Detectability of bovine TB using the tuberculin skin test does not vary significantly according to pathogen genotype within Northern Ireland

Strains of many infectious diseases differ in parameters that influence epidemic spread, for example virulence, transmissibility, detectability and host specificity. Knowledge of inter-strain variation can be exploited to improve management and decrease disease incidence. Bovine tuberculosis (bTB) is increasingly prevalent among farmed cattle in the UK, exerting a heavy economic burden on the farming industry and government. We aimed to determine whether strains of Mycobacterium bovis (the causative agent of bTB) identified and classified using genetic markers (spoligotyping and multi-locus VNTR analysis) varied in response to the tuberculin skin test; this being the primary method of bTB detection used in the UK. Inter-strain variation in detectability of M. bovis could have important implications for disease control. The skin test is based on a differential delayed type hypersensitivity (DTH) response to intradermal injections of purified protein derivative (PPD) from M. bovis (PPD-B) and Mycobacterium avium (PPD-A). We searched for an association between skin test response (PPD-B skin rise minus PPD-A skin rise) and M. bovis genotype at the disclosing test in culture-confirmed cases using a field dataset consisting of 21,000 isolates belonging to 63 genotypes of M. bovis from cattle in Northern Ireland. We found no substantial variation among genotypes (estimated responses clustered tightly around the mean) controlling for animal sex, breed and test effects. We also estimated the ratio of skin test detected to undetected cases (i.e. cases only detected at abattoir). The skin test detection ratio varied among abattoirs with some detecting a greater proportion of cases than others but this variation was unrelated to the community composition of genotypes within each abattoir catchment. These two lines of evidence indicate that M. bovis genotypes in Northern Ireland have similar detectability using the skin test.

Enrichment of low-abundance proteins from bovine and porcine serum samples for proteomic studies

One of the main applications of serum proteomics is the identification of new biomarkers for animal disease or animal production. However, potential obstacles to these studies are the poor performance of affinity serum depletion methods based on human antigens when using animal samples, and loss of minor serum components bound to albumin and other proteins. In the present study, we have analyzed the efficiency and reproducibility of the ProteoMiner® beads with bovine and porcine serum samples, and compared to a traditional immunoaffinity-based albumin and IgG depletion system specific for human samples. The ProteoMiner kit is based on the use of a combinatorial peptide binding library and intends to enrich low-abundance proteins.

Genome-wide association study identifies novel loci associated with resistance to bovine tuberculosis

Tuberculosis (TB) caused by Mycobacterium bovis is a re-emerging disease of livestock that is of major economic importance worldwide, as well as being a zoonotic risk there is significant heritability for host resistance to bovine TB (bTB) in dairy cattle. To identify resistance loci for bTB, we undertook a genome-wide association study in female Holstein-Friesian cattle with 592 cases and 559 age-matched controls from case herds. Cases and controls were categorised into distinct phenotypes: skin test and lesion positive vs skin test negative on multiple occasions, respectively these animals were genotyped with the Illumina BovineHD 700K BeadChip. Genome-wide rapid association using linear and logistic mixed models and regression (GRAMMAR), regional heritability mapping (RHM) and haplotype-sharing analysis identified two novel resistance loci that attained chromosome-wise significance, protein tyrosine phosphatase receptor T (PTPRT; P=4.8 × 10 -7) and myosin IIIB (MYO3B; P=5.4 × 10 -6). We estimated that 21% of the phenotypic variance in TB resistance could be explained by all of the informative single-nucleotide polymorphisms, of which the region encompassing the PTPRT gene accounted for 6.2% of the variance and a further 3.6% was associated with a putative copy number variant in MYO3B the results from this study add to our understanding of variation in host control of infection and suggest that genetic marker-based selection for resistance to bTB has the potential to make a significant contribution to bTB control.

Improved Detection of Mycobacterium bovis in Bovine Tissues Using Immunomagnetic Separation Approaches

Immunomagnetic separation (IMS) represents a simple but effective method of selectively capturing and concentrating Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), from tissue samples. It is a physical cell separation technique that does not impact cell viability, unlike traditional chemical decontamination prior to culture. IMS is performed with paramagnetic beads coated with M. bovis-specific antibody and peptide binders. Once captured by IMS, M. bovis cells can be detected by either PCR or cultural detection methods. Increased detection rates of M. bovis, particularly from non-visibly lesioned lymph node tissues from bTB reactor animals, have recently been reported when IMS-based methods were employed.

