overall accuracy of 83%. No significant risk of cross-reactivity with serum samples from cattle infected with most relevant species of mycobacteria other than M. bovis was found. The DPP BovidTB IgM/IgG assay may be suitable for use in multi-test algorithms to improve current strategies for bTB surveillance.

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.

Interferon-γ Response of Mycobacterium avium subsp. paratuberculosis Infected Goats to Recombinant and Synthetic Mycobacterial Antigens.

Despite its potential for early diagnosis of Mycobacterium avium subsp. paratuberculosis (MAP) infection, the IFN-γ release assay is not used routinely, because of low specificity of the established crude antigen preparation Johnin (PPDj). Limited data are available assessing the potential of MAP-derived protein and lipopeptide antigens to replace PPDj in assays for goats, while cattle and sheep have been studied more extensively. Furthermore, MAP infection is claimed to interfere with the diagnosis of bovine tuberculosis when other crude antigen preparations (PPDb, PPDa) are applied. In this study, the diagnostic potential of MAP-derived recombinant protein antigens, synthetic MAP lipopentapeptides and of Mycobacterium bovis-specific peptide cocktails was assessed compared to crude mycobacterial antigen preparations in experimentally infected goats. Goats were inoculated with MAP, or Mycobacterium avium subsp. hominissuis (MAH) as surrogate for environmental mycobacteria, non-exposed animals served as controls. Mycobacterium avium Complex-specific antibody and PPDj-induced IFN-γ responses were monitored in vivo. Infection status was assessed by pathomorphological findings and bacteriological tissue culture at necropsy 1 year after inoculation. The IFN-γ response to 13 recombinant protein antigens of MAP, two synthetic MAP lipopentapeptides and three recombinant peptide cocktails of Mycobacterium bovis was investigated at three defined time points after infection. At necropsy, MAP or MAH infection was confirmed in all inoculated goats, no signs of infection were found in the controls. Antibody formation was first detected 3-6 weeks post infection (wpi) in MAH-inoculated and 11-14 wpi in the MAP-inoculated goats. Maximum PPDj-induced IFN-γ levels in MAH and MAP exposed animals were recorded 3-6 and 23-26 wpi, respectively. Positive responses continued with large individual variation. Antigens Map 0210c, Map 1693c, Map 2020, Map 3651cT(it), and Map 3651c stimulated increased whole blood IFN-γ levels in several MAP-inoculated goats compared to MAH inoculated and control animals. These IFN-γ levels correlated with the intensity of the PPDj-induced responses. The two synthetic lipopentapeptides and the other MAP-derived protein antigens had no discriminatory potential. Stimulation with Mycobacterium bovis peptide cocktails ESAT6-CFP10, Rv3020c, and Rv3615c did not elicit IFN-γ production. Further work is required to investigate if test sensitivity will increase when mixtures of the MAP-derived protein antigens are applied.

Evaluation of the Efficacy of BCG in Protecting Against Contact Challenge With Bovine Tuberculosis in Holstein-Friesian and Zebu Crossbred Calves in Ethiopia.

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.

Novel polyprotein antigens designed for improved serodiagnosis of bovine tuberculosis.

Recent studies have demonstrated potential for serologic assays to improve surveillance and control programs for bovine tuberculosis. Due to the animal-to-animal variation of the individual antibody repertoires observed in bovine tuberculosis, it has been suggested that serodiagnostic sensitivity can be maximized by use of multi-antigen cocktails or genetically engineered polyproteins expressing immunodominant B-cell epitopes. In the present study, we designed three novel multiepitope polyproteins named BID109, TB1f, and TB2f, with each construct representing a unique combination of four full-length peptides of Mycobacterium bovis predominantly recognized in bovine tuberculosis. Functional performance of the fusion antigens was evaluated using multi-antigen print immunoassay (MAPIA) and Dual Path Platform (DPP) technology with panels of monoclonal and polyclonal antibodies generated against individual proteins included in the fusion constructs as well as with serum samples from M. bovis-infected and non-infected cattle, American bison, and domestic pigs. It was shown that epitopes of each individual protein were expressed in the fusion antigens and accessible for efficient binding by the respective antibodies. The three fusion antigens demonstrated stronger immunoreactivity in MAPIA than that of single protein antigens. Evaluation of the fusion antigens in DPP assay using serum samples from 125 M. bovis-infected and 57 non-infected cattle showed the best accuracy (∼84 %) for TB2f antigen composed of MPB70, MPB83, CFP10, and Rv2650c proteins. Thus, the study results suggest a potential for the multiepitope polyproteins to improve diagnostic sensitivity of serologic assays for bovine tuberculosis.

RNA-Seq Transcriptome Analysis of Peripheral Blood From Cattle Infected With Mycobacterium bovis Across an Experimental Time Course.

Bovine tuberculosis, caused by infection with members of the Mycobacterium tuberculosis complex, particularly Mycobacterium bovis, is a major endemic disease affecting cattle populations worldwide, despite the implementation of stringent surveillance and control programs in many countries. The development of high-throughput functional genomics technologies, including RNA sequencing, has enabled detailed analysis of the host transcriptome to M. bovis infection, particularly at the macrophage and peripheral blood level. In the present study, we have analysed the transcriptome of bovine whole peripheral blood samples collected at -1 week pre-infection and +1, +2, +6, +10, and +12 weeks post-infection time points. Differentially expressed genes were catalogued and evaluated at each post-infection time point relative to the -1 week pre-infection time point and used for the identification of putative candidate host transcriptional biomarkers for M. bovis infection. Differentially expressed gene sets were also used for examination of cellular pathways associated with the host response to M. bovis infection, construction of de novo gene interaction networks enriched for host differentially expressed genes, and time-series analyses to identify functionally important groups of genes displaying similar patterns of expression across the infection time course. A notable outcome of these analyses was identification of a 19-gene transcriptional biosignature of infection consisting of genes increased in expression across the time course from +1 week to +12 weeks post-infection.

Screening of predicted secreted antigens from Mycobacterium bovis reveals the immunodominance of the ESAT-6 protein family.

Results of previous studies utilizing bioinformatic approaches in antigen-mining experiments revealed that secreted proteins are among the most frequently recognized antigens from Mycobacterium bovis. Thus, we hypothesized that the analysis of secreted proteins is likely to reveal additional immunogenic antigens that can be used to increase the specificity of diagnostic tests or be suitable vaccination candidates for mycobacterial infections. To test this hypothesis, 382 pools of overlapping peptides spanning 119 M. bovis secreted and potentially secreted proteins were screened for the ability to stimulate a gamma interferon response in vitro using whole blood from tuberculin-positive reactor (TB reactor) cattle. Of the 119 proteins screened, 70 (59%) induced positive responses in the TB reactor animals to various degrees. Strikingly, all but one of the 15 ESAT-6 proteins tested were recognized by at least 30% of the TB reactor animals, with 12 of the 22 most commonly recognized antigens belonging to this protein family. Further analysis of these data demonstrated that there was no significant difference in immunogenicity between the ESAT-6 proteins that were components of potentially intact ESX secretory systems and those corresponding to additional partial esx loci. Importantly for vaccine design, antigenic epitopes in some highly conserved regions shared by numerous ESAT-6 proteins were identified. However, despite this considerable homology, peptide-mapping experiments also revealed that immunodominant peptides were located in regions of amino acid variability.