Genome-Wide Association Study Reveals Quantitative Trait Loci and Candidate Genes Associated with High Interferon-gamma Production in Holstein Cattle Naturally Infected with Mycobacterium Bovis.

Mycobacterium bovis (Mb) is the causative agent of bovine tuberculosis (bTb). Genetic selection aiming to identify less susceptible animals has been proposed as a complementary measure in ongoing programs toward controlling Mb infection. However, individual animal phenotypes for bTb based on interferon-gamma (IFNÉ£) and its use in bovine selective breeding programs have not been explored. In the current study, IFNÉ£ production was measured using a specific IFNÉ£ ELISA kit in bovine purified protein derivative (bPPD)-stimulated blood samples collected from Holstein cattle. DNA isolated from the peripheral blood samples collected from the animals included in the study was genotyped with the EuroG Medium Density bead Chip, and the genotypes were imputed to whole-genome sequences. A genome-wide association analysis (GWAS) revealed that the IFNÉ£ in response to bPPD was associated with a specific genetic profile (heritability = 0.23) and allowed the identification of 163 SNPs, 72 quantitative trait loci (QTLs), 197 candidate genes, and 8 microRNAs (miRNAs) associated with this phenotype. No negative correlations between this phenotype and other phenotypes and traits included in the Spanish breeding program were observed. Taken together, our results define a heritable and distinct immunogenetic profile associated with strong production of IFNÉ£ in response to Mb.

Neuropathological lesions in intravenous BCG-stimulated K18-hACE2 mice challenged with SARS-CoV-2.

In the wake of the COVID-19 pandemic caused by SARS-CoV-2, questions emerged about the potential effects of Bacillus Calmette-Guérin (BCG) vaccine on the immune response to SARS-CoV-2 infection, including the neurodegenerative diseases it may contribute to. To explore this, an experimental study was carried out in BCG-stimulated and non-stimulated k18-hACE2 mice challenged with SARS-CoV-2. Viral loads in tissues determined by RT-qPCR, histopathology in brain and lungs, immunohistochemical study in brain (IHC) as well as mortality rates, clinical signs and plasma inflammatory and coagulation biomarkers were assessed. Our results showed BCG-SARS-CoV-2 challenged mice presented higher viral loads in the brain and an increased frequency of neuroinvasion, with the greatest differences observed between groups at 3-4 days post-infection (dpi). Histopathological examination showed a higher severity of brain lesions in BCG-SARS-CoV-2 challenged mice, mainly consisting of neuroinflammation, increased glial cell population and neuronal degeneration, from 5 dpi onwards. This group also presented higher interstitial pneumonia and vascular thrombosis in lungs (3-4 dpi), BCG-SARS-CoV-2 mice showed higher values for TNF-α and D-dimer values, while iNOS values were higher in SARS-CoV-2 mice at 3-4 dpi. Results presented in this study indicate that BCG stimulation could have intensified the inflammatory and neurodegenerative lesions promoting virus neuroinvasion and dissemination in this experimental model. Although k18-hACE2 mice show higher hACE2 expression and neurodissemination, this study suggests that, although the benefits of BCG on enhancing heterologous protection against pathogens and tumour cells have been broadly demonstrated, potential adverse outcomes due to the non-specific effects of BCG should be considered.

Non-specific protection against severe COVID-19 associated to typhoid fever and DTP vaccination.

