Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for Johne’s disease (JD), a contagious granulomatous gastroenteritis with global distribution. This study investigated the expression of miRNAs and their potential regulatory mechanisms in Canadian Holstein cows with different MAP infection status, including 4 infected (MAPI), 5 tolerant to MAP infection (MAPT) and 5 healthy controls (HC). Intestinal tissues, jejunum (JE), jejunal lymph node (JELN) and caecal Peyer’s patches (CPP) were collected for miRNA-sequencing, differential expression and functional analysis. A total of 318, 281 and 251 miRNAs were identified in JE, JELN, and CPP, respectively. Out of these, 28, 46, and 24 were highly expressed in JE, JELN and CPP, respectively, including 23 miRNAs highly expressed across all tissues. Their functional enrichment indicates overrepresentation in mostly immune related functions. More differentially expressed (DE) miRNAs were identified in CPP (39, 21 and 11) followed by the JELN (25, 8 and 13) and JE (9, 9 and 15) for the comparisons MAPI vs HC, MAPT vs HC, and MAPI vs MAPT, respectively. Six common DE miRNAs (bta-miR-125a, bta-miR-146a, bta-miR-146b, bta-miR-21-5p, bta-miR-320a, bta-miR-370) (FDR < 0.1) in the JELN and CPP of MAPI cows are implicated in the immune response, suggesting roles in MAP infection. Similarly, more functional annotations were recorded for the CPP (1284) than JELN (377) and JE (four). DE miRNAs in JELN and CPP were enriched in gene ontology (GO) terms (e.g. lymphocyte activation, lymphocyte homeostasis, leukocyte differentiation, regulation of cell differentiation, T cell receptor complex, alpha–beta T cell activation) and KEGG pathways (e.g. T/B cell receptor signaling pathways, Chemokine signaling pathway, Leukocyte transendothelial migration, Th17 cell differentiation, MAPK signaling pathway, etc.) with immune related functions indicative of participation in the regulatory mechanisms of the host immune response to MAP infection. The comparison MAPI vs HC revealed a more pronounced immune response in JELN and CPP indicative of a heightened immune response, but fewer DE miRNAs and less pronounced immune activation for MAPT vs HC indicative of the development of tolerance to MAP presence. MiRNA expression and functional enrichment in JE, JELN, and CPP highlightes tissue-specific regulation of immune pathways and cellular functions during JD. Moreover, largely a different set of DE miRNAs, biological processes and pathways were driving the MAPI and MAPT phenotypes. These findings underscore the complex interplay between miRNAs and MAP in the studied tissues and provide a foundation for further exploration of miRNAs as potential biomarkers for the management of JD.

This study aimed to develop a multiplex quantitative PCR (qPCR) assay for the detection of Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of paratuberculosis disease, a chronic and endemic infectious disease affecting ruminant livestock worldwide. Infected animals may remain asymptomatic for years while intermittently shedding MAP into their environment through feces, contributing to ongoing transmission. To develop a robust multiplex qPCR assay, we reviewed all TaqMan qPCR studies published since 1990 and selected 18 primer-probe combinations targeting the MAP-specific F 57 gene and the repetitive sequence elements ISMAP 02 and IS 900 . In samples with moderate to high MAP levels, all combinations performed well, with only minor differences in analytical performance. However, in low-abundance samples, several TaqMan designs yielded unreliable results, indicating limited specificity in complex matrices. Among the evaluated assays, the IS 900 -Herthnek design demonstrated significantly higher diagnostic sensitivity, detecting MAP in 81% of low-abundance samples, compared to 0% and 3% for the IS 900 -Kim and IS 900 -Slana assays, respectively. For ISMAP 02 , only the ISMAP 02 -Sevilla assay produced reliable results. For F 57 , Herthnek provided the most consistent and accurate quantification. Similar trends were observed in environmental sample testing. Based on these findings, we recommend a multiplex qPCR assay incorporating the IS 900 -Herthnek, ISMAP 02 -Sevilla, and F 57 -Herthnek TaqMan designs for the detection of MAP in fecal and environmental samples. This combination offers high analytical sensitivity and specificity, making it a valuable and accurate tool for the diagnosis of paratuberculosis and for environmental surveillance on dairy farms to identify herds potentially harboring MAP-infected animals. Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease (JD) in ruminant livestock industries and has been associated with Crohn’s disease in humans. Emerging scientific evidence also links MAP to other human conditions, including inflammatory bowel disease, autoimmune disorders, colorectal cancer, and Alzheimer’s disease. This potential public health threat has intensified interest in developing more sensitive diagnostic tools and effective control strategies to eradicate MAP from dairy herds. Infected ruminants typically remain in the subclinical stage of the disease for 2–5 years, during which they shed MAP in their feces and contaminate the environment. Diagnosis during this stage is particularly challenging, as the pathogen evades the host’s immune response, rendering serological tests insufficiently sensitive. In contrast, fecal PCR offers greater sensitivity than serum ELISA and traditional culture methods. Multiplex quantitative PCR is especially promising due to its high specificity and sensitivity for detecting MAP-infected animals and identifying herds with active shedders. Herd-level environmental screening, followed by individual animal testing, represents a robust national biosecurity strategy. This approach is a critical step toward reducing MAP transmission and improving herd health within the dairy industry.

