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Bovine paratuberculosis (PTB) is a chronic inflammatory disease caused by Mycobacterium avium susbp. paratuberculosis (MAP). Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) significantly associated with susceptibility to bovine PTB. The main objective of this study was to identify quantitative trait loci (QTLs) associated with MAP infection in Spanish Holstein cows (N = 983) using combinations of diagnostic tests and imputed whole-genome sequence (WGS) data. The infection status of these animals was defined by three diagnostic methods including ELISA for MAP-antibodies detection, and tissue culture and PCR for MAP detection. The 983 cows included in this study were genotyped with the Bovine MD SNP50 Bead Chip, and the corresponding genotypes were imputed to WGS using the 1,000 Bull genomes reference population. In total, 33.77 million SNP variants per animal were identified across the genome. Linear mixed models were used to calculate the heritability (h 2 ) estimates for each diagnostic test and test combinations. Next, we performed a case-control GWAS using the imputed WGS datasets and the phenotypes and combinations of phenotypes with h 2 estimates > 0.080. After performing the GWAS, the test combinations that showed SNPs with a significant association (P FDR ≤ 0.05), were the ELISA-tissue PCR-tissue culture, ELISA-tissue culture, and ELISA-tissue PCR. A total of twelve quantitative trait loci (QTLs) highly associated with MAP infection status were identified on the Bos taurus autosomes (BTA) 4, BTA5, BTA11, BTA12, BTA14, BTA23, BTA24, and BTA28, and some of these QTLs were linked to immune-modulating genes. The identified QTLs on BTA23 spanning from 18.81 to 22.95 Mb of the Bos taurus genome overlapped with several QTLs previously found to be associated with PTB susceptibility, bovine tuberculosis susceptibility, and clinical mastitis. The results from this study provide more clues regarding the molecular mechanisms underlying susceptibility to PTB infection in cattle and might be used to develop national genetic evaluations for PTB in Spain.

The single and comparative intradermal tuberculin tests (SITT and CITT) are official in vivo tests for bovine tuberculosis (TB) diagnosis using bovine and avian purified protein derivatives (PPD-B and PPD-A). Infection with bacteria other than Mycobacterium tuberculosis complex (MTC) can result in nonspecific reactions to these tests. We evaluated the performance of the skin test with PPDs and new defined antigens in the guinea pig model. A standard dose (SD) of Rhodococcus equi , Nocardia sp., M . nonchromogenicum , M . monacense , M . intracellulare , M . avium subsp. paratuberculosis , M . avium subsp. avium , M . avium subsp. hominissuis , M . scrofulaceum , M. persicum , M . microti , M . caprae and M . bovis , and a higher dose (HD) of M . nonchromogenicum , M . monacense , M . intracellulare , M . avium subsp. paratuberculosis were tested using PPD-B, PPD-A, P22, ESAT-6-CFP-10-Rv3615c peptide cocktail long (PCL) and fusion protein (FP). The SD of R . equi , Nocardia sp., M . nonchromogenicum , M . monacense , M . intracellulare and M . avium subsp. paratuberculosis did not cause any reactions. The HD of M . nonchromogenicum , M . monacense , M . intracellulare , and M . avium subsp. paratuberculosis and the SD of M . avium subsp. hominissuis , M . scrofulaceum and M . persicum , caused nonspecific reactions (SIT). A CITT interpretation would have considered M . avium complex and M . scrofulaceum groups negative, but not all individuals from M . nonchromogenicum HD, M . monacense HD and M . persicum SD groups. Only animals exposed to M. bovis and M. caprae reacted to PCL and FP. These results support the advantage of complementing or replacing PPD-B to improve specificity without losing sensitivity.

