Development of cattle TB vaccines based on heterologous prime-boosting strategies

AbstractDevelopment of a TB vaccine for cattle is a research priority in Great Britain. Two challenges need to be addressed. Firstly, vaccine strategies enhancing the efficacy of M. bovis bacille Calmette Guérin (BCG), currently the only potentially available TB vaccine, and secondly the development of a diagnostic test to be used alongside vaccination to differentiate vaccinated and infected animals (DIVA test). Significant progress in developing TB vaccines for cattle has been made over the last 7 years. Specifically: (i) DNA, protein, or viral subunit subunit vaccines used in combination with BCG have been shown to give superior protection against experimental challenge in cattle than BCG (heterologous prime-boost), (ii) neonatal BCG vaccination provides protection, (iii) prototype reagents that allow discrimination between vaccinated and infected animals have been developed; and (iv) and correlates of disease severity have been identified that can predict the success or failure of vaccination. The present overview provides details of some of these advances. [Ethiop.J.Health Dev. 2008;22(Special Issue):100-104

Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR

BACKGROUND: We have evaluated a sensitive screening assay for Mycobacterium tuberculosis (MTB) complex organisms and a specific assay for detecting Mycobacterium bovis DNA in lymph nodes taken from cattle with evidence of bovine tuberculosis. Underlying these series of experiments was the need for a versatile DNA extraction protocol which could handle tissue samples and with the potential for automation. The target for the screening assay was the multi-copy insertion element IS1081, present in 6 copies in the MTB complex. For confirmation of M. bovis we used primers flanking a specific deletion in the genome of M. bovis known as region of difference 4 (RD4). The sensitivity and specificity of these PCRs has been tested on genomic DNA from MTB complex reference strains, mycobacteria other than tuberculosis (MOTT), spiked samples and on clinical material. RESULTS: The minimum detection limits of the IS1081 method was < I genome copy and for the RD4 PCR was 5 genome copies. Both methods can be readily adapted for quantitative PCR with the use of SYBR Green intercalating dye on the RotorGene 3000 platform (Corbett Research). Initial testing of field samples of bovine lymph nodes with visible lesions (VL, n = 109) highlighted two shortfalls of the molecular approach. Firstly, comparison of IS1081 PCR with the "gold standard" of culture showed a sensitivity of approximately 70%. The sensitivity of the RD4 PCR method was 50%. Secondly, the success rate of spoligotyping applied directly to clinical material was 51% compared with cultures. A series of further experiments indicated that the discrepancy between sensitivity of detection found with purified mycobacterial DNA and direct testing of field samples was due to limited mycobacterial DNA recovery from tissue homogenates rather than PCR inhibition. The resilient mycobacterial cell wall, the presence of tissue debris and the paucibacillary nature of some cattle VL tissue may all contribute to this observation. Any of these factors may restrict application of other more discriminant typing methods. A simple means of increasing the efficiency of mycobacterial DNA recovery was assessed using a further pool of 95 cattle VL. Following modification of the extraction protocol, detection rate with the IS1081 and RD4 methods increased to 91% and 59% respectively. CONCLUSION: The IS1081 PCR is a realistic screening method for rapid identification of positive cases but the sensitivity of single copy methods, like RD4 and also of spoligotyping will need to be improved to make these applicable for direct testing of tissue extracts

Development of a BCG challenge model for the testing of vaccine candidates against tuberculosis in cattle

Vaccination is being considered as part of a sustainable strategy for the control of bovine tuberculosis (BTB) in the UK. The live attenuated Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been used experimentally to vaccinate cattle against BTB. However, BCG confers partial protection against BTB and therefore, there is a need to develop improved vaccines. BTB vaccine efficacy experiments require the use of biosafety level 3 facilities which are expensive to maintain, generally oversubscribed and represent a bottle neck for the testing of vaccine candidates. One indicator of the induction of protective responses would be the ability of the host's immune response to control/kill mycobacteria. In this work we have evaluated an intranodal BCG challenge for the selection of vaccine candidates at biosafety level 2 which are capable of inducing mycobactericidal responses. To our knowledge, this is the first such report. Whilst BCG only confers partial protection, it is still the standard against which other vaccines are judged. Therefore we tested the BCG intranodal challenge in BCG (Danish strain) vaccinated cattle and showed that vaccinated cattle had lower BCG cfu counts than naïve cattle at 14 and 21 days after intranodal challenge with BCG (Tokyo strain). This model could help prioritize competing TB vaccine candidates and exploration of primary and secondary immune responses to mycobacteria