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease (JD), a chronic enteritis infection affecting ruminants with significant economic implications for the dairy industry. The molecular regulatory mechanisms governing host responses to MAP infection remain unclear. This study elucidated the roles of microRNAs (miRNAs) in the ileum (IL), ileal lymph node (ILLN), and liver of cows during subclinical MAP infection. Tissues (IL, ILLN, and liver) were collected from four JD-positive (JDP), five MAP-tolerant (MAPT), and five healthy control (HC) cows. MiRNA transcriptomes were analyzed using miRNA-sequencing, differential miRNA expression, correlation, and functional analyses. A total of 441, 405, and 201 miRNAs were identified in IL, ILLN, and liver, respectively, including 12 miRNAs highly expressed across all three tissues. ILLN exhibited more differentially expressed (DE) miRNAs, including 20 (JDP vs. HC), 21 (MAPT vs. HC), and 15 (JDP vs. MAPT) than the IL: 17 (JDP vs. HC), 8 (MAPT vs. HC), and 6 (JDP vs. MAPT). MiRNAs DE in both IL and ILLN (bta-miR-146b, bta-miR-375, bta-miR-21-5p, bta-miR-146a, bta-miR-125a, bta-miR-100, bta-miR-147, bta-miR-99a-5p, bta-miR-320 from JDP vs. HC, and bta-miR-146a, bta-let-7b from MAPT vs. HC comparisons) have roles in the immune process suggesting their potential impacts on the host response to MAP infection. The target genes of IL DE miRNAs were overrepresented in more gene ontology terms (68) while those of the ILLN were overrepresented in more pathways (20) demonstrating tissue specificity. In addition, the majority of DE miRNAs and overrepresented functional terms were unique to comparison groups and phenotypes while few were shared by two or more comparison groups and phenotype or infection status. Most of the functional terms such as cytokine receptor activity, regulation of leukocyte mediated immunity, T cell receptor signaling pathway, positive regulation of interleukin-4 production among others, have immune related functions revealing important roles of DE miRNAs in the host immune response to MAP infection. In summary, these findings underscore the pivotal regulatory roles of the identified miRNAs in shaping the host response of cows to JD. Moreover, the results indicated that mostly different sets of miRNAs, biological process and pathways were driving the JDP and MAPT phenotypes, as well as emphasize the tissue-specific nature of the miRNA response to JD, shedding light on the nuanced intricacies of host-pathogen interactions in response to MAP infection.