The complexity of bovine tuberculosis (TB) epidemiology, especially in extensive livestock systems with wildlife reservoirs, calls for novel control strategies such as vaccination. This study evaluated the comparative efficacy of homologous and heterologous inactivated vaccines against that of the live Bacille Calmette–Guérin (BCG) vaccine on M. bovis isolation, gross TB lesions, and immune responses. Data from four controlled vaccination and challenge trials involving 41 calves were analysed. Animals were vaccinated with live BCG or heat-inactivated M. bovis via oral or parenteral routes, and immunologic and post-mortem analyses were conducted to evaluate vaccine performance. Compared with nonvaccinated controls, all vaccination strategies significantly reduced M. bovis bacterial loads in the lungs (up to 99%). However, bacterial loads in lymphoid tissues increased, confirming that tuberculosis is primarily a lymphatic disease. Diagnostic interference varied by vaccine type and administration route, with oral administration resulting in lower interference. Gross lesion scores were inconsistent across groups, suggesting limited utility as a measure of vaccine efficacy. Immune responses revealed increased detection of infection, particularly with inactivated vaccines. The lung bacterial load appeared to be decoupled from cellular immune responses, lymph node lesions and bacterial load, which were negatively correlated. Inactivated vaccines can offer a safe and effective means of reducing the bovine TB reproductive rate (R₀) even without total bacterial clearance. These findings highlight the need for revised evaluation criteria and support the integration of inactivated vaccines into TB control strategies.

Genome-wide association studies (GWAS) have identified host genetic variants associated with paratuberculosis (PTB) susceptibility. Most of the GWAS-identified SNPs are in non-coding regions. Connecting these non-coding variants and downstream affected genes is a challenge and, up to date, only a few functional mutations or expression quantitative loci (cis-eQTLs) associated with PTB susceptibility have been identified. In the current study, the associations between imputed whole-genome sequence genotypes and whole RNA-Sequencing data from peripheral blood (PB) and ileocecal valve (ICV) samples of Spanish Holstein cows ( N  = 16) were analyzed with TensorQTL . This approach allowed the identification of 88 and 37 cis-eQTLs regulating the expression levels of 90 and 37 genes in PB and ICV samples, respectively (False discorey rate, FDR ≤ 0.05). Next, we applied summary-based data Mendelian randomization (SMR) to integrate the cis- eQTL dataset with GWAS data obtained from a cohort of 813 culled cattle that were classified according to the presence or absence of PTB-associated histopathological lesions in gut tissues. After multiple testing corrections (FDR ≤ 0.05), we identified two novel cis-eQTLs affecting the expression of the early growth response factor 4 ( EGR4 ) and the bovine neuroblastoma breakpoint family member 6-like protein isoform 2 ( MGC134040 ) that showed pleiotropic associations with the presence of multifocal and diffuse lesions in gut tissues; P  = 0.002 and P  = 0.017, respectively. While EGR4 acts as a brake on T-cell proliferation and cytokine production through interaction with the nuclear factor Kappa β ( NF-κß), MGC134040 is a target gene of NF-κß . Our findings provide a better understanding of the genetic factors influencing PTB outcomes, confirm that the multifocal lesions are localized/confined lesions that have different underlying host genetics than the diffuse lesions, and highlight regulatory SNPs and regulated-gene targets to design future functional studies.

Tuberculosis BCG vaccination induced non-specific protective effects in humans led to postulate the concept of trained immunity (TRAIM) as an innate type of immune mechanism that triggered by a pathogen, protects against others. Killed vaccines have been considered not to be effective. However, field efficacy of a commercial vaccine against paratuberculosis, as well as of a recently developed M. bovis heat-inactivated vaccine (HIMB) prompted to test whether it could also induce TRAIM. To this, we used a sarcoptic mange rabbit model. Twenty-four weaned rabbits were treated orally or subcutaneously with a suspension of either HIMB (10 7 UFC) or placebo. Eighty-four days later the animals were challenged with approximately 5000 S. scabiei mites on the left hind limb. Skin lesion extension was measured every 2 weeks until 92 days post-infection (dpi). Two animals were killed at 77 dpi because of extensive skin damage. The rest were euthanized and necropsied and the lesion area and the mite burden per squared cm were estimated. Specific humoral immune responses to S. scabiei and to M. bovis were investigated with the corresponding specific ELISA tests. Subcutaneously and orally HIMB vaccinated animals compared with placebo showed reduced lesion scores (up to 74% and 62%, respectively) and mite counts (−170% and 39%, respectively). This, together with a significant positive correlation (r = 0.6276, p  = 0.0031) between tuberculosis-specific antibodies and mite count at 92 dpi supported the hypothesis of non-specific effects of killed mycobacterial vaccination. Further research is needed to better understand this mechanism to maximize cross protection.

