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

17 páginas, 5 tablas, 5 figuras.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 (IF127). 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.This work was supported by grants from the Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA) and by European Funds for Regional Development (FEDER) (INIA RTA2014-00009-C02 and RTA2018-094192). The study is partially funded by the Principado de Asturias (PCTI 2018-2020, GRUPIN: IDI2018-000237). Maria Canive and Cristina Blanco-Vazquez are recipients of INIA fellowships. The authors thank ASTEGA Veterinary services for their assistance on sample collection. We are also grateful to Kyle Hearn for the careful editing of the manuscript

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

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

[EN] 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 immunotherapeuticsSIThis work was supported by grants from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and by European Funds for Regional Development (FEDER) (INIA RTA2014-00009-C02 and RTA2018-094192). Te study is partially funded by the Principado de Asturias (PCTI 2018–2020, GRUPIN: IDI2018-000237). Maria Canive and Cristina Blanco-Vazquez are recipients of INIA fellowships. Te authors thank ASTEGA Veterinary services for their assistance on sample collection. We are also grateful to Kyle Hearn for the careful editing of the manuscrip

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

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

[EN] 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 methodsSIThis study was part of the I+D+i project (RTI2018- 094192-R-C22) and was funded by the Spanish MCIN/AEI/10.13039/501100011033/ Ministry of Science, Innovation and the European Regional Development Funds (FEDER Una manera de hacer Europa) and by the Gobierno del Principado de Asturias, Regional funds PCTI 2021– 2023 (GRUPIN: IDI2021-000102) co-funded by FEDER. We acknowledge the National Institute for Agricultural Research (INIA) for the scholarships of CB-V (Ayuda CPD2016-0142 financiada por MCIN/AEI/ 10.13039/501100011033 y FSE El FSE invierte en tu future) and M

Increased lytic efficiency of bovine macrophages trained with killed mycobacteria

Innate immunity is evolutionarily conserved in multicellular organisms and was considered to lack memory until very recently. One of its more characteristic mechanisms is phagocytosis, the ability of cells to engulf, process and eventually destroy any injuring agent. We report the results of an ex vivo experiment in bovine macrophages in which improved clearance of Mycobacterium bovis (M. bovis) was induced by pre-exposure to a heat killed M. bovis preparation. The effects were independent of humoral and cellular adaptive immune responses and lasted up to six months. Specifically, our results demonstrate the existence of a training effect in the lytic phase of phagocytosis that can be activated by killed mycobacteria, thus suggesting a new mechanism of vaccine protection. These findings are compatible with the recently proposed concept of trained immunity, which was developed to explain the observation that innate immune responses provide unspecific protection against pathogens including other than those that originally triggered the immune response.Funding for these studies was provided by the EU project WildTBVac (Contract #613799) and by grants from the Instituto Nacional de Investigación y Tecnología Agraria y alimentaria (INIA, RTA2011-00049) and the Ministry of Science (MINECO, AGL2014-56305) and European Funds Regional Development (FEDER).Peer Reviewe

Association between combinations of genetic polymorphisms and epidemiopathogenic forms of bovine paratuberculosis

[EN] Control of major mycobacterial diseases affecting livestock is a challenging issue that requires different approaches. The use of genetic markers for improving resistance to Mycobacterium avium subsp. paratuberculosis infection in cattle has been explored as a promising population strategy We performed paratuberculosis epidemiopathogenic phenotypic and genotypic characterization involving 24 SNPs in six candidate genes (NOD2, CD209, SLC11A1, SP110, TLR2 and TLR4) on 502 slaughtered Friesian cows. In the current study, we investigate whether recently proposed paratuberculosis (PTB) epidemiopathogenic (EP) forms (apparently free-AF, latent-LAT and patent-PAT) could be associated with some combination of these 24 SNPs. Best EP form grouping was obtained using a combination of 5 SNPs in four genes (CD209: rs210748127; SLC11A1: rs110090506; SP110: rs136859213 and rs110480812; and TLR2: rs41830058). These groups were defined according to the level of infection progression risk to patent epidemiopathogenic forms and showed the following distributions: LOWIN (low) with 39 (8%) cases (94.9% AF/5.1% LAT/0% PAT); LATIN (low) with 17 (3%) cases (5.9% AF/94.1% LAT/0% PAT); AVERIN (average) with 413 (82%) cases (52.1% AF/38.5% LAT/9.4% PAT) and PATIN (patent) with 33 (7%) cases (36.4% AF/24.2% LAT/39.4% PAT). Age of slaughter was significantly higher for LATIN (88.3 months) compared to AVERIN (65.3 months; p = 0.0007) and PATIN (59.1 months; p = 0.0004), and for LOWIN (73.9 months) compared to PATIN (p = 0.0233), and nearly significant compared to AVERIN (p = 0.0572) These results suggest that some selected genetic polymorphisms have a potential use as markers of PTB EP forms and thus add a new tool for the control of this widespread infectionSIThis work was supported by the Ministry of Economy and Competitiveness (MINECO) (projects AGL2006-14315-C02 and RTA2014-00009), Basque Government (GV/EJ) (SAIOTEK program: SA-2010/00102), European Regional Development Fund (ERDF), and European Social Fund (ESF) is also gratefully acknowledged. Patricia V azquez was holder of a graduate fellowship award (FPI) (BES-2007-17170) from the Spanish MINEC

Evaluation of the bovine ATP-binding cassette subfamily A member 13 (ABCA13) as a potential biomarker for sensitive detection of animals with focal pathological forms of subclinical paratuberculosis

Trabajo presentado al: ICP 15th International Association for Paratuberculous, Dublín, 12-16 Junio. 2022.Peer reviewe