Mycobacterium Bovis Infection In Cattle Research Articles

Tuberculosis in wild animals in India.

India is renowned for its complex megadiverse ecosystems and abundant biodiversity. Bovine tuberculosis (bTB) often remains synonymous with Mycobacterium bovis infection in cattle. The domain of tuberculosis (TB) among wild animals, induced by members of the Mycobacterium tuberculosis complex organisms (MTBC), is often underexplored and underreported in India. Within this context, instances of wild animal tuberculosis (wTB) have manifested across both captive and free-roaming animals. The sources contributing to wTB in animals can be human, animal, or environmental factors, thus illuminating the complex transmission pathways. The diagnosis of wTB continues to pose a formidable challenge, a consequence of the expansive taxonomic diversity in both the host and the pathogen. Complications inherent in acquiring samples from wildlife, the absence of standardized diagnostic protocols, limited insights into infection prevalence, and resource constraints compound diagnosis. Amidst these, adopting the comprehensive One Health paradigm surfaces as an imperative, accentuating the interconnectedness bridging human, animal, and environmental health. Recognizing key stakeholders and fostering intersectoral collaboration to provide enhanced diagnostic techniques driven by skilled personnel and advanced infrastructure play pivotal roles in a comprehensive strategy. Additionally, leveraging vaccination efforts contributes to effective control. A national wTB surveillance program is a cornerstone, ensuring an integrated and holistic approach to disease management. Through this review, we delve into the current landscape of wTB in India, unveiling its multifaceted challenges, and further explore the multifarious strategies that the One Health approach proffers in this dynamic endeavor.

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.

Spatial association of Mycobacterium bovis infection in cattle and badgers at the pasture interface in an endemic area in France.

Despite control and surveillance programmes, Mycobacterium bovis, the main aetiologic agent of bovine tuberculosis (bTB), is still detected on cattle farms and in wildlife populations in France, especially in badgers in the French Côte-d'Or département. The aim of our study was to find out if infected badgers were trapped significantly closer to pastures of infected farms than non-infected badgers and, if so, to determine the most efficient distance around those pastures for badger trapping, particularly for surveillance purposes. We studied two subareas (southern and northern), chosen based on natural barriers to badger movements and according to the presence of pastures belonging to infected farms (POIFs) and infected or non-infected badgers. In each subarea, we computed the shortest distances D0 and D between badgers trapped a given year n between 2015 and 2019 (n=59 infected and n=1535 non-infected badgers for D0; n=53 infected and n=1476 non-infected badgers for D) and POIFs designated as infected between the year n - 4 and n+1 (respectively n=373 and n=388 POIFs). D0 was calculated without considering spoligotypes, while D was calculated considering the possible epidemiological link between infected badgers and POIFs by using bTB spoligotype information. Then, we computed the observed mean and median of the D0 and D distances and used a bootstrap analysis to test if infected badgers were found significantly closer to POIFs than non-infected badgers. We observed that infection of badgers was not independent of distance from POIF in both subareas but distances (D0 or D) were different between the northern and southern subarea. In the northern subarea, which displays a mosaic landscape (mean and median D distances were respectively 612m and 303m for infected badgers), infected badgers indeed were trapped closer to POIFs, considering D0 and D. In the southern subarea, predominantly forested, infected badgers were significantly closer to POIFs than non-infected badgers when considering D0 but not for D (mean and median D distances were respectively 7148m and 4831m for infected badgers). These results will help to determine the most efficient distance from POIFs to trap badgers to determine their infection status in countryside landscapes. They also highlight the need to better understand the epidemiological systems at play in more forested landscapes where badgers may behave differently or other susceptible sympatric wild species might play a more important role in the circulation of M. bovis, both phenomena contributing to badger infection at greater distances from POIFs.

