Mycobacterium bovis Strain Ravenel Is Attenuated in Cattle.

Mycobacterium tuberculosis variant bovis (MBO) has one of the widest known mammalian host ranges, including humans. Despite the characterization of this pathogen in the 1800s and whole genome sequencing of a UK strain (AF2122) nearly two decades ago, the basis of its host specificity and pathogenicity remains poorly understood. Recent experimental calf infection studies show that MBO strain Ravenel (MBO Ravenel) is attenuated in the cattle host compared to other pathogenic strains of MBO. In the present study, experimental infections were performed to define attenuation. Whole genome sequencing was completed to identify regions of differences (RD) and single nucleotide polymorphisms (SNPs) to explain the observed attenuation. Comparative genomic analysis of MBO Ravenel against three pathogenic strains of MBO (strains AF2122-97, 10-7428, and 95-1315) was performed. Experimental infection studies on five calves each, with either MBO Ravenel or 95-1315, revealed no visible lesions in all five animals in the Ravenel group despite robust IFN-γ responses. Out of 486 polymorphisms in the present analysis, 173 were unique to MBO Ravenel among the strains compared. A high-confidence subset of nine unique SNPs were missense mutations in genes with annotated functions impacting two major MBO survival and virulence pathways: (1) Cell wall synthesis & transport [espH (A103T), mmpL8 (V888I), aftB (H484Y), eccC5 (T507M), rpfB (E263G)], and (2) Lipid metabolism & respiration [mycP1(T125I), pks5 (G455S), fadD29 (N231S), fadE29 (V360G)]. These substitutions likely contribute to the observed attenuation. Results from experimental calf infections and the functional attributions of polymorphic loci on the genome of MBO Ravenel provide new insights into the strain's genotype-disease phenotype associations.

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.

Mycobacterium bovis Transmission between Cattle and a Farmer in Central Poland.

INTRODUCTION: Zoonoses have recently become an increasing public health problem. Zoonoses are estimated to account for 60% of all emerging infectious diseases. One particularly important zoonosis is human tuberculosis, especially tuberculosis due to Mycobacterium bovis (M. bovis), which is naturally resistant to pyrazinamide (PZA). MATERIAL AND METHODS: The patient had a pulmonary form of tuberculosis accompanied by a cough and fever. At the same time, the disease was also confirmed in 20 out of 25 cattle on the farm. The clinical specimen (sputum) was examined in accordance with the European Union (EU) laboratories' methodology. Tissue materials from cattle were verified in the National Veterinary Research Institute (NVRI), in the Bovine tuberculosis (BTB) Reference Laboratory, Pulawy, Poland and tested in accordance with the guidelines for the laboratory diagnosis of BTB. RESULTS: All M. bovis isolates represented one spoligotype, SB0120. The results of mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) evaluation showed the same genetic pattern. CONCLUSIONS: Findings from this study suggest the first confirmed interspecific transmission of Mycobacterium bovis, between a farmer and his cattle, in Poland. Present findings support the increasing concern regarding zoonotic TB that has been highlighted elsewhere.

Potential for improved detection of bovine tuberculosis by targeting combined blood biomarkers in multi-test algorithms.

Bovine tuberculosis (bTB) control programs can be improved by combined use of tests for humoral and cell-mediated immune responses targeting multiple biomarkers of Mycobacterium bovis. To further the diagnostic benefits of this approach, we used Dual Path Platform (DPP) technology to test sera from cattle with naturally acquired bTB in the United States (US) and Spain for the presence of M. bovis antigen, IgM and/or IgG antibodies to MPB70/MPB83 fusion antigen in conjunction with tuberculin skin tests (TST) or interferon-gamma release assays (IGRA). When TST was complemented with detection of IgM and IgG antibodies, the diagnostic sensitivity increased from 85.4% to 95.1% in the US and from 64.2% to 81.5% in Spain. Likewise, adding the DPP assays enhanced IGRA diagnostic sensitivity from 82.7% to 93.8% in Spain. Detection of circulating M. bovis antigen showed added value when used in combination with the DPP antibody assays but it was limited when analyzed in the context of TST or IGRA results. Present findings support the benefits of a multi-test approach for the ante-mortem diagnosis of bTB in cattle.

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.

Use of blood matrices and alternative biological fluids for antibody detection in animal tuberculosis.

