Alternative splicing of pre-mRNA modulates the immune response in Holstein cattle naturally infected with Mycobacterium avium subsp. paratuberculosis.

Little is known about the role of alternative splicing (AS) in regulating gene expression in Mycobacteria-infected individuals in distinct stages of infection. Pre-mRNA AS consists of the removal of introns and the assembly of exons contained in eukaryotic genes. AS events can influence transcript stability or structure with important physiological consequences. Using RNA-Seq data from peripheral blood (PB) and ileocecal valve (ICV) samples collected from Holstein cattle with focal and diffuse paratuberculosis (PTB)-associated histopathological lesions in gut tissues and without lesions (controls), we detected differential AS profiles between the infected and control groups. Four of the identified AS events were experimentally validated by reverse transcription-digital droplet PCR (RT-ddPCR). AS events in several genes correlated with changes in gene expression. In the ICV of animals with diffuse lesions, for instance, alternatively spliced genes correlated with changes in the expression of genes involved in endocytosis, antigen processing and presentation, complement activation, and several inflammatory and autoimmune diseases in humans. Taken together, our results identified common mechanisms of AS involvement in the pathogenesis of PTB and human diseases and shed light on novel diagnostic and therapeutic interventions to control these diseases.

MicroRNAs modulate immunological and inflammatory responses in Holstein cattle naturally infected with Mycobacterium avium subsp. paratuberculosis.

MicroRNAs (miRNAs) regulate the post-transcriptional expression of genes by binding to their target mRNAs. In this study, whole miRNA sequencing was used to compare the expression of miRNAs in ileocecal valve (ICV) and peripheral blood (PB) samples of cows with focal or diffuse paratuberculosis (PTB)-associated lesions in gut tissues versus (vs) control cows without lesions. Among the eight miRNAs differentially expressed in the PB samples from cows with diffuse lesions vs controls, three (miR-19a, miR-144, miR32) were also down-regulated in cows with diffuse vs focal lesions. In the ICV samples, we identified a total of 4, 5, and 18 miRNAs differentially expressed in cows with focal lesions vs controls, diffuse lesions vs controls, and diffuse vs focal lesions, respectively. The differential expression of five microRNAs (miR-19a, miR-144, miR-2425-3p, miR-139, miR-101) was confirmed by RT-qPCR. Next, mRNA target prediction was performed for each differentially expressed miRNA. A functional analysis using the predicted gene targets revealed a significant enrichment of the RNA polymerase and MAPK signaling pathways in the comparison of cows with focal vs no lesions and with diffuse vs focal lesions, respectively. The identified miRNAs could be used for the development of novel diagnostic and therapeutical tools for PTB control.

Lateral-flow assays for bovine paratuberculosis diagnosis.

Mycobacterium avium subsp. paratuberculosis (MAP) causes bovine paratuberculosis (PTB). PTB is responsible for significant economic losses in dairy herds around the word. PTB control programs that rely on testing and culling of test-positive cows have been developed. Current diagnostics, such as ELISA for detecting MAP antibodies in serum samples and PCR detecting MAP DNA in feces, have inadequate sensitivity for detecting subclinical animals. Innovative "omics" technologies such as next-generation sequencing (NGS) technology-based RNA-sequencing (RNA-Seq), proteomics and metabolomics can be used to find host biomarkers. The discovered biomarkers (RNA, microRNAs, proteins, metabolites) can then be used to develop new and more sensitive approaches for PTB diagnosis. Traditional approaches for measuring host antibodies and biomarkers, such as ELISAs, northern blotting, quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR), cDNA microarrays, and mass spectrometry are time-consuming, expensive, and sometimes exhibit poor sensitivity. With the rapid development of nanotechnology, low-cost monitoring devices for measuring antibodies against MAP proteins in point-of-care (POC) settings have been developed. Lateral flow assays (LFAs), in particular, are thought to be appropriate for the on-site detection of antibodies to MAP antigens and/or host biomarkers. This review aims to summarize LFAs that have recently been developed to accurately detect antibodies against MAP antigens, as well as the benefits that host biomarkers linked with MAP infection give to PTB diagnosis. The identification of these novel biomarkers could be the basis for the development of new LFAs. The dairy industry and producers are likely to benefit from reliable and rapid technologies capable of detecting MAP infection in situ to establish a quick and sensitive PTB diagnosis.

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.

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.

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.

The Upregulation of Cathepsin G Is Associated with Resistance to Bovine Paratuberculosis.

An in silico genomic-transcriptomic combined approach allowed the identification of a polymorphism (cis-eQTL-rs41976219) in the Bos taurus genome associated with the CTSG mRNA expression in bovine blood samples, which suggests that individual genetic variation might modulate the CTSG transcriptional response. In the current study, a sandwich ELISA is used to measure the CTSG protein levels in supernatants of monocyte-derived macrophages (MDMs) isolated from cows with the AA (n = 5) and AC (n = 11) genotypes for the rs41976219 and infected ex vivo with MAP. Cows with the AC genotype have significantly higher CTSG protein levels (1.85 ng/mL) in the supernatants of enriched CD14+-MDMs after 2 h of infection when compared with infected CD14+-MDMs from cows with the AA genotype (1.68 ng/mL). Statistically significant differences in the intracellular MAP load at 7 d p.i. are observed between animals with the AA (2.16 log CFUs) and AC (1.44 log CFUs) genotypes. Finally, the association between the rs41976219 allelic variants and resistance to PTB is tested in a larger cattle population (n = 943) classified according to the presence (n = 442) or absence (n = 501) of PTB-associated lesions. The presence of the two minor alleles in the rs41976219 (CC) is more frequent among healthy cows than in cows with PTB-associated lesions in gut tissues (2.2% vs. 1.4%, OR = 0.61). In agreement with this, the CTSG levels in plasma samples of cows without lesions in gut tissues and with the CC (n = 8) genotype are significantly higher than in the plasmas of cows with the AA + AC (n = 36) genotypes.

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.

Genome-wide association studies for the identification of cattle susceptible and resilient to paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease or paratuberculosis (PTB), with important animal health and economic implications. There are no therapeutic strategies to control this disease, and vaccination with inactivated vaccines is limited in many countries because it can interfere with the intradermal test used for bovine tuberculosis detection. Thus, infected animals either get culled after a positive ELISA or fecal PCR result or die due to clinical disease. In this study, we review recent studies aimed to discover genetic markers which could help to identify and select cattle less susceptible and more resilient to PTB. In recent years, the genotyping and subsequent imputation to whole-genome sequence (WGS) has allowed the identification of single-nucleotide polymorphisms (SNPs), quantitative trait loci (QTL), and candidate genes in the Bos taurus genome associated with susceptibility to MAP infection. In most of these genome-wide association studies (GWAS), phenotypes were based on ante-mortem test results including serum ELISA, milk ELISA, and detection of MAP by fecal PCR and bacteriological culture. Cattle infected with MAP display lesions with distinct severity but the associations between host genetics and PTB-associated pathology had not been explored until very recently. On the contrary, the understanding of the mechanisms and genetic loci influencing pathogen resistance, and disease tolerance in asymptomatic individuals is currently very limited. The identification of long-time asymptomatic cattle that is able to resist the infection and/or tolerate the disease without having their health and milk production compromised is important for disease control and breeding purposes.

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.