Relationship between ambient temperature at sampling and the interferon gamma test result for bovine tuberculosis in cattle

Bovine tuberculosis (bTB) is a disease of significant economic and zoonotic importance, therefore, optimising tests for the identification of Mycobacterium bovis infected cattle is essential. The Interferon Gamma (IFN-γ) Release Assay (IGRA) can diagnose M. bovis infected cattle at an early stage, is easy to perform and can be used alongside skin tests for confirmatory purposes or to increase diagnostic sensitivity. It is known that IGRA performance is sensitive to environmental conditions under which samples are taken and transported. In this study, the association between the ambient temperature on the day of bleeding and the subsequent IGRA result for bTB was quantified using field samples from Northern Ireland (NI). Results of 106,434 IGRA results (2013–2018) were associated with temperature data extracted from weather stations near tested cattle herds. Model dependent variables were the levels of IFN-γ triggered by avian purified protein derivative (PPDa), M. bovis PPD (PPDb), their difference (PPD(b-a)) as well as the final binary outcome (positive or negative for M. bovis infection). IFN-γ levels after both PPDa and PPDb stimulation were lowest at the extremes of the temperature distribution for NI. The highest IGRA positive probability (above 6%) was found on days with moderate maximum temperatures (6–16 °C) or moderate minimum temperatures (4–7 °C). Adjustment for covariates did not lead to major changes in the model estimates. These data suggest that IGRA performance can be affected when samples are taken at high or low temperatures. Whilst it is difficult to exclude physiological factors, the data nonetheless supports the temperature control of samples from bleeding through to laboratory to help mitigate post-collection confounders.

Novel functional sequences uncovered through a bovine multiassembly graph

Many genomic analyses start by aligning sequencing reads to a linear reference genome. However, linear reference genomes are imperfect, lacking millions of bases of unknown relevance and are unable to reflect the genetic diversity of populations. This makes reference-guided methods susceptible to reference-allele bias. To overcome such limitations, we build a pangenome from six reference-quality assemblies from taurine and indicine cattle as well as yak. The pangenome contains an additional 70,329,827 bases compared to the Bos taurus reference genome. Our multiassembly approach reveals 30 and 10.1 million bases private to yak and indicine cattle, respectively, and between 3.3 and 4.4 million bases unique to each taurine assembly. Utilizing transcriptomes from 56 cattle, we show that these nonreference sequences encode transcripts that hitherto remained undetected from the B. taurus reference genome. We uncover genes, primarily encoding proteins contributing to immune response and pathogen-mediated immunomodulation, differentially expressed between Mycobacterium bovis-infected and noninfected cattle that are also undetectable in the B. taurus reference genome. Using whole-genome sequencing data of cattle from five breeds, we show that reads which were previously misaligned against the Bos taurus reference genome now align accurately to the pangenome sequences. This enables us to discover 83,250 polymorphic sites that segregate within and between breeds of cattle and capture genetic differentiation across breeds. Our work makes a so-far unused source of variation amenable to genetic investigations and provides methods and a framework for establishing and exploiting a more diverse reference genome.

Occurrence of Mycobacterium bovis infection in cattle in Kaduna metropolis and its environs

The tedious nature of tuberculin tests in Nigeria, warrants the need for an alternative rapid animal-side means of diagnosis. The aim of this study was to provide information on the epidemiology of Mycobacterium bovis infection in cattle. An experiment was conducted to evaluate the occurrence of M. bovis infection in cattle in Kaduna metropolis and it's environ. A total 239 heads of cattle from 8 herds were randomly selected and tuberculin tested, two herds being from each of the four local government areas involved and pooled milk samples from the eight herds were tested for acid fastness. Also, all heads of cattle totaling 2978 slaughtered at the 2 abattoirs during the month of May, 2012 were examined at postmortem and tissues with lesions compatible with lesions of were collected and tested for acid fastness. The results of the experiment showed that only 23 (9.6%) of the animals tested were classified as reactors and these came from only one of the eight herds studied. A clear swelling was seen in each of the positive cases. Out of the eight herds tested, 6 (75%) were settled and the remaining 2 (25%) were semi- settled. Similarly, 6 (75%) of the herds were for mixed production system and 2(25%) were kept solely as dairy herds. Positive tuberculin test was only observed from among one of the six settled herds 1(17%) while cattle in the remaining 5(83%} settled herds all tested negative. All the two semi-settled herds (100%) tested negative. The tuberculin reaction among different production systems showed that only 1 (50%) of the dairy herds tested positive and all the 6 (100%) mixed herds tested negative. Atuberculin positive reaction of 19 (7.9%) was observed among the females and 4 (1.67%) among the males. The chi-square test of significance between the tuberculin reaction and sex of the animals showed no significant effect (P > 0.05). Hence, it can be concluded that, Mycobacterium bovis was neither cultured nor isolated and therefore, could not be confirmed responsible for the observed reactions. However, its involvement is strongly suggested by both its higher values compared to those for Mycobacterium avium in the single intradermal comparative tuberculin test and the observed in the stained smears of both the milk samples collected and the bovine tissues from the abattoirs.