Trained immunity (TRAIM) or the enhanced non-specific immune response after primary stimulation by infection or vaccination is a recent but well-recognized concept. To verify its predictions, our objective was to determine the effects of two bacterial vaccines, typhoid fever (TFV) and diphtheria-tetanus-pertussis (DTP) on the infection, hospitalization and death frequencies associated to COVID-19 in a retrospective study on subjects vaccinated or not with TFV and DTP in the 4 years prior to the start of COVID-19 pandemia in the Basque Country (Spain). The studied outcome records were split into two periods according to COVID-19 vaccination, the pre-vaccination (ACV) from March to December 2020 and the post-vaccination (PCV) from September 2021 to June 2022). In total, 13,673 subjects were vaccinated against TFV and 42,997 against DTP. A total of 2,005,084 individual records were studied in the ACV period and 1,436,693 in the PCV period. The proportion of infection, hospitalization and death associated to COVID-19 among controls in ACV was 4.97 %, 7.14 % and 3.54 %, respectively vs. 7.20 %, 2.24 % and 0.10 % among TFV subjects. Regarding DTP, the proportions were 4.97 %, 7.12 % and 3.58 % for controls and 5.79 %, 5.79 % and 0.80 % for vaccinees. In the PCV period, the proportion of infection, hospitalization and death among controls was 21.89 %, 2.62 % and 0.92 %, respectively vs. 31.19 %, 0.76 %, 0.00 % among TFV. For DTP, infection, hospitalization and death proportions were 21.89 %, 2.62 % and 0.92 %, respectively, among controls vs. 32.03 %, 1.85 % and 0.24 % among vaccinated subjects. The corresponding combined ACV and PCV odds ratios (OR) for SARS-CoV2 infection were 1.505 (95%CI 1.455-1.558; p < 0.0001; reduction -41.85 %) and 1.633 (95%CI 1.603-1.662; p < 0.0001; reduction -51.74 %), for TFV and DTP, respectively. Regarding COVID-19 associated hospitalization, the OR were 0.295 (95%CI 0.220-0.396; p = 0.0001; reduction 69.74 %) and 0.667 (95%CI 0.601-0.741; p = 0.0001; reduction 32.44 %), for TFV and DTP, respectively). COVID-19 associated death OR were 0.016 (95%CI 0.002-0.113, p < 0.0001; reduction 98.38 %) and 0.212 (95%CI 0.161-0.280; p = 0.0001; reduction 78.52 %), for TFV and DTP, respectively. We conclude that TRAIM effects by TFV and DTP vaccination in the four years prior to the pandemic SARS-CoV2 were supported by slightly increased infection rates, but strongly reduced COVID-19 associated hospitalization and death rates.

Non-specific effects of inactivated Mycobacterium bovis oral and parenteral treatment in a rabbit scabies model.

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 (107 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.

Summary-data based Mendelian randomization identifies gene expression regulatory polymorphisms associated with bovine paratuberculosis by modulation of the nuclear factor Kappa ß (NF-κß)-mediated inflammatory response.

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 ß (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.

Efficacy of heat-inactivated Mycobacterium bovis vaccine delivered to European badgers (Meles meles) through edible bait.

Badgers (Meles meles) are a major tuberculosis (TB) reservoir in Europe, with the potential to transmit infection to cattle. Here we assessed whether a recently described oral tuberculosis vaccine based on heat-inactivated Mycobacterium bovis (HIMB), delivered as edible baits, can protect badgers from infection. Eight badgers were given individually five baits, each one consisting of a ball of peanut butter, natural peanut and oat flakes including a dose of the vaccine containing 5 × 107 colony-forming units. In parallel, a control group of seven badgers did not receive the vaccine. One month and a half later a second dose of the vaccine was offered to the vaccinated group. Ninety-four days after the second dose, all badgers were challenged with M. bovis (103 colony-forming units per animal) delivered endobronchially to the right middle lung lobe. Clinical, immunological, pathological and bacteriological variables were measured throughout the whole study to assess the efficacy of the vaccine. Two vaccinated animals showed high bacterial load of M. bovis and worsening of pathological lesions of TB. Conversely, the other six vaccinated animals showed slight improvement in bacterial load and pathology with respect to the control group. These results suggest that delivering the TB vaccine via food bait can partially protect wild badger populations, although vaccination can lead to either protection or tolerization, likely depending on the animal's immune status and general condition at the time of vaccination. Further optimization of the vaccination trial/strategy is needed to reduce the rate of tolerization, such as altering vaccine dose, number of doses, type of bait, use of adjuvants or route of administration.

Association between High Interferon-Gamma Production in Avian Tuberculin-Stimulated Blood from Mycobacterium avium subsp. paratuberculosis-Infected Cattle and Candidate Genes Implicated in Necroptosis.