Bovine tuberculosis (bTB) control programs can be improved by implementation of advanced ante-mortem testing algorithms. Serodiagnostic methods using traditional blood or blood-derived specimens may benefit from the use of less invasive alternative biological fluids, provided those mirror systemic antibody responses. In the present study, we used Dual Path Platform (DPP) and Multiantigen Print Immunoassay (MAPIA) to compare antibody levels in ten sample types including whole blood (fresh and hemolyzed), plasma (fresh and leftover from Bovigam testing), serum, saliva, broncho-alveolar lavage, urine, diaphragm extract, and bile collected from cattle aerosol-infected with Mycobacterium bovis. High correlation (r = 0.97-0.99) in measurements of IgG antibodies to MPB70/MPB83 fusion antigen by DPP assay was found between all blood-derived specimens, supporting matrix equivalency. Broncho-alveolar lavage and diaphragm extract yielded positive results in all the infected animals tested, showing high correlation with matching serum data (r = 0.94 and r = 0.95, respectively) and suggesting their potential use in antibody assays. Characterized by MAPIA, the antigen reactivity patterns obtained with paired sera and alternative specimens were nearly identical, with slight differences in intensity. Antibodies were also found by DPP assay in saliva, urine, and bile from some of the infected animals, but the titers were relatively low, thus reducing the diagnostic value of such specimens. The proposed approach was evaluated in a pilot field study on warthogs diagnosed with M. bovis infection. Relative levels of antibody in tissue fluid obtained from lymph nodes or lungs were consistent with those detected in sera and detectable in all infected warthogs. The findings support the diagnostic utility of non-traditional biological fluids and tissue samples when used as alternative test specimens in serologic assays for bTB.

Comparative cellular immune responses in calves after infection with Mycobacterium avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii and M. bovis.

In the present study, calves were infected with Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. avium (M. avium), Mycobacterium kansasii (M. kansasii), or Mycobacterium bovis (M. bovis) to determine differences in cellular immunity. Comparative cellular responses were assessed upon stimulation of cells with mycobacterial whole cell sonicates respective of each infection group. Antigen-specific whole blood interferon gamma (IFN-γ) responses were observed in all infection groups compared to noninfected control calves, however, responses were more robust for M. bovis calves. Upon antigen stimulation of PBMCs, secretion of IFN-γ and IL-10 was higher for M. bovis calves compared to other infection groups. In contrast, IL-12 secretion was lower for M. bovis calves compared to MAP infected calves. Within the total PBMC population, higher numbers of CD4+, CD8+, and γδ TCR + T cells were observed for MAP and M. avium calves compared to M. bovis calves. This aligned with higher expression of CD26 on these subpopulations for MAP and M. avium calves, as well. In contrast, greater expression of CD25 was observed on CD4+ and γδ TCR + T cells and natural killer cells for M. bovis calves. Overall, similarities in cellular immune responses were observed between the closely related MAP and M. avium during infection of calves. In contrast, significant differences were noted between calves infected with MAP and M. bovis. This suggests that host immune responses to different mycobacteria may impact interpretation of diagnostic tools based upon their cellular immunity.

Antibody responses in European bison (Bison bonasus) naturally infected with Mycobacterium caprae.

Mycobacterium caprae, a member of the Mycobacterium tuberculosis complex, infects humans and animals causing lesions and disease like that of Mycobacterium bovis. The aim of this study was to evaluate antibody responses in European Bison (EB, Bison bonasus; a vulnerable species) naturally infected with M. caprae using dual path platform (DPP) BovidTB test and multi-antigen print immunoassay (MAPIA). Study cohorts consisted of naturally M. caprae-infected EB (n = 4), M. caprae-exposed but uninfected (n = 3), EB infected with non-tuberculous mycobacteria or other respiratory pathogens (n = 3), and negative controls (n = 19). M. caprae-infected EB were seropositive by both DPP and MAPIA; 3/4 were seropositive by DPP; and 4/4 were seropositive by MAPIA. One M. caprae-infected animal that developed generalized disease with most advanced gross lesions in the group produced the most robust antibody response. All 25 EB with no culture-confirmed M. caprae infection, including three animals exposed to M. caprae and three other animals infected with non-tuberculous pathogens, were seronegative on both tests. Antibody responses to M. caprae infection included IgM antibodies against MPB70/MPB83 and IgG antibodies to both MPB70/MPB83 and CFP10/ESAT-6. This study demonstrates the potential for use of serological assays in the ante-mortem diagnosis of M. caprae infection in EB.