Bovine paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic granulomatous enteritis that affects cattle worldwide. According to their severity and extension, PTB-associated histological lesions have been classified into the following groups; focal, multifocal, and diffuse. It is unknown whether these lesions represent sequential stages or divergent outcomes. In the current study, the associations between host genetic and pathology were explored by genotyping 813 Spanish Holstein cows with no visible lesions (N = 373) and with focal (N = 371), multifocal (N = 33), and diffuse (N = 33) lesions in gut tissues and regional lymph nodes. DNA from peripheral blood samples of these animals was genotyped with the bovine EuroG MD Bead Chip, and the corresponding genotypes were imputed to whole-genome sequencing (WGS) data using the 1000 Bull genomes reference population. A genome-wide association study (GWAS) was performed using the WGS data and the presence or absence of each type of histological lesion in a case–control approach. A total of 192 and 92 single nucleotide polymorphisms (SNPs) defining 13 and 9 distinct quantitative trait loci (QTLs) were highly-associated (P ≤ 5 × 10 −7 ) with the multifocal (heritability = 0.075) and the diffuse (heritability = 0.189) lesions, respectively. No overlap was seen in the SNPs controlling these distinct pathological outcomes. The identified QTLs overlapped with some QTLs previously associated with PTB susceptibility, bovine tuberculosis susceptibility, clinical mastitis, somatic cell score, bovine respiratory disease susceptibility, tick resistance, IgG level, and length of productive life. Pathway analysis with candidate genes overlapping the identified QTLs revealed a significant enrichment of the keratinization pathway and cholesterol metabolism in the animals with multifocal and diffuse lesions, respectively. To test whether the enrichment of SNP variants in candidate genes involved in the cholesterol metabolism was associated with the diffuse lesions; the levels of total cholesterol were measured in plasma samples of cattle with focal, multifocal, or diffuse lesions or with no visible lesions. Our results showed reduced levels of plasma cholesterol in cattle with diffuse lesions. Taken together, our findings suggested that the variation in MAP-associated pathological outcomes might be, in part, genetically determined and indicative of distinct host responses.

Mycobacterium avium subsp. paratuberculosis (MAP) infection causes a chronic granulomatous inflammatory regional enteritis in ruminants. Cell-mediated immune responses are assumed to be protective and therefore, to be associated with its more delimited lesion types, while humoral responses are mainly associated with diffuse histopathological lesions. However, this duality of immune responses has been recently questioned. The aim of this study was to assess the relationship between both types of immunological responses and the type and extension of intestinal lesions and the presence of MAP in bovine tissues. Standard histopathological examinations, two microbiological procedures (culture and real time PCR (rtPCR)), as well as MAP specific antibody and interferon gamma (IFN-γ) release assays (IGRA) were performed on tissues and blood of 333 slaughtered Holstein-Friesian animals. Paratuberculous lesions were observed in 176 (52.9%) of the animals and overall MAP detection rates were estimated at 13.5% and 28.5% for tissue culture and rtPCR, respectively. Unlike the relatively constant non-specific IFN-γ release, both the antibody levels and the specific IFN-γ release significantly increased with tissue damage. Delimited immunopathological forms, which accounted for 93.2% of all forms, were mostly related to positive testing in the IGRA (38.4%) whereas diffuse ones (6.8%) were associated with antibody seropositivity (91.7%). However, since the frequency of positive immune responses in both tests increased as the lesions severity increased, polarization of Th1/Th2 responses was less prominent than expected. MAP was detected in the majority of ELISA-positive animals (culture+: 90%, rtPCR+: 85%) but the bacteria was only confirmed in the 36.1% of IGRA-positive animals by any of the two microbiological tests. In terms of diagnosis, the antibody test was a good indicator of advanced tissue damage (diffuse forms), but the IGRA did not associate well with more delimited forms or with MAP detection.