The Brain–Cognitive Behavior Problem: A Retrospective

ABSTRACT Cross-reactive responses elicited by exposure to nontuberculous mycobacteria often confound the interpretation of antemortem tests for Mycobacterium bovis infection of cattle. The use of specific proteins (e.g., ESAT-6, CFP-10, and MPB83), however, generally enhances the specificity of bovine tuberculosis tests. While genes for these proteins are absent from many nontuberculous mycobacteria, they are present in M. kansasii. Instillation of M. kansasii into the tonsillar crypts of calves elicited delayed-type hypersensitivity and in vitro gamma interferon and nitrite concentration responses of leukocytes to M. avium and M. bovis purified protein derivatives (PPDs). While the responses of M. kansasii-inoculated calves to M. avium and M. bovis PPDs were approximately equivalent, the responses of M. bovis-inoculated calves to M. bovis PPD exceeded their respective responses to M. avium PPD. The gamma interferon and nitrite responses of M. kansasii-inoculated calves to recombinant ESAT-6-CFP-10 (rESAT-6-CFP-10) exceeded corresponding responses of noninoculated calves as early as 15 and 30 days after inoculation, respectively, and persisted throughout the study. The gamma interferon and nitrite responses of M. bovis-inoculated calves to rESAT-6-CFP-10 exceeded the corresponding responses of M. kansasii-inoculated calves beginning 30 days after inoculation. By using a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum antibodies were detected as early as 50 days after challenge with M. kansasii. By a multiantigen print immunoassay and immunoblotting, serum antibodies to MPB83, but not ESAT-6 or CFP-10, were detected in M. kansasii-inoculated calves; however, responses to MPB83 were notably weaker than those elicited by M. bovis infection. These findings indicate that M. kansasii infection of calves elicits specific responses that may confound the interpretation of bovine tuberculosis tests.

Enzyme-linked immunosorbent assay as complement of intradermal skin test for the detection of mycobacterium bovis infection in cattle

Diagnostic tests based on cell-mediated immunity are used in programs for the control and eradication of bovine tuberculosis (bTB), which is mainly caused by Mycobacterium bovis. Additional serological assays could be performed as an ancillary method to detect an infected animal that fails to produce an immune response against the intradermal reaction (IDR), the official bTB test. In this study, we evaluated the effectiveness of an enzyme-linked immunosorbent assay (ELISA) that uses bovine PPD as a capture antigen as a complement to the IDR in herds with confirmed cases of bTB. The study was conducted in two stages. First, a panel of 200 serum samples was analyzed by ELISA. The sensitivity and specificity obtained were 60% and 99%, respectively. The subsequent stage consisted of evaluating 7,494 bovines from 14 selected dairy farms. The number of animals yielding a IDR negative/ELISA positive result were 200. A necropsy analysis of 33 of these IDR negative/ELISA positive animals revealed that 30 (91%) presented granulomatous lesions and positive M. bovis isolation. This finding confirmed bTB in most cases. Altogether, the results obtained in the present study suggest that the combined use of IDR and ELISA is an effective strategy to improve the control of bTB in endemic herds.