Background. DNA methylation contributes to the elaboration of phenotypes and is hypothesized to account for interindividual variations in farm animals. Currently, methodologies available to investigate DNA methylation in cattle rely on high throughput sequencing, which cannot be applied to large cohorts. To enable large scale DNA methylation analysis, we developed the RUMIGEN EpiChip, the first DNA methylation array specifically designed for cattle. Results. Manufactured by Illumina, the EpiChip contains 43,317 CpG markers allowing analysis of phenotypes of agronomical interest as well as the study of regulatory processes. The assay design drew on data from numerous studies achieved by the scientific community, and includes CpGs where methylation varies with health status, physiological stage, fertility, and environmental challenges, as well as CpGs located in functional genomic elements (promoters, CTCF binding sites, expression quantitative trait loci). The technical performances of the EpiChip were tested on several semen and blood DNA samples and in two laboratories, showing excellent repeatability, accuracy and interoperability. Methylation values were also concordant with those obtained by reduced representation bisulfite sequencing. The EpiChip has demonstrated a good ability to explore biological processes such as genomic imprinting and differences between cell types, opening the possibility of inferring blood cell composition. Finally, analysis of longitudinal ear biopsies allowed accurate age prediction, suggesting the potential of the array to refine epigenetic clocks. Conclusions. The RUMIGEN EpiChip is a cost-effective and versatile resource that opens new opportunities for the study of regulatory mechanisms underlying phenotypic variation and for large-scale association analyses in cattle. ### Competing Interest Statement The authors have declared no competing interest. European Union’s Horizon 2020, 101000226 Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-21-CE20-0021 APIS-GENE

This study aimed to determine the impact of supplementations of copper, vitamins A and D (ADCU), and a bovine colostrum extract (BC) on the micronutrient status, antioxidant status, and intestinal microbiota of piglets until the post-weaning period. Twenty-three sows were fed conventional gestation and lactation diets, and twenty-four sows were fed conventional diets supplemented with ADCU. For each litter, all piglets received one of four treatments during lactation: no supplementation; ADCU; BC; and ADCU + BC. Within each litter, one low (LW) and one high birth weight (HW) piglet were euthanized before and after weaning to collect liver and intestinal samples. Serum vitamin D, liver retinol, and liver Cu were greater in ADCU piglets (p < 0.01), mostly before weaning. After weaning, liver Cu decreased markedly with a drop of 75% in all treatments, despite high levels of Cu in their post-weaning diets. The antioxidant status of piglets was not globally altered by treatments (p > 0.05). For microbiota composition, sow supplementation increased (p < 0.01) richness in bacterial species in the piglet colon, either before or shortly after weaning. Short-chain fatty acids in caecal digesta were increased by sow supplementation in LW piglets before weaning at 16 days of age (p < 0.05). In conclusion, oral supplementations to piglets increased postnatal micronutrient statuses during lactation, but this did not generally persist after weaning. Treatments to sows or piglets did not improve the response of piglets to oxidative stress, but supplementation to sows favoured gut microbiota diversity, particularly in LW piglets.

Bacteria encounter various stressful conditions within a variety of dynamic environments, which they must overcome for survival. One way they achieve this is by developing phenotypic heterogeneity to introduce diversity within their population. Such distinct subpopulations can arise through endogenous fluctuations in regulatory components, wherein bacteria can express diverse phenotypes and switch between them, sometimes in a heritable and reversible manner. This switching may also lead to antigenic variation, enabling pathogenic bacteria to evade the host immune response. Therefore, phenotypic heterogeneity plays a significant role in microbial pathogenesis, immune evasion, antibiotic resistance, host niche tissue establishment, and environmental persistence. This heterogeneity can result from stochastic and responsive switches, as well as various genetic and epigenetic mechanisms. The development of phenotypic heterogeneity may create clonal populations that differ in their level of virulence, contribute to the formation of biofilms, and allow for antibiotic persistence within select morphological variants. This review delves into the current understanding of the molecular switching mechanisms underlying phenotypic heterogeneity, highlighting their roles in establishing infections caused by select bacterial pathogens.