Tuberculin skin testing boosts interferon gamma responses to DIVA reagents in Mycobacterium bovis-Infected cattle

ABSTRACT 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. </jats:p

Admixture mapping of tuberculosis and pigmentation-related traits in an African–European hybrid cattle population

Admixture mapping affords a powerful approach to genetic mapping of complex traits and may be particularly suited to investigation in cattle where many breeds and populations are hybrids of the two divergent ancestral genomes, derived from Bos taurus and Bos indicus. Here we design a minimal genome wide SNP panel for tracking ancestry in recent hybrids of Holstein Friesian and local Arsi zebu in a field sample from a region of high bovine tuberculosis endemicity in the central Ethiopian highlands. We first demonstrate the utility of this approach by mapping the red coat color phenotype, uncovering a highly significant peak over the MC1R gene and a second peak with no previously known candidate gene. Secondly, we exploit the described differential susceptibility to bovine tuberculosis between the ancestral strains to identify a region in which Bos taurus ancestry associates, at suggestive significance, with skin test positivity. Interestingly, this association peak contains the toll-like receptor gene cluster on chromosome 6. With this work we have shown the potential of admixture mapping in hybrid domestic animals with divergent ancestral genomes, a recurring condition in domesticated species

Characterization of two in vivo-expressesd methyltransferases of the Mycobacterium tuberculosis complex:Antigenicity and genetic regulation

Genome sequencing of Mycobacterium tuberculosis complex members has accelerated the search for new disease-control tools. Antigen mining is one area that has benefited enormously from access to genome data. As part of an ongoing antigen mining programme, we screened genes that were previously identified by transcriptome analysis as upregulated in response to an in vitro acid shock for their in vivo expression profile and antigenicity. We show that the genes encoding two methyltransferases, Mb1438c/Rv1403c and Mb1440c/Rv1404c, were highly upregulated in a mouse model of infection, and were antigenic in M. bovis-infected cattle. As the genes encoding these antigens were highly upregulated in vivo, we sought to define their genetic regulation. A mutant was constructed that was deleted for their putative regulator, Mb1439/Rv1404; loss of the regulator led to increased expression of the flanking methyltransferases and a defined set of distal genes. This work has therefore generated both applied and fundamental outputs, with the description of novel mycobacterial antigens that can now be moved into field trials, but also with the description of a regulatory network that is responsive to both in vivo and in vitro stimuli

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

Funder: The Defense Science and Technology LaboratoryFunder: Medical Research Council; Id: http://dx.doi.org/10.13039/501100000265Funder: Economic and Social Research Council; Id: http://dx.doi.org/10.13039/501100000269Funder: Department for International Development, UK Government; Id: http://dx.doi.org/10.13039/501100000278Funder: Biotechnology and Biological Sciences Research Council; Id: http://dx.doi.org/10.13039/501100000268Bovine 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

Inferring Mycobacterium bovis transmission between cattle and badgers using isolates from the Randomised Badger Culling Trial.

Mycobacterium bovis (M. bovis) is a causative agent of bovine tuberculosis, a significant source of morbidity and mortality in the global cattle industry. The Randomised Badger Culling Trial was a field experiment carried out between 1998 and 2005 in the South West of England. As part of this trial, M. bovis isolates were collected from contemporaneous and overlapping populations of badgers and cattle within ten defined trial areas. We combined whole genome sequences from 1,442 isolates with location and cattle movement data, identifying transmission clusters and inferred rates and routes of transmission of M. bovis. Most trial areas contained a single transmission cluster that had been established shortly before sampling, often contemporaneous with the expansion of bovine tuberculosis in the 1980s. The estimated rate of transmission from badger to cattle was approximately two times higher than from cattle to badger, and the rate of within-species transmission considerably exceeded these for both species. We identified long distance transmission events linked to cattle movement, recurrence of herd breakdown by infection within the same transmission clusters and superspreader events driven by cattle but not badgers. Overall, our data suggests that the transmission clusters in different parts of South West England that are still evident today were established by long-distance seeding events involving cattle movement, not by recrudescence from a long-established wildlife reservoir. Clusters are maintained primarily by within-species transmission, with less frequent spill-over both from badger to cattle and cattle to badger