Strong antibody responses to Mycobacterium bovis infection in domestic pigs and potential for reliable serodiagnostics.

Mycobacterium bovis (M. bovis), the main cause of animal tuberculosis (TB), can infect a wide variety of domestic and wild animal species, including suids. Suids may serve as reservoir hosts or disease sentinels in different scenarios. Accurate detection of M. bovis infection in pigs is important for TB control programs. Although previous studies have shown the value of serological assays for screening animal populations, the diagnostic accuracy was considered suboptimal. In this study, we used Dual Path Platform (DPP) technology and multi-antigen print immunoassay (MAPIA) to characterize antigen recognition profiles and temporal antibody responses. Four M. bovis experimentally infected pigs developed an early antibody response to antigen MPB83, with a peak in IgG levels starting around 4-6 weeks post-inoculation, although none of the pigs developed antibodies to fusion protein CFP10/ESAT6 within 16 weeks of the experiment. Three of four experimentally infected pigs developed antibody responses before detectable antigen-specific interferon gamma responses. Naturally infected pigs with gross lesions containing viable M. bovis showed IgM (19/40 infected animals) and IgG (39/40) antibody responses to both MPB70/MPB83 (39/40) and CFP10/ESAT6 (34/40). Using MPB70/MPB83 antigen alone to measure IgG antibody levels by DPP assay, an estimated test sensitivity was 97.5 % (95 % CI: 85.3-99.9 %). None of the 57 negative control samples had detectable IgM or IgG antibodies to either of the two test antigens in DPP assay, suggesting an estimated specificity of 100 % (95 % CI: 92.1-100.0 %) in pigs. MAPIA showed robust IgG reactivity to multiple protein antigens of M. bovis in the naturally infected pigs. The results demonstrate that serological assays which detect IgG antibodies to MPB83 have high sensitivity and specificity for accurate detection of M. bovis infection in pigs. Further investigations should be done to validate anti-MPB70/MPB83 antibodies as a reliable serodiagnostic biomarker for TB diagnosis in pigs.

Severity of bovine tuberculosis is associated with innate immune-biased transcriptional signatures of whole blood in early weeks after experimental Mycobacterium bovis infection.

Mycobacterium bovis, the causative agent of bovine tuberculosis, is a pathogen that impacts both animal and human health. Consequently, there is a need to improve understanding of disease dynamics, identification of infected animals, and characterization of the basis of immune protection. This study assessed the transcriptional changes occurring in cattle during the early weeks following a M. bovis infection. RNA-seq analysis of whole blood-cell transcriptomes revealed two distinct transcriptional clusters of infected cattle at both 4- and 10-weeks post-infection that correlated with disease severity. Cattle exhibiting more severe disease were transcriptionally divergent from uninfected animals. At 4-weeks post-infection, 25 genes had commonly increased expression in infected cattle compared to uninfected cattle regardless of disease severity. Ten weeks post-infection, differential gene expression was only observed when severely-affected cattle were compared to uninfected cattle. This indicates a transcriptional divergence based on clinical status following infection. In cattle with more severe disease, biological processes and cell type enrichment analyses revealed overrepresentation of innate immune-related processes and cell types in infected animals. Collectively, our findings demonstrate two distinct transcriptional profiles occur in cattle following M. bovis infection, which correlate to clinical status.

Memory B cells and tuberculosis.

Immunological memory is a central feature of adaptive immunity. Memory B cells are generated upon stimulation with antigen presented by follicular dendritic cells in the peripheral lymphoid tissues. This process typically involves class-switch recombination and somatic hypermutation and it can be dependent or independent on germinal centers or T cell help. The mature B cell memory pool is generally characterized by remarkable heterogeneity of functionally and phenotypically distinct sub-populations supporting multi-layer immune plasticity. Memory B cells found in human patients infected with Mycobacterium tuberculosis include IgD+ CD27+ and IgM+ CD27+ subsets. In addition, expansion of atypical memory B cells characterized by the lack of CD27 expression and by inability to respond to antigen-induced re-activation is documented in human tuberculosis. These functionally impaired memory B cells are believed to have adverse effects on host immunity. Human and animal studies demonstrate recruitment of antigen-activated B cells to the infection sites and their presence in lung granulomas where proliferating B cells are organized into discrete clusters resembling germinal centers of secondary lymphoid organs. Cattle studies show development of IgM+, IgG+, and IgA+ memory B cells in M. bovis infection with the ability to rapidly differentiate into antibody-producing plasma cells upon antigen re-exposure. This review discusses recent advances in research on generation, re-activation, heterogeneity, and immunobiological functions of memory B cells in tuberculosis. The role of memory B cells in post-skin test recall antibody responses in bovine tuberculosis and implications for development of improved immunodiagnostics are also reviewed.