Animal tuberculosis (TB) is a complex animal health problem that causes disruption to trade and significant economic losses. TB involves a multi-host system where sheep, traditionally considered a rare host of this infection, have been recently included. The aims of this study were to develop an experimental TB infection model in sheep with a Mycobacterium caprae field strain isolated from a tuberculous diseased ewe, and to use this to evaluate the safety and efficacy of two vaccines against TB in sheep, the live-attenuated M. bovis BCG vaccine (Danish strain) and a heat-inactivated M. bovis (HIMB) vaccine. Eighteen 2 month-old lambs were experimentally challenged with M. caprae by the endotracheal route (1.5 × 103 CFU). They were separated per treatment group into parenterally vaccinated with a live BCG Danish strain vaccine (n = 6), orally vaccinated with a suspension of HIMB (n = 6) and unvaccinated controls (n = 6). Clinical, immunological, pathological and bacteriological parameters of infection were measured. All lambs were successfully infected and developed gross TB lesions in the respiratory system. The BCG vaccine conferred considerable protection against experimental TB in lambs, as measured by a reduction of the gross lesion volumes and bacterial load. However, HIMB vaccinated animals did not show protection. This study proposes a reliable new experimental model for a better understanding of tuberculosis in sheep. BCG vaccination offers an effective prospect for controlling the disease. Moreover alternative doses and/or routes of administration should be considered to evaluate the efficacy of the HIMB vaccine candidate.

This study aimed to assess the efficacy of a heat-inactivated Mycobacterium caprae (HIMC) vaccine in goats experimentally challenged with the same strain of M. caprae . Twenty-one goats were divided into three groups of seven: vaccinated with heat-inactivated Mycobacterium bovis (HIMB), with HIMC and unvaccinated. At 7 weeks post-vaccination all animals were endobronchially challenged with M. caprae . Blood samples were collected for immunological assays and clinical signs were recorded throughout the experiment. All goats were euthanized at 9 weeks post-challenge. Gross pathological examination, analysis of lung pathology using computed tomography, and bacterial load quantification in pulmonary lymph nodes (LN) by qPCR were carried out. Only HIMC vaccinated goats showed a significant reduction of lung lesions volume and mycobacterial DNA load in LN compared to unvaccinated controls. Both vaccinated groups showed also a significant reduction of the other pathological parameters, an improved clinical outcome and a higher proportion of IFN-γ-producing central memory T cells after vaccination. The results indicated that homologous vaccination of goats with HIMC induced enhanced protection against M. caprae challenge by reducing lung pathology and bacterial load compared to the heterologous vaccine (HIMB). Further large-scale trials are necessary to assess the efficacy of autovaccines under field conditions.

Innate immunity is evolutionarily conserved in multicellular organisms and was considered to lack memory until very recently. One of its more characteristic mechanisms is phagocytosis, the ability of cells to engulf, process and eventually destroy any injuring agent. We report the results of an ex vivo experiment in bovine macrophages in which improved clearance of Mycobacterium bovis (M. bovis) was induced by pre-exposure to a heat killed M. bovis preparation. The effects were independent of humoral and cellular adaptive immune responses and lasted up to six months. Specifically, our results demonstrate the existence of a training effect in the lytic phase of phagocytosis that can be activated by killed mycobacteria, thus suggesting a new mechanism of vaccine protection. These findings are compatible with the recently proposed concept of trained immunity, which was developed to explain the observation that innate immune responses provide unspecific protection against pathogens including other than those that originally triggered the immune response.