This study aimed to develop a multiplex quantitative PCR (qPCR) assay for the detection of Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of paratuberculosis disease, a chronic and endemic infectious disease affecting ruminant livestock worldwide. Infected animals may remain asymptomatic for years while intermittently shedding MAP into their environment through feces, contributing to ongoing transmission. To develop a robust multiplex qPCR assay, we reviewed all TaqMan qPCR studies published since 1990 and selected 18 primer-probe combinations targeting the MAP-specific F57 gene and the repetitive sequence elements ISMAP02 and IS900. In samples with moderate to high MAP levels, all combinations performed well, with only minor differences in analytical performance. However, in low-abundance samples, several TaqMan designs yielded unreliable results, indicating limited specificity in complex matrices. Among the evaluated assays, the IS900-Herthnek design demonstrated significantly higher diagnostic sensitivity, detecting MAP in 81% of low-abundance samples, compared to 0% and 3% for the IS900-Kim and IS900-Slana assays, respectively. For ISMAP02, only the ISMAP02-Sevilla assay produced reliable results. For F57, Herthnek provided the most consistent and accurate quantification. Similar trends were observed in environmental sample testing. Based on these findings, we recommend a multiplex qPCR assay incorporating the IS900-Herthnek, ISMAP02-Sevilla, and F57-Herthnek TaqMan designs for the detection of MAP in fecal and environmental samples. This combination offers high analytical sensitivity and specificity, making it a valuable and accurate tool for the diagnosis of paratuberculosis and for environmental surveillance on dairy farms to identify herds potentially harboring MAP-infected animals.IMPORTANCEMycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease (JD) in ruminant livestock industries and has been associated with Crohn's disease in humans. Emerging scientific evidence also links MAP to other human conditions, including inflammatory bowel disease, autoimmune disorders, colorectal cancer, and Alzheimer's disease. This potential public health threat has intensified interest in developing more sensitive diagnostic tools and effective control strategies to eradicate MAP from dairy herds. Infected ruminants typically remain in the subclinical stage of the disease for 2-5 years, during which they shed MAP in their feces and contaminate the environment. Diagnosis during this stage is particularly challenging, as the pathogen evades the host's immune response, rendering serological tests insufficiently sensitive. In contrast, fecal PCR offers greater sensitivity than serum ELISA and traditional culture methods. Multiplex quantitative PCR is especially promising due to its high specificity and sensitivity for detecting MAP-infected animals and identifying herds with active shedders. Herd-level environmental screening, followed by individual animal testing, represents a robust national biosecurity strategy. This approach is a critical step toward reducing MAP transmission and improving herd health within the dairy industry.

DNA methylation has been documented to play vital roles in diseases and biological processes. In bovine, little is known about the regulatory roles of DNA methylation alterations on production and health traits, including mastitis. Here, we employed whole-genome DNA methylation sequencing to profile the DNA methylation patterns of milk somatic cells from sixteen cows with naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis and ten healthy control cows. We observed abundant DNA methylation alterations, including 3,356,456 differentially methylated cytosines and 153,783 differential methylation haplotype blocks (dMHBs). The DNA methylation in regulatory regions, including promoters, first exons and first introns, showed global significant negative correlations with gene expression status. We identified 6435 dMHBs located in the regulatory regions of differentially expressed genes and significantly correlated with their corresponding genes, revealing their potential effects on transcriptional activities. Genes harboring DNA methylation alterations were significantly enriched in multiple immune- and disease-related pathways, suggesting the involvement of DNA methylation in regulating host responses to S. aureus subclinical mastitis. In addition, we found nine discriminant signatures (differentiates cows with S. aureus subclinical mastitis from healthy cows) representing the majority of the DNA methylation variations related to S. aureus subclinical mastitis. Validation of seven dMHBs in 200 cows indicated significant associations with mammary gland health (SCC and SCS) and milk production performance (milk yield). In conclusion, our findings revealed abundant DNA methylation alterations in milk somatic cells that may be involved in regulating mammary gland defense against S. aureus infection. Particularly noteworthy is the identification of seven dMHBs showing significant associations with mammary gland health, underscoring their potential as promising epigenetic biomarkers. Overall, our findings on DNA methylation alterations offer novel insights into the regulatory mechanisms of bovine subclinical mastitis, providing further avenues for the development of effective control measures.