Immunogenetic responses in calves to intranasal delivery of bovine respiratory syncytial virus (BRSV) epitopes encapsulated in poly (DL-lactide-co-glycolide) microparticles

Bovine respiratory syncytial virus (BRSV) is the principal aetiological agent of the bovine respiratory disease complex. A BRSV subunit vaccine candidate consisting of two synthetic peptides representing putative protective epitopes on BRSV surface glycoproteins in soluble form or encapsulated in poly(lactide-co-glycolide) (PLG) microparticles were prepared. Calves (10 weeks old) with diminishing levels of BRSV-specific maternal antibody were intranasally administered a single dose of the different peptide formulations. Peptide-specific local immune responses (nasal secretion IgA), but not systemic humoral (serum IgG) or cellular responses (serum IFN-γ), were generated by all forms of peptide. There was a significant reduction in occurrence of respiratory disease in the animals inoculated with all peptide formulations compared to animals given PBS alone. Furthermore no adverse effects were observed in any of the animals post vaccination. These results suggest that intranasal immunisation with the peptide subunit vaccine does induce an as yet unidentified protective immune response.

The application of metabolomic biomarker screening tools for improved Bovine Respiratory Disease diagnosis and management

Bovine Respiratory Disease (BRD) is considered to be one of the most significant causes of economic loss in cattle worldwide. The disease has multifactorial aetiology, where viral induced respiratory damage can predispose animals to developing secondary bacterial infections. Accurate identification of viral infected animals prior to the onset of bacterial infection is necessary to reduce the overuse of antimicrobial treatments and minimize further economic losses from reduced production capacity and death. This research focuses on Bovine Parainfluenza Virus Type 3 (BPIV-3), one of the viruses involved in generating BRD. Vaccination measures for BPIV-3 can induce a level of immunity preventing disease progression, however, not all animals respond equally and immunization can complicate disease diagnosis. Alternative diagnostic approaches are required to identify animals which fail to respond to vaccination during infection outbreaks and are therefore likely to be more susceptible to secondary bacterial infections. Mass spectrometry based metabolomics was employed to identify plasma markers capable of differentiating between vaccinated and non-vaccinated calves after challenge with BPIV-3. Differentiation of vaccinated and non-vaccinated study groups (n=6) was possible as early as day 2 post-BPIV-3 challenge up until day 20 using a panel of potential metabolite markers. This study illustrates the potential for metabolomics to provide more detailed information on animal vaccination status that could be used to develop tools for improved herd health management, reduce economic loss through rapid identification and isolation of animals without immune protection (improving herd level immunity) and help reduce the usage of antimicrobial therapeutic treatments in animals.

Herd-level bovine tuberculosis risk factors: assessing the role of low-level badger population disturbance

Bovine TB (bTB) is endemic in Irish cattle and has eluded eradication despite considerable expenditure, amid debate over the relative roles of badgers and cattle in disease transmission. Using a comprehensive dataset from Northern Ireland (>10,000 km²; 29,513 cattle herds), we investigated interactions between host populations in one of the first large-scale risk factor analyses for new herd breakdowns to combine data on both species. Cattle risk factors (movements, international imports, bTB history, neighbours with bTB) were more strongly associated with herd risk than area-level measures of badger social group density, habitat suitability or persecution (sett disturbance). Highest risks were in areas of high badger social group density and high rates of persecution, potentially representing both responsive persecution of badgers in high cattle risk areas and effects of persecution on cattle bTB risk through badger social group disruption. Average badger persecution was associated with reduced cattle bTB risk (compared with high persecution areas), so persecution may contribute towards sustaining bTB hotspots; findings with important implications for existing and planned disease control programmes.

Use of bacterial whole-genome sequencing to investigate local persistence and spread in bovine tuberculosis

Mycobacterium bovis is the causal agent of bovine tuberculosis, one of the most important diseases currently facing the UK cattle industry. Here, we use high-density whole genome sequencing (WGS) in a defined sub-population of M. bovis in 145 cattle across 66 herd breakdowns to gain insights into local spread and persistence. We show that despite low divergence among isolates, WGS can in principle expose contributions of under-sampled host populations to M. bovis transmission. However, we demonstrate that in our data such a signal is due to molecular type switching, which had been previously undocumented for M. bovis. Isolates from farms with a known history of direct cattle movement between them did not show a statistical signal of higher genetic similarity. Despite an overall signal of genetic isolation by distance, genetic distances also showed no apparent relationship with spatial distance among affected farms over distances <5 km. Using simulations, we find that even over the brief evolutionary timescale covered by our data, Bayesian phylogeographic approaches are feasible. Applying such approaches showed that M. bovis dispersal in this system is heterogeneous but slow overall, averaging 2 km/year. These results confirm that widespread application of WGS to M. bovis will bring novel and important insights into the dynamics of M. bovis spread and persistence, but that the current questions most pertinent to control will be best addressed using approaches that more directly integrate WGS with additional epidemiological data.