The mechanisms underlying host resistance to Mycobacterium avium subsp. paratuberculosis (MAP) infection are largely unknown. In the current study, we hypothesize that cows with an ability to produce higher levels of interferon-gamma (IFNÉ£) might control MAP infection more successfully. To test this hypothesis, IFNÉ£ production was measured using a specific IFNÉ£ ELISA kit in avian purified protein derivative (aPPD)-stimulated blood samples collected from 152 Holstein cattle. DNA isolated from peripheral blood samples of the animals included in the study was genotyped with the EuroG Medium-Density Bead Chip, and the genotypes were imputed to whole-genome sequencing. A genome-wide association analysis (GWAS) revealed that high levels of IFNÉ£ in response to the aPPD were associated with a specific genetic profile (heritability = 0.64) and allowed the identification of 71 SNPs, 40 quantitative trait loci (QTL), and 104 candidate genes. A functional analysis using the 104 candidate genes revealed a significant enrichment of genes involved in the innate immune response and, more specifically, in necroptosis. Taken together, our results define a heritable and distinct immunogenetic profile associated with the production of high IFNÉ£ levels and with the capacity of the host to lyse MAP-infected macrophages by necroptosis.

Oral immunization with heat-inactivated Mycobacterium bovis reduces local parasite dissemination and hepatic granuloma development in mice infected with Leishmania amazonensis.

Aiming to explore whether oral immunization with heat-inactivated Mycobacterium bovis (HIMB) protects mice against Leishmania infection, 18 female BALB/c mice were randomly assigned to the immunized group, that received oral HIMB, or the control group, and were infected by inoculation of 10,000 Leishmania amazonensis promastigotes in the footpad. Spleen culture was positive in 55.55% of immunized mice and in 100% of control mice (p = 0.082). The number of immunolabeled amastigotes number in the popliteal lymph node was lower in the immunized group (p = 0.009). The immunized group presented fewer mature granulomas in the liver (p = 0.005) and more Lys + macrophages (p = 0.002) and fewer CD3+ T lymphocytes (p < 0.001) per hepatic granuloma. We conclude that immunization with HIMB via the oral route limited local parasite dissemination and hepatic granuloma development in mice challenged with Leishmania amazonensis through stimulation of macrophages, which is compatible with trained immunity.

Differences in skin test reactions to official and defined antigens in guinea pigs exposed to non-tuberculous and tuberculous bacteria.

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.

A humoral diagnostic test outperforms cellular tests in a farm with a latent tuberculosis outbreak caused by a new Mycobacterium tuberculosis complex spoligotype that affected sheep but not goats.

Tuberculosis (TB) is a disease caused by members of the M. tuberculosis complex (MTC) that affects numerous species. M. caprae, a member of the complex which is close to M. bovis, is emerging and affects several different hosts that include goats, cattle, sheep, pigs, rabbits, wild boar, red deer, foxes and also humans. A new M. caprae spoligotype (SB2737) was isolated from an outbreak of sheep tuberculosis affecting a mixed sheep (323)-goat (29) farm in 2021. The index case was detected by the La Rioja slaughterhouse veterinary inspection. Tracing back to the farm of origin, both species were submitted to Comparative Intradermal Tuberculin Test (CITT) and M. bovis-specific antibody ELISA tests. A subsample was also examined by IFN-γ release assay (IGRA) and all positives were slaughtered and pathologically and microbiologically investigated. Only 1.2% of sheep and no goat were positive in the CITT, and 11.4% in the IGRA sheep subsample, while up to 36.8% were positive in two consecutive M. bovis-specific antibody ELISA tests. Goats had always tested negative in annual intradermal follow-up since 2013. Upon confirmation of the immunologically positive sheep at slaughter, all the remaining negative animals were killed and 29.2% of sheep were still found infected. This raised the final overall prevalence to 37.5%. Antibody ELISA was the most sensitive (81.4%) in vivo detection method still showing a 85.0% specificity relative to pathological and microbiological tuberculosis status. It was nearly 10 times more sensitive than skin test and had an 86.8% positive predictive value. Notwithstanding a possible singular pathogenesis of the new spoligotype, this outbreak adds up to previous reports suggesting that sheep tuberculosis could be huge reservoir of infection worldwide overlooked by skin test low sensitivity or simply lack of investigation. This makes it urgent to extend the use antibody tests to address the Trojan horse of hidden M. tuberculosis complex infections on bovine TB control programs.

Evaluation of a droplet digital PCR assay for quantification of Mycobacterium avium subsp. paratuberculosis DNA in whole-blood and fecal samples from MAP-infected Holstein cattle.