Twenty-six nulliparous sows were fed conventional gestation and lactation diets supplemented (N = 13) or not (N = 13) with extra daily supplements of 25-hydroxy-cholecalciferol (25-OH-D3; 4 kappa IU), beta-carotene (24 kappa IU), and copper (Cu)-proteinate (45 mg) from day 90 of gestation to 21 d of lactation (L21). In each litter, 10 piglets were divided into 5 pairs received, at 2 (L2) and 8 d (L8) of age, one of the five combinations of micronutrient sources and routes of administration (N = 260 piglets total). These neonatal treatments (N = 26 pairs or 52 piglets each) consisted of oral vitamin D3, retinol acetate and CuSO4 (T1); oral 25-OH-D3, beta-carotene, and Cu proteinate (T2); exposure to ultraviolet light (UVB), oral retinol palmitate and Cu gluconate (T3); intramuscular vitamin D3 and retinyl propionate and oral Cu acetate (T4); oral saline (CTRL). Oral or intramuscular provisions corresponded to 12 mg of Cu and 70 and 12 kappa IU of vitamins A and D, respectively. Blood samples were collected from all piglets at L2, L8, and L21 for determination of serum Cu, retinol, and 25-OH-D3. Body weight was measured at birth, L2, L8, and L21. Piglets were weaned at L21, and liver and blood samples were collected 2 d later to evaluate oxidative enzymes in blood and liver and hepatic ATP concentrations and expression of genes associated with antioxidant status. Sow treatments had marginal or no impacts on Cu, retinol, 25-OH-D3, or antioxidant status in piglet blood serum and liver. However, when supplements were given to piglets, hepatic Cu was 38% greater in for all treated piglets compared to CTRL (P < 0.01), hepatic retinol was 3 times higher in T1 than in CTRL (P < 0.01) and intermediate for other treatments whereas serum 25-OH-D3 was markedly increased with T2 and T3 at L8 and L21, respectively, compared to CTRL (Piglet treatment x Age interaction, P < 0.01). Concerning antioxidant activities, glutathione peroxidase, and superoxide dismutase were increased (P < 0.03) in plasma of T2 piglets whereas the highest values (P < 0.03) for indicators of oxidative damage to proteins were observed in T4 piglets. The study revealed that oral Cu proteinate from T2, oral retinol acetate from T1, oral 25-hydroxy-cholecalciferol from T2, and UVB light exposure from T3 were the most efficient ways of increasing the postnatal status of these micronutrients in suckling piglets and this may have some impacts on their peri-weaning antioxidant status. Lay Summary Pre- and postnatal transfers of copper and vitamins A and D are limited in nursing piglets. This study aimed to determine the efficiency of neonatal strategies of supplementations in these micronutrients. Five pairs of piglets were selected within litters of nulliparous sows fed conventional gestation and lactation diets supplemented (N = 13) or not (N = 13) with these micronutrients. In each litter, each pair received, at 2 and 8 d of age, one combination of micronutrient sources and routes of administration as follows: conventional sources given orally (T1), non-conventional sources given orally (T2); UVB light exposure and non-conventional sources of copper and vitamin A given orally (T3); vitamins A and D given intramuscularly and non-conventional sources of copper given orally (T4); oral saline (CTRL). Dietary supplements to sows had no or slight impacts on the copper, vitamin A and vitamin D statuses of piglets or on their antioxidant competence. However, for supplements directly administered to piglets, oral copper proteinate from T2, oral retinol acetate from T1, oral 25-hydroxy-cholecalciferol from T2 and UVB light exposure from T3 were the most efficient ways for increasing the postnatal status of these micronutrients in piglets along with impacts on their antioxidant competence.