English

Bovine tuberculosis causes huge economic losses to the dairy industry in terms of poor growth, reduce milk production potential of infected animals, emaciation and poor quality of carcass. Bovine tuberculosis due to Mycobacterium bovis is a major threat to public health. Therefore, this study was aimed at to know the frequency of Mycobacterium bovis infection in cattle and buffaloes. A total of 265 animals, including adult cattle (132) and buffaloes (133) of different age and sex were screened for tuberculosis infection with tuberculin test. Equal numbers of milk and nasal swab samples were collected from tuberculin reactor and non-reactor cattle and buffaloes for the isolation and identification of the causative organism. The infectious agent was identified and confirmed through procedures such as culture characteristics, ZN (Ziehl-Neelsen) staining, and PCR (Polymerase Chain Reaction) was also performed. The study revealed an overall 10.56% (95% C.I. 7.28 to 14.71) prevalence of bovine tuberculosis, the frequency of infectious agent was higher in buffaloes (11.04%) than cattle (9.76%). TB (Tuberculosis) was present in 100% herds. Multivariate logistic regression analysis with the backward elimination procedure revealed that, age (OR=1.564), body weight (OR=1.008) and status (OR=5.72) showed a significant association with the occurrence of tuberculosis. Nasal samples yielded more positive PCR results than milk samples. By considering PCR as a gold standard, ZN was more sensitive and PPD was a more specific test. Among all tests, PCR proved most accurate and fast test for the confirmation of bovine tuberculosis

Molecular epidemiology of cattle tuberculosis in Mexico through whole-genome sequencing and spoligotyping.

Mycobacterium bovis infection in cattle persists in Mexico, posing a threat to human health. Control of bovine tuberculosis, through the National Program Against Bovine Tuberculosis, has led to the decrease of disease prevalence in most of the country, except for high dairy production regions. Genotyping of M. bovis has been performed mainly by spoligotyping and variable number tandem repeats (VNTR), but higher resolution power can be useful for a finer definition of the spread of the disease. Whole genome sequencing and spoligotyping was performed for a set of 322 M. bovis isolates from different sources in Mexico: Baja California, Coahuila, Estado de Mexico, Guanajuato, Hidalgo, Jalisco, Queretaro and Veracruz, from dairy and beef cattle, as well as humans. Twelve main genetic clades were obtained through WGS and genetic diversity analysis. A clear differentiation of the Baja California isolates was seen as they clustered together exclusively. However, isolates from the central states showed no specific clustering whatsoever. Although WGS proves to have higher resolving power than spoligotyping, and since there was concordance between WGS and spoligotyping results, we consider that the latter is still an efficient and practical method for monitoring bovine tuberculosis in developing countries, where resources for higher technology are scarce.

The prevalence estimates of Mycobacterium bovis infection in cattle with ELISA

Tuberculosis is an infectious bacterial disease of Bovidae and the causative agent is Mycobacterium bovis. It is responsible for remarkable economic losses among cattle herds with widely dispersion. Prompt and consistent diagnosis of tuberculosis especially in countries where the disease is endemic as in Turkey is of great importance to detect and identify infectious cases for strengthening control measures. In the present study, it was aimed to detect true animal and herd prevalence (within-herd, and between-herd) of antibodies against M. bovis in cattle herds. A serologic survey for antibody detection against the M. bovis was conducted by using an ELISA kit. Thirty three cattle herds were randomly selected from different farms and totally 460 cattle over five years of age were sampled. The true animal, within-herd, and between-herd prevalences found were 5.9% (95% CI = 3.0 to 8.8), 11.1% (95 CI = 6.5 to 15.8) and 73.4% (95 CI = 51.2 to 95.6), respectively. Results will provide useful information about the status of M. bovis infection and will contribute to the disease control practices.

Effect of Mycobacterium bovis infection in cattle on changes in appetite-related hormones