Bovine paratuberculosis (PTB) is an infectious disease that affects ruminants worldwide and is a burden on the dairy industry. PTB control measures include culling of Mycobacterium avium subsp. paratuberculosis (MAP)-infected animals from the herd and the enhancement of farm-biosecurity measures. Diagnostics tools for the direct detection of MAP are fecal real-time qPCR and bacteriological culture, the last one being considered the gold standard. However, both show limitations for detecting subclinical MAP-infected cattle with low bacterial load in feces and gut tissues. Droplet digital polymerase chain reaction (ddPCR) is a third-generation PCR method that shows high reproducibility for the quantification of low DNA copy numbers. The objective of this study was to design a ddPCR assay to detect and quantify a fragment of the F57 MAP-specific sequence in samples of naturally MAP-infected Holstein cattle. DNA was isolated from whole-blood and fecal samples from control cows with a negative ELISA and qPCR result (N = 75) and from cows with PTB-associated focal (N = 32), multifocal (N = 21), and diffuse lesions (N = 17) in gut tissues. After ddPCR, the DNA extracted from fecal samples of cows with diffuse lesions showed higher mean copies per microliter (13,791.2 copies/µl) than samples from cows with multifocal lesions (78.8 copies/µl), focal lesions (177.1 copies/µl) or control cows (4.8 copies/µl) (P ≤ 0.05). Significant differences in mean DNA copies/µl were also observed in the blood samples from cows with focal lesions (47.7 copies/µl) when compared with cows with multifocal and diffuse lesions; 18.1 and 12.4 copies/µl, respectively. Using a principal component analysis, the results of the fecal ddPCR clustered together with the results of a commercial ELISA for the specific detection of MAP antibodies, fecal and tissue qPCR, and bacteriological culture results. In contrast, blood ddPCR results clustered together with the results of an ELISA for the detection of a biomarker of subclinical PTB, the ABCA13 transporter. Blood ddPCR was the most sensitive tool (sensitivity 71%, specificity 100%) of all the quantitative methods used in the study for the detection of subclinical cows with focal lesions.

Heat inactivated mycobacteria, alpha-Gal and zebrafish: Insights gained from experiences with two promising trained immunity inductors and a validated animal model.

Trained immunity (TRAIM) may be defined as a form of memory where innate immune cells such as monocytes, macrophages, dendritic and natural killer (NK) cells undergo an epigenetic reprogramming that enhances their primary defensive capabilities. Cross-pathogen protective TRAIM can be triggered in different hosts by exposure to live microbes or microbe-derived products such as heat-inactivated Mycobacterium bovis or with the glycan α-Gal to elicit protective responses against several pathogens. We review the TRAIM paradigm using two models representing distinct scales of immune sensitization: the whole bacterial cell and one of its building blocks, the polysaccharides or glycans. Observations point out to macrophage lytic capabilities and cytokine regulation as two key components in non-specific innate immune responses against infections. The study of the TRAIM response deserves attention to better characterize the evolution of host-pathogen cooperation both for identifying the aetiology of some diseases and for finding new therapeutic strategies. In this field, the zebrafish provides a convenient and complete biological system that could help to deepen in the knowledge of TRAIM-mediated mechanisms in pathogen-host interactions.

Corrigendum: Overview of Cattle Diseases Listed Under Category C, D or E in the Animal Health Law for Which Control Programmes Are in Place Within Europe.

[This corrects the article DOI: 10.3389/fvets.2021.688078.].

Use of ATP-Binding Cassette Subfamily A Member 13 (ABCA13) for Sensitive Detection of Focal Pathological Forms of Subclinical Bovine Paratuberculosis.

Bovine paratuberculosis (PTB) is a chronic enteritis caused by Mycobacterium avium subspecies paratuberculosis (Map) that causes a heavy economic impact worldwide. Map infected animals can remain asymptomatic for years while transmitting the mycobacteria to other members of the herd. Therefore, accurate detection of subclinically infected animals is crucial for disease control. In a previous RNA-Seq study, we identified several mRNAs that were overexpressed in whole blood of cows with different PTB-associated histological lesions compared with control animals without detected lesions. The proteins encoded by two of these mRNAs, ATP binding cassette subfamily A member 13 (ABCA13) and Matrix Metallopeptidase 8 (MMP8) were significantly overexpressed in whole blood of animals with focal histological lesions, the most frequent pathological form in the subclinical stages of the disease. In the current study, the potential of sensitive early diagnostic tools of commercial ELISAs, based on the detection of these two biomarkers, was evaluated in serum samples of 704 Holstein Friesian cows (566 infected animals and 138 control animals from PTB-free farms). For this evaluation, infected animals were classified into three groups, according to the type of histological lesions present in their gut tissues: focal (n = 447), multifocal (n = 59), and diffuse (n = 60). The ELISA based on the detection of ABCA13 was successfully validated showing good discriminatory power between animals with focal lesions and control animals (sensitivity 82.99% and specificity 80.43%). Conversely, the MMP8-based ELISA showed a poor discriminatory power between the different histological groups and non-infected controls. The ABCA13-based ELISA showed a higher diagnostic value (0.822) than the IDEXX ELISA (0.517), the fecal bacterial isolation (0.523) and the real-time PCR (0.531) for the detection of animals with focal lesions. Overall, our results indicate that this ABCA13 ELISA greatly improves the identification of subclinically infected animals with focal lesions that are undetectable using current diagnostic methods.

A Genome-Wide Association Study for Tolerance to Paratuberculosis Identifies Candidate Genes Involved in DNA Packaging, DNA Damage Repair, Innate Immunity, and Pathogen Persistence.

Although the genetic susceptibility to diseases has been extensively studied, the genetic loci and the primary molecular and cellular mechanisms that control disease tolerance are still largely unknown. Bovine paratuberculosis (PTB) is an enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). PTB affects cattle worldwide and represents a major issue on animal health. In this study, the associations between host genetic and PTB tolerance were investigated using the genotypes from 277 Spanish Holstein cows with two distinct phenotypes: cases) infected animals with positive PCR and bacteriological culture results but without lesions in gut tissues (N= 24), and controls) animals with negative PCR and culture results but with PTB-associated lesions (N= 253). DNA from peripheral blood of the study population was genotyped with the Bovine EuroG MD Bead Chip, and the corresponding genotypes were imputed to whole-genome sequencing (WGS) data. A genome-wide association study was performed using the WGS data and the defined phenotypes in a case-control approach. A total of 142 single nucleotide polymorphisms (SNPs) were associated (false discovery rate ≤ 0.05, P values between 1.5 × 10-7 and 5.7 × 10-7) with tolerance (heritability= 0.55). The 40 SNPs with P-values < 5 × 10-7 defined 9 QTLs and 98 candidate genes located on BTA4, BTA9, BTA16, BTA25, and BTA26. Some of the QTLs identified in this study overlap with QTLs previously associated with PTB, bovine tuberculosis, mastitis, somatic cell score, bovine diarrhea virus persistent infection, tick resistance, and length of productive life. Two candidate genes with important roles in DNA damage response (ERCC4 and RMI2) were identified on BTA25. Functional analysis using the 98 candidate genes revealed a significant enrichment of the DNA packaging process (TNP2/PRMI1/PRM2/PRM3). In addition, the TNF-signaling (bta04668; TRAF5/CREB5/CASP7/CHUK) and the toxoplasmosis (bta05145; TGFß2/CHUK/CIITA/SOCS1) pathways were significantly enriched. Interestingly, the nuclear Factor NF-κß Inhibitor Kinase Alpha (CHUK), a key molecule in the regulation of the NF-κB pathway, was enriched in both pathways. Taken together, our results define a distinct immunogenetic profile in the PTB-tolerant animals designed to control bacterial growth, modulate inflammation, limit tissue damage and increase repair, thus reducing the severity of the disease.

Identification of loci associated with pathological outcomes in Holstein cattle infected with Mycobacterium avium subsp. paratuberculosis using whole-genome sequence data.

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.

Identification of loci associated with susceptibility to Mycobacterium avium subsp. paratuberculosis infection in Holstein cattle using combinations of diagnostic tests and imputed whole-genome sequence data.

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 (h2) 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 h2 estimates > 0.080. After performing the GWAS, the test combinations that showed SNPs with a significant association (PFDR ≤ 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.