Differential antigen recognition by serum antibodies from three bovid hosts of Mycobacterium bovis infection.

Cattle, bison and buffaloes are susceptible to Mycobacterium bovis, the causative agent for bovine tuberculosis. Accurate and timely identification of infected animals is critical for improved management and control of disease in these species. Bovids develop humoral immune responses to M. bovis infection making serological tests attractive for tuberculosis screening. However, optimization and validation of antibody assays designed for various animal species require understanding of antigen recognition patterns in each target host. The objective of this study was to characterize serological reactivity profiles generated by cattle, American bison, and African buffaloes in tuberculosis. Serum samples from M. bovis-infected animals were tested for the presence of IgM and IgG antibodies to MPB70/MPB83 and CFP10/ESAT6 chimeric proteins using Dual-Path Platform technology. All three host species showed IgG responses of higher magnitude and frequency than IgM responses; further, IgM seroreactivity was limited to MPB70/MPB83, whereas IgG antibodies recognized both test antigens. In cattle, the IgM and IgG responses were elicited mainly by MPB70/MPB83, whereas bison and buffaloes showed similar IgG seroreactivity rates for MPB70/MPB83 and CFP10/ESAT6 antigens. The findings demonstrate distinct patterns of predominant antigen recognition by different bovid species in M. bovis infection.

Vaccination of white-tailed deer (Odocoileus virginianus) with Mycobacterium bovis bacille Calmette-Guérin (BCG) results in positive tuberculin skin test results in a dose-dependent fashion.

Mycobacterium bovis is the cause of tuberculosis in most mammalian species, most notably cattle and other members of the family Bovidae; however, many species of the family Cervidae are also susceptible. In North America, tuberculosis has been identified in both captive and free-ranging cervids. Captive cervids are tested for tuberculosis following many of the same guidelines applied to cattle, including intradermal tuberculin testing using M. bovis purified protein derivative (PPD). Both captive and free-ranging deer and elk have been implicated as the source of infection for many cattle herds. Vaccination with the human vaccine M. bovis BCG has been considered as one possible tool to aid in eradication of tuberculosis from cattle and both captive and free-ranging cervids. Studies in cattle have demonstrated that BCG vaccination can induce false positive intradermal tuberculin test reactions in some cattle. Similar findings have been reported for red deer. We orally vaccinated white-tailed deer with BCG and showed that vaccination can induce false positive skin test reactions in some deer and that the rate of false positive reactions is greater with a higher vaccine dose.

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.

Characterization of γδ T Cell Effector/Memory Subsets Based on CD27 and CD45R Expression in Response to Mycobacterium bovis Infection.

Tuberculosis (TB) remains a leading cause of death from infectious diseases worldwide. Mycobacterium bovis is the causative agent of bovine TB and zoonotic TB infection. γδ T cells are known to participate in the immune control of mycobacterial infections. Data in human and nonhuman primates suggest that mycobacterial infection regulates memory/effector phenotype and adaptive immune functions of γδ T cells. To date, the impact of M. bovis infection on bovine γδ T cells and their effector and memory differentiation remains unknown. In this study, we show that circulating γδ T cells from M. bovis-infected cattle can be differentiated based on the expression of CD27, which is indicative of their capacity to respond to virulent M. bovis infection: CD27+ γδ T cells proliferated in response to M. bovis Ag and, thus, may comprise the adaptive γδ T cell compartment in cattle. We further show that bovine M. bovis-specific γδ T cells express surface markers characteristic of central memory T cells (CD45R-CD27+CD62Lhi) and that M. bovis-specific CD4 and γδ T cells both upregulate the expression of the tissue-homing receptors CXCR3 and CCR5 during infection. Our studies contribute significantly to our understanding of γδ T cell differentiation during TB infection and provide important insights into the link between phenotypic and functional subsets in the bovine. Accurate characterization of γδ T cell effector and memory-like responses induced during mycobacterial infection will contribute to improved strategies for harnessing the γδ T cell response in protection against TB for humans and animals.