Mycobacterium avium ssp. paratuberculosis (MAP) is the bacterium responsible for causing Johne's disease (JD), which is endemic to dairy cattle and also implicated in the etiology of Crohn's disease. The difficulty in diagnosing asymptomatic cows for JD makes this disease hard to control. Johne's disease is considered a priority under the One Health approach to prevent the spread of the causative agent to humans. Environmental screening is a strategic approach aimed at identifying dairy herds with animals infected with MAP. It serves as the initial step toward implementing more intensive actions to control the disease. Quantitative PCR (qPCR) technology is widely used for diagnosis. Given that genome sequencing is now much more accessible than ever before, it is possible to target regions of the MAP genome that allow for the greatest diagnostic sensitivity and specificity. The aim of this study was to identify among the published qPCR assays targeting IS900 the more cost-effective options to detect MAP and to validate them in the diagnostic context of JD. Mycobacterium avium ssp. paratuberculosis IS900 is a prime target because it is a multicopy genetic element. A total of 136 publications have reported on the use of IS900 qPCR assays over the past 3 decades. Among these records, 29 used the SYBR Green chemistry, and 107 used TaqMan technology. Aside from the 9 reports using commercial assays, 72 TaqMan reports cited previously published work, leaving us with 27 TaqMan qPCR designs. Upon closer examination, 5 TaqMan designs contained mismatches in primer or probe sequences. Additionally, others exhibited high similarity to environmental microorganisms or non-MAP mycobacteria. We assessed the performance of 6 IS900 qPCR designs and their sensitivity when applied to clinical or environmental samples, which varied from 4 to 56 fold overall. Additionally, we provide recommendations for testing clinical and environmental samples, as certain strategies used previously should be avoided due to poor qPCR design (e.g., the presence of mismatches) or a lack of specificity.

Supplemental Table S1 Detail information on cows selected for whole genome DNA methylation sequencing. Supplemental Table S2 General statistics on whole genome DNA methylation sequencing read statistics Supplemental Table S3 Comparisons of methylation level between cows with S. aureus subclinical mastitis and healthy control cows at a scope of the whole genome, chromosomes and gene features. Supplemental Table S4 List of differentially methylated cytosines in the context of CpG Supplemental Table S5 List of differentially methylated cytosines in the context of CHG and CHH Supplemental Table S6 Distribution of differentially methylated cytosines among chromosomes and genetic features Supplemental Table S7 Count of differentially methylated cytosines (DMCs) in the context of CpG, CHG and CHH in different gene features per gene Supplemental Table S8 Functional enrichment of differentially expressed genes harboring differentially methylated cytosines at different genomic functional regions Supplemental Table S9 Predicted methylation level for LINE-1 and tRNA-derived SINEs and comparisons between cows with S. aureus subclinical mastitis and healthy control cows Supplemental Table S10 Differential methylation haplotype blocks and their colocation with QTLs related to mastitis and immune capacity Supplemental Table S11 Differential genes with significant changes in general methylation level of regulatory regions and gene expression changes Supplemental Table S12 Functional enrichment for differential genes with significant changes in general methylation level of regulatory regions and gene expression changes Supplemental Table S13 Correlation between dMHBs located at regulatory regions and their overlapped differentially expressed genes Supplemental Table S14 Motif enrichment and identification in GE-dMHBs Supplemental Table S15 Functional enrichment for GE-dMHBs (dMHBs overlapped with genes and significantly correlated with the expression of corresponding genes) Supplemental Table S16 Candidate discriminate signatures selected from GE-dMHBs (pro-GE-dMHBs) and their overlapped genes Supplemental Table S17 Information about samples and primers used for TBAS and results of seven GE-dMHBs analyzed in 200 cows

