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.

Control of Mycobacterium avium subsp. paratuberculosis load within infected bovine monocyte-derived macrophages is associated with host genetics.

The genetic loci influencing individual resistance to Mycobacterium avium subsp. paratuberculosis (MAP) infection are still largely unknown. In the current study, we searched for genetic loci associated with resistance to MAP infection by evaluating the performance of monocyte-derived macrophages (MDMs) isolated from the peripheral blood of 75 healthy Holsteins cows and infected ex vivo with MAP. Bacterial load (log colony-forming units, log CFUs) within MDMs was quantified at 2 h and 7 days p. i. using a BACTEC MGIT 960 instrument. In addition, the expression levels of some genes with important roles in the innate immune response including epiregulin (EREG), complement component C3 (C3), galectin-9 (Gal9), and nitric oxide (NO-) were measured in the supernatant of the infected cells. DNA from peripheral blood samples of the animals included in the study was isolated and genotyped with the EuroG MD bead Chip (44,779 single nucleotide-polymorphisms, SNPs). Linear mixed models were used to calculate the heritability (h2 ) estimates for each indicator of MDM performance, MAP load within MDMs and EREG, C3, Gal9, and NO-expression. After performing a genome-wide association study, the only phenotypes that showed SNPs with a significant association were the bacterial load within MDMs at 2 h (h2 = 0. 87) and 7 days (h2 = 0.83) p.i. A total of 6 SNPs, 5 candidate genes, and one microRNA on the Bos taurus chromosomes BTA2, BTA17, BTA18, and BTA21 were associated with MAP load at 2 h p.i. Overlap was seen in two SNPs associated with the log CFUs at 2 h and 7 d p.i. The identified SNPs had negative regression coefficients, and were, therefore, associated with a low bacterial load within MDMs. Some of the identified SNPs were located within QTLs previously associated with longevity, reproductive, and udder health traits. Some of the identified candidate genes; Oxysterol Binding Protein Like 6, Cysteine and Serine Rich Nuclear Protein 3, and the Coiled-Coil Domain Containing 92 regulate cellular cholesterol trafficking and efflux, apoptosis, and interferon production, respectively. Taken together, our results define a heritable and distinct immunogenetic profile in MAP-infected macrophages designed to limit bacterial load early after infection.

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.

Bovine Intelectin 2 Expression as a Biomarker of Paratuberculosis Disease Progression.

Paratuberculosis (PTB), a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), is responsible for important economic losses in the dairy industry. Our previous RNA-sequencing (RNA-Seq) analysis showed that bovine intelectin 2 (ITLN2) precursor gene was overexpressed in ileocecal valve (ICV) samples of animals with focal (log2 fold-change = 10.6) and diffuse (log2 fold-change = 6.8) PTB-associated lesions compared to animals without lesions. This study analyzes the potential use of ITLN2, a protein that has been described as fundamental in the innate immune response to infections, as a biomarker of MAP infection. The presence of ITLN2 was investigated by quantitative immunohistochemical analysis of ICV samples of 20 Holstein Friesian cows showing focal (n = 5), multifocal (n = 5), diffuse (n = 5) and no histological lesions (n = 5). Significant differences were observed in the mean number of ITLN2 immunostained goblet and Paneth cells between the three histopathological types and the control. The number of immunolabelled cells was higher in the focal histopathological type (116.9 ± 113.9) followed by the multifocal (108.7 ± 140.5), diffuse (76.5 ± 97.8) and control types (41.0 ± 81.3). These results validate ITLN2 as a post-mortem biomarker of disease progression.

Identification 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-α2 (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.

Detection of latent forms of Mycobacterium avium subsp. paratuberculosis infection using host biomarker-based ELISAs greatly improves paratuberculosis diagnostic sensitivity.

Bovine paratuberculosis (PTB) is a chronic granulomatous enteritis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), responsible for important economic losses in the dairy industry. Current diagnostic methods have low sensitivities for detection of latent forms of MAP infection, defined by focal granulomatous lesions and scarce humoral response or MAP presence. In contrast, patent infections correspond to multifocal and diffuse types of enteritis where there is increased antibody production, and substantial mycobacterial load. Our previous RNA-Seq analysis allowed the selection of five candidate biomarkers overexpressed in peripheral blood of MAP infected Holstein cows with focal (ABCA13 and MMP8) and diffuse (FAM84A, SPARC and DES) lesions vs. control animals with no detectable PTB-associated lesions in intestine and regional lymph nodes. The aim of the current study was to assess the PTB diagnostic potential of commercial ELISAs designed for the specific detection of these biomarkers. The ability of these ELISAs to identify animals with latent and/or patent forms of MAP infection was investigated using serum from naturally infected cattle (n = 88) and non-infected control animals (n = 67). ROC analysis revealed that the ABCA13-based ELISA showed the highest diagnostic accuracy for the detection of infected animals with focal lesions (AUC 0.837, sensitivity 79.25% and specificity 88.06%) and with any type of histological lesion (AUC 0.793, sensitivity 69.41% and specificity 86.57%) improving on the diagnostic performance of the popular IDEXX ELISA and other conventional diagnostic methods. SPARC and MMP8 showed the highest diagnostic accuracy for the detection of animals with multifocal (AUC 0.852) and diffuse lesions (AUC 0.831), respectively. In conclusion, our results suggest that quantification of ABCA13, SPARC and MMP8 by ELISA has the potential for implementation as a diagnostic tool to reliably identify MAP infection, greatly improving early detection of MAP latent infections when antibody responses and fecal shedding are undetectable using conventional diagnostic methods.

RNA-Seq analysis of ileocecal valve and peripheral blood from Holstein cattle infected with Mycobacterium avium subsp. paratuberculosis revealed dysregulation of the CXCL8/IL8 signaling pathway.

Paratuberculosis is chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Whole RNA-sequencing (RNA-Seq) is a promising source of novel biomarkers for early MAP infection and disease progression in cattle. Since the blood transcriptome is widely used as a source of biomarkers, we analyzed whether it recapitulates, at least in part, the transcriptome of the ileocecal valve (ICV), the primary site of MAP colonization. Total RNA was prepared from peripheral blood (PB) and ICV samples, and RNA-Seq was used to compare gene expression between animals with focal or diffuse histopathological lesions in gut tissues versus control animals with no detectable signs of infection. Our results demonstrated both shared, and PB and ICV-specific gene expression in response to a natural MAP infection. As expected, the number of differentially expressed (DE) genes was larger in the ICV than in the PB samples. Among the DE genes in the PB and ICV samples, there were some common genes irrespective of the type of lesion including the C-X-C motif chemokine ligand 8 (CXCL8/IL8), apolipoprotein L (APOLD1), and the interferon inducible protein 27 (IFI27). The biological processes (BP) enriched in the PB gene expression profiles from the cows with diffuse lesions included the killing of cells of other organism, defense response, immune response and the regulation of neutrophil chemotaxis. Two of these BP, the defense and immune response, were also enriched in the ICV from the cows with diffuse lesions. Metabolic analysis of the DE genes revealed that the N-glycan biosynthesis, bile secretion, one-carbon pool by folate and purine metabolism were significantly enriched in the ICV from the cows with focal lesions. In the ICV from cows with diffuse lesions; the valine, leucine and isoleucine degradation route, purine metabolism, vitamin digestion and absorption and the cholesterol routes were enriched. Some of the identified DE genes, BP and metabolic pathways will be studied further to develop novel diagnostic tools, vaccines and immunotherapeutics.