There are limited studies available for appetite-related hormonal changes in bovine tuberculosis, therefore the purpose of this study was to determine the changes in appetite-related hormones (leptin, ghrelin, and nesfatin -1) of cattle with tuberculosis and to clarify their possible roles. A total of 30 cattle, comprising 15 clinically healthy cattle and 15 cattle infected with tuberculosis were enrolled in the study. The diagnosis of tuberculosis was performed by tuberculin skin test, and changes in the concentrations of ghrelin, leptin, nesfatin-1, lipid profile and muscular enzymes in cattle with tuberculosis were determined. It was found that nesfatin-1, ghrelin and muscular enzymes concentrations were higher in bovine infected with tuberculosis than in the healthy controls. On the other hand, leptin, triglyceride, low density lipoprotein (LDL), high density lipoprotein (HDL), and total cholesterol levels were all lower in the tuberculosis group when compared to the control group. In this study, we detected serum nesfatin-1 concentrations for the first time in Mycobacterium bovis infection in addition to the other known appetite-related hormones. The data suggest that increased concentrations of nesfatin-1 may play a role in weight loss in bovine tuberculosis, but there is a need for further studies to determine its exact role in the etiopathogenesis of bovine tuberculosis. Key words : bovine tuberculosis, ghrelin, leptin, nesfatin-1.

Fine-mapping host genetic variation underlying outcomes to Mycobacterium bovis infection in dairy cows

BackgroundSusceptibility to Mycobacterium bovis infection in cattle is governed in part by host genetics. However, cattle diagnosed as infected with M. bovis display varying signs of pathology. The variation in host response to infection could represent a continuum since time of exposure or distinct outcomes due to differing pathogen handling. The relationships between host genetics and variation in host response and pathological sequelae following M. bovis infection were explored by genotyping 1966 Holstein-Friesian dairy cows at 538,231 SNPs with three distinct phenotypes. These were: single intradermal cervical comparative tuberculin (SICCT) test positives with visible lesions (VLs), SICCT-positives with undetected visible lesions (NVLs) and matched controls SICCT-negative on multiple occasions.ResultsRegional heritability mapping identified three loci associated with the NVL phenotype on chromosomes 17, 22 and 23, distinct to the region on chromosome 13 associated with the VL phenotype. The region on chromosome 23 was at genome-wide significance and candidate genes overlapping the mapped window included members of the bovine leukocyte antigen class IIb region, a complex known for its role in immunity and disease resistance. Chromosome heritability analysis attributed variance to six and thirteen chromosomes for the VL and NVL phenotypes, respectively, and four of these chromosomes were found to explain a proportion of the phenotypic variation for both the VL and NVL phenotype. By grouping the M. bovis outcomes (VLs and NVLs) variance was attributed to nine chromosomes. When contrasting the two M. bovis infection outcomes (VLs vs NVLs) nine chromosomes were found to harbour heritable variation. Regardless of the case phenotype under investigation, chromosome heritability did not exceed 8% indicating that the genetic control of bTB resistance consists of variants of small to moderate effect situated across many chromosomes of the bovine genome.ConclusionsThese findings suggest the host genetics of M. bovis infection outcomes is governed by distinct and overlapping genetic variants. Thus, variation in the pathology of M. bovis infected cattle may be partly genetically determined and indicative of different host responses or pathogen handling. There may be at least three distinct outcomes following M. bovis exposure in dairy cattle: resistance to infection, infection resulting in pathology or no detectable pathology.

IL-10 suppression of IFN-γ responses in tuberculin-stimulated whole blood from Mycobacterium bovis infected cattle

The measurement of bovine interferon-gamma (IFN-γ) forms the basis of a diagnostic test for bovine tuberculosis where Mycobacterium bovis sensitised effector T cells produce IFN-γ following in vitro stimulation with tuberculin antigens. In cattle infected with M. bovis it is also known that the anti-inflammatory IL-10 cytokine can inhibit in vitro production of IFN-γ leading to a reduced response in the IFN-γ diagnostic test. In order to investigate this in greater detail, whole blood samples from tuberculin skin test positive and negative cattle were stimulated with bovine and avian tuberculin antigens and in parallel with a neutralising anti-IL-10 monoclonal antibody. The results showed that IFN-γ protein levels increased when IL-10 activity was suppressed by Anti − IL-10. By using a standard diagnostic interpretation, the elevated levels of IFN-γ were shown to change the level of agreement between the performance of the single intradermal comparative tuberculin test (SICTT) and IFN-γ assay, depending on the tuberculin treatment. A transcriptomic analysis using RT-qPCR investigated the influence of IL-10 activity on expression of a suite of cytokine genes (IFNG, IL12B, IL10 and CXCL10) associated with antigen-stimulated production of IFN-γ. The IFNG and IL12B genes both experienced significant increases in expression in the presence of Anti-IL-10, while the expression of IL10 and CXCL10 remained unaffected.