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's Disease (JD) in ruminants, which is responsible for significant economic loss to the global dairy industry. Mixed strain infection (MSI) refers to the concurrent infection of a susceptible host with genetically distinct strains of a pathogen, whereas within-host changes in an infecting strain leading to genetically distinguishable progeny is called microevolution. The two processes can influence host-pathogen dynamics, disease progression and outcomes, but not much is known about their prevalence and impact on JD. Therefore, we obtained up to 10 MAP isolates each from 14 high-shedding animals and subjected them to whole-genome sequencing. Twelve of the 14 animals examined showed evidence for the presence of MSIs and microevolution, while the genotypes of MAP isolates from the remaining two animals could be attributed solely to microevolution. All MAP isolates that were otherwise isogenic had differences in short sequence repeats (SSRs), of which SSR1 and SSR2 were the most diverse and homoplastic. Variations in SSR1 and SSR2, which are located in ORF1 and ORF2, respectively, affect the genetic reading frame, leading to protein products with altered sequences and computed structures. The ORF1 gene product is predicted to be a MAP surface protein with possible roles in host immune modulation, but nothing could be inferred regarding the function of ORF2. Both genes are conserved in Mycobacterium avium complex members, but SSR1-based modulation of ORF1 reading frames seems to only occur in MAP, which could have potential implications on the infectivity of this pathogen. IMPORTANCE Johne's disease (JD) is a major problem in dairy animals, and concerns have been raised regarding the association of Mycobacterium avium subsp. paratuberculosis (MAP) with Crohn's disease in humans. MAP is an extremely slow-growing bacterium with low genome evolutionary rates. Certain short sequence repeats (SSR1 and SSR2) in the MAP chromosome are highly variable and evolve at a faster rate than the rest of the chromosome. In the current study, multiple MAP isolates with genetic variations such as single-nucleotide polymorphisms, and more noticeably, diverse SSRs, could simultaneously infect animals. Variations in SSR1 and SSR2 affect the products of the respective genes containing them. Since multiple MAP isolates can infect the same animal and the possibility that the pathogen undergoes further changes within the host due to unstable SSRs, this could provide a compensative mechanism for an otherwise slow-evolving pathogen to increase phenotypic diversity for overcoming host responses.

Staphylococcus chromogenes (SC) is a common coagulase-negative staphylococcus described as an emerging mastitis pathogen and commonly found in dairy farms. This study investigated the potential involvement of DNA methylation in subclinical mastitis caused by SC. The whole-genome DNA methylation patterns and transcriptome profiles of milk somatic cells from four cows with naturally occurring SC subclinical mastitis (SCM) and four healthy cows were characterized by next-generation sequencing, bioinformatics, and integration analyses. Comparisons revealed abundant DNA methylation changes related to SCM, including differentially methylated cytosine sites (DMCs, n = 2,163,976), regions (DMRs, n = 58,965), and methylation haplotype blocks (dMHBs, n = 53,098). Integration of methylome and transcriptome data indicated a negative global association between DNA methylation at regulatory regions (promoters, first exons, and first introns) and gene expression. A total of 1486 genes with significant changes in the methylation levels of their regulatory regions and corresponding gene expression showed significant enrichment in biological processes and pathways related to immune functions. Sixteen dMHBs were identified as candidate discriminant signatures, and validation of two signatures in more samples further revealed the association of dMHBs with mammary gland health and production. This study demonstrated abundant DNA methylation changes with possible involvement in regulating host responses and potential as biomarkers for SCM.

Staphylococcus aureus is one of the most prevalent contagious bacterial pathogen of bovine mastitis. The subclinical mastitis it causes has long-term economic implications and it is difficult to control. To further understanding of the genetic basis of mammary gland defense against S. aureus infection, the transcriptomes of milk somatic cells from 15 cows with persistent natural S. aureus infection (S. aureus-positive, SAP) and 10 healthy control cows (HC) were studied by deep RNA-sequencing technology. Comparing the transcriptomes of SAP to HC group revealed 4,077 differentially expressed genes (DEG; 1,616 up- and 2,461 downregulated). Functional annotation indicated enrichment of DEG in 94 Gene Ontology (GO) and 47 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Terms related to the immune response and disease processes were mostly enriched for by upregulated DEG, whereas biological process terms related to cell adhesion, cell movement and localization, and tissue development were mostly enriched for by downregulated DEG. Weighted gene co-expression network analysis grouped DEG into 7 modules, the most important module (colored turquoise by software and here referred to as Turquoise module) was positively significantly correlated with S. aureus subclinical mastitis. The 1,546 genes in the Turquoise module were significantly enriched in 48 GO terms and 72 KEGG pathways, with 80% of them being disease- and immune-related terms [e.g., immune system process (GO:0002376), cytokine-cytokine receptor interaction (bta04060) and S. aureus infection (bta05