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.

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.

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.

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.

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.

Correlations between single nucleotide polymorphisms in bovine CD209, SLC11A1, SP110 and TLR2 genes and estimated breeding values for several traits in Spanish Holstein cattle.

Little is known about the correlations between the genetic susceptibility/resistance to Mycobacterium avium subsp. paratuberculosis (Map) infection and the estimated breeding values for type, production and functional traits. Previously, we identified 70 combinations of five single nucleotide polymorphisms (SNPs) in four bovine innate immune genes (SLC11A1, SP110, TLR2, CD209) that are associated with the genetic risk of paratuberculosis (PTB) or Johne's disease progression, which can be graded as low (LOWIN), latent (LATIN), or patent (PATIN) risk. Other possible combinations of these 5 SNPs were grouped in the average group (AVERIN). In the current study, differences in estimated breeding values (EBVs) for several traits were analyzed using linear models in a large cohort of Holstein cows (N = 15656) genotyped across Spain in 2016 or 2017. After the assignment of each genotyped cow to a risk group, cows within the PATIN risk group (N = 1448) had a superior combined genetic index (2797.57), type genetic index (524.62), milk yield (653.92 kg), protein yield (21.77 kg), fat yield (24.82 kg) and economic merit index (125 Euros) compared with the other three risk groups. Statistically significant differences in the longevity scores between the cows that were included in the PATIN risk group (108.85) and the LOWIN (107.82) and AVERIN (107.92) groups were also observed. The associations between the genetic risk groups and PTB diagnostic results were validated in a population of 99 cows from a Spanish farm with a high prevalence of PTB. Significant differences in ELISA readings between the PATIN (65.49 %) and the AVERIN (15.97 %), LATIN (2.11 %), and LOWIN (3.27 %) groups were observed. In addition, significant differences in Map DNA copies/gram of feces were observed between the PATIN and the other three risk groups. These results together with the substantial economic impact of PTB in dairy cattle support the selection of the animals with less susceptibility to PTB in the Spanish breeding program.

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.