A haplotype at intron 8 of PTPRT gene is associated with resistance to Brucella infection in Argentinian creole goats

Brucellosis is the leading zoonosis on a worldwide scale and constitutes a major public health threat in many regions of the world. Several molecular markers associated with natural resistance to intracellular bacterial infection have been identified. Recently seven single-nucleotide polymorphisms (SNPs) located in the PTPRT gene were associated with resistance to Mycobacterium bovis infection in cattle. Here, we perform a case-control study to test if polymorphisms at PTPRT intron 8 might influence the resistance or susceptibility to Brucella infection in goats. DNA samples from 22 seropositive (cases) and 22 seronegative (controls) for brucellosis, unrelated female creole goats, were included in the present study. Four previously reported polymorphisms (SNP1: rs643551276, SNP2: rs651618967, SNP3: rs662137815 and SNP4: rs657542977) and a new SNP (SNP5: chr13: 691695526) were detected by PCR-DNA sequencing method. Genotypic and allelic frequencies differed significantly between cases and controls at SNPs 1, 2, 4 and 5 (p≤0.001). Indeed, the SNP1 TT, SNP2 TT, SNP4 CC and SNP5 TT genotypes were associated with absence of Brucella-specific antibodies (ORs=0.019 to 0.045). Moreover, haplotype association analysis revealed a significant association of the TTCCT haplotype with protection to Brucella infection (p≤1×10−4; OR=18), including the major allelic variants associated with resistance. These results represent the first evidence of genetic association between polymorphisms in the PTPRT gene and absence of brucellosis in goats.

Early Detection of Circulating Antigen and IgM-Associated Immune Complexes during Experimental Mycobacterium bovis Infection in Cattle.

The presence of circulating antigen in cattle experimentally infected with Mycobacterium bovis was demonstrated using dual-path platform (DPP) technology. The antigen capture immunoassays employed rabbit polyclonal antibody recognizing predominantly M. tuberculosis complex-specific epitopes and were able to detect soluble substances and whole cells of mycobacteria. The antigen found in serum appeared to be mostly bound to IgM, but not to IgG, within the immune complexes formed at early stages of M. bovis infection. The antigen was also detected in bile and urine, indicating possible clearance pathways. The data correlation analyses supported the idea of the role of IgM responses in antigen persistence during M. bovis infection. The antigen was detectable in serum months prior to detectable antibody seroconversion. This proof-of-concept study suggested the potential for improved immunodiagnostics for bovine tuberculosis.

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.

The CD4+ T cell methylome contributes to a distinct CD4+ T cell transcriptional signature in Mycobacterium bovis-infected cattle

We hypothesised that epigenetic regulation of CD4+ T lymphocytes contributes to a shift toward a dysfunctional T cell phenotype which may impact on their ability to clear mycobacterial infection. Combined RNA-seq transcriptomic profiling and Reduced Representation Bisulfite Sequencing identified 193 significantly differentially expressed genes and 760 differentially methylated regions (DMRs), between CD4+ T cells from M. bovis infected and healthy cattle. 196 DMRs were located within 10 kb of annotated genes, including GATA3 and RORC, both of which encode transcription factors that promote TH2 and TH17 T helper cell subsets respectively. Gene-specific DNA methylation and gene expression levels for the TNFRSF4 and Interferon-γ genes were significantly negatively correlated suggesting a regulatory relationship. Pathway analysis of DMRs identified enrichment of genes involved in the anti-proliferative TGF-β signaling pathway and TGFB1 expression was significantly increased in peripheral blood leukocytes from TB-infected cattle. This first analysis of the bovine CD4+ T cell methylome suggests that DNA methylation directly contributes to a distinct gene expression signature in CD4+ T cells from cattle infected with M. bovis. Specific methylation changes proximal to key inflammatory gene loci may be critical to the emergence of a non-protective CD4+ T cell response during mycobacterial infection in cattle.

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.