Fatal Tuberculosis in a Free-Ranging African Elephant and One Health Implications of Human Pathogens in Wildlife.

Tuberculosis (TB) in humans is a global public health concern and the discovery of animal cases of Mycobacterium tuberculosis (Mtb) infection and disease, especially in multi-host settings, also has significant implications for public health, veterinary disease control, and conservation endeavors. This paper describes a fatal case of Mtb disease in a free-ranging African elephant (Loxodonta africana) in a high human TB burden region. Necropsy revealed extensive granulomatous pneumonia, from which Mtb was isolated and identified as a member of LAM3/F11 lineage; a common lineage found in humans in South Africa. These findings are contextualized within a framework of emerging Mtb disease in wildlife globally and highlights the importance of the One Health paradigm in addressing this anthroponotic threat to wildlife and the zoonotic implications.

Evaluating the cytokine profile of the WC1+ γδ T cell subset in the ileum of cattle with the subclinical and clinical forms of MAP infection.

In the present study, we evaluated expression of IFN-γ, IL-17, TNF-α, IL-10 and TGF-ß by mucosal cells, including WC1+ γδ T cells, in ileal tissues taken from non-infected cattle and cattle naturally infected with Mycobacterium avium subsp paratuberculosis (MAP). Infected cattle were either in the subclinical or clinical stage of infection. We hypothesized that the cytokine profile of the WC1+ γδ T cell subset would be different between subclinical and clinical cattle. Our data indicate a significant increase in the numbers of WC1+ γδ T cells expressing IL-10 in clinical cattle compared to subclinical and non-infected cattle. We observed a significant increase in TGF-ß expression by non-WC1+ cells in clinically infected cattle. Expression of IFN-γ, IL-17 and TNF-α in mucosal cells, including the WC1+ γδ T cell subset, was identified in all examined groups. However, our data indicate that the stage of infection did not significantly influence expression of these proinflammatory cytokines. This study demonstrates changes in the cytokine mRNA expression profile of mucosal cells in the ileum, and specifically WC1+ γδ T cells, as cattle progress to the clinical disease. The change is characterized by an increase in expression of anti-inflammatory cytokines.

Spectrum of antibody profiles in tuberculous elephants, cervids, and cattle.

Using multi-antigen print immunoassay and DPP® VetTB Assay approved in the United States for testing captive cervids and elephants, we analyzed antibody recognition of MPB83 and CFP10/ESAT-6 antigens in Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis and in white-tailed deer (Odocoileus virginianus), fallow deer (Dama dama), elk (Cervus elaphus), and cattle (Bos taurus) infected with Mycobacterium bovis. Serum IgG reactivity to MPB83 was found in the vast majority of tuberculous cattle and cervid species among which white-tailed deer and elk also showed significant CFP10/ESAT-6 recognition rates with added serodiagnostic value. In contrast, the infected elephants developed antibody responses mainly to CFP10/ESAT-6 with MPB83 reactivity being relatively low. The findings demonstrate distinct patterns of predominant antigen recognition by different animal hosts in tuberculosis.

Measuring bovine γδ T cell function at the site of Mycobacterium bovis infection.

Bovine γδ T cells are amongst the first cells to accumulate at the site of Mycobacterium bovis infection; however, their role in the developing lesion remains unclear. We utilized transcriptomics analysis, in situ hybridization, and a macrophage/γδ T cell co-culture system to elucidate the role of γδ T cells in local immunity to M. bovis infection. Transcriptomics analysis revealed that γδ T cells upregulated expression of several novel, immune-associated genes in response to stimulation with M. bovis antigen. BCG-infected macrophage/γδ T cell co-cultures confirmed the results of our RNAseq analysis, and revealed that γδ T cells from M. bovis-infected animals had a significant impact on bacterial viability. Analysis of γδ T cells within late-stage M. bovis granulomas revealed significant expression of IFN-γ and CCL2, but not IL-10, IL-22, or IL-17. Our results suggest γδ T cells influence local immunity to M. bovis through cytokine secretion and direct effects on bacterial burden.