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

[EN]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 x 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.Financial support for this study was provided by a Grant from the Spanish Ministry of Science, Innovation, and Universities (RTI2018-094192-R-C21) and by European Regional Development Funds (FEDER) to MAH. MC and GBB have been awarded fellowships from the National Institute for Agricultural Research (INIA) and the Spanish Ministry of Science, Innovation and Universities programs, respectively. This work has been possible thanks to the support of the computing infrastructure of the i2BASQUE Research and Academic Network. We gratefully acknowledge the Bull Genomes Consortium for providing accessibility to the WGS data that was used in this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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.Financial support for this study was provided by a grant from the Spanish Ministry of Science, Innovation, and Universities (MICINN, project code: RTI2018-094192-R-C21) and by European Regional Development Funds (FEDER) to MAH. MC and GBB have been awarded fellowships from the National Institute for Agricultural Research (INIA) and MICINN, respectivel

Identifcation of Loci Associated with Susceptibility to Bovine Paratuberculosis and with the Dysregulation of the MECOM, eEF1A2, and U1 Spliceosomal RNA Expression

Although genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with the susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection, only a few functional mutations for bovine paratuberculosis (PTB) have been characterized. Expression quantitative trait loci (eQTLs) are genetic variants typically located in gene regulatory regions that alter gene expression in an allele-specific manner. eQTLs can be considered as functional links between genomic variants, gene expression, and ultimately phenotype. In the current study, peripheral blood (PB) and ileocecal valve (ICV) gene expression was quantified by RNA-Seq from fourteen Holstein cattle with no lesions and with PTB-associated histopathological lesions in gut tissues. Genotypes were generated from the Illumina LD EuroG10K BeadChip. The associations between gene expression levels (normalized read counts) and genetic variants were analyzed by a linear regression analysis using R Matrix eQTL 2.2. This approach allowed the identification of 192 and 48 cis-eQTLs associated with the expression of 145 and 43 genes in the PB and ICV samples, respectively. To investigate potential relationships between these cis-eQTLs and MAP infection, a case-control study was performed using the genotypes for all the identified cis-eQTLs and phenotypical data (histopathology, ELISA for MAP-antibodies detection, tissue PCR, and bacteriological culture) of 986 culled cows. Our results suggested that the heterozygous genotype in the cis-eQTL-rs43744169 (T/C) was associated with the up-regulation of the MDS1 and EVI1 complex (MECOM) expression, with positive ELISA, PCR, and bacteriological culture results, and with increased risk of progression to clinical PTB. As supporting evidence, the presence of the minor allele was associated with higher MECOM levels in plasma samples from infected cows and with increased MAP survival in an ex-vivo macrophage killing assay. Moreover, the presence of the two minor alleles in the cis-eQTL-rs110345285 (C/C) was associated with the dysregulation of the eukaryotic elongation factor 1-alpha2 (eEF1A2) expression and with increased ELISA (OD) values. Finally, the presence of the minor allele in the cis-eQTL rs109859270 (C/T) was associated with the up-regulation of the U1 spliceosomal RNA expression and with an increased risk of progression to clinical PTB. The introduction of these novel functional variants into marker-assisted breeding programs is expected to have a relevant effect on PTB control.Financial support for this study was provided by a grant from the Spanish Ministry of Science, Innovation, and Universities (MICINN, https://sede.micinn.gob.es/, project code: RTI2018-094192-R-C21) and by European Regional Development Funds (FEDER) to MAH. This study was co-funded by a grant from the Plan of Science, Technology, and Innovation of the Principality of Asturias, Regional funds PCTI 2018–2020 (www.ficyt.es/pcti/), project code: IDI2018-000237. MC and CBV have been awarded fellowships from the National Institute for Agricultural Research (INIA) progra

Latent infections are the most frequent form of paratuberculosis in slaughtered Friesian cattle

Paratuberculosis is a chronic mycobacterial infection causing granulomatous enteritis in ruminants, whose pathogenesis and epidemiology poses numerous challenges, including latency and reactivation. The most recent and complete classification of paratuberculosis immunopathological types in cattle recognized five categories. In this study, 1031 slaughtered Friesian cattle were submitted to serological, microbiological and pathological examinations with the aim of maximizing the rate of case detection. In most cases, infected animals had minimal lesions and almost no other proof of infection (38.9%), while the more characteristic types with the whole constellation of microbiological and immunological evidences accounted for a lower proportion (7.7%). As these findings in cattle suggest similarities with the epidemiology of tuberculosis in humans, we propose to re-group the original immunopathological types into two broader paratuberculosis epidemio-pathogenic forms or states: latent and patent. The former term would define infections with focal lesions and might constitute an apparent resilience status representing a difficult to detect reservoir of infection whose role could become critical if later immune-compromising factors lead to re-activation. The latter would group those cases with multifocal and diffuse inflammatory lesions with higher mycobacterial load and viability suggestive of a more immediate epidemiological risk. Interestingly, the relative frequency of presentation of each profile varied with age. The proportion of latent forms remained relatively constant between 33.8% and 54.3% through adulthood from 3 years of age, while patent forms were more frequent during the first years of age and tended to decrease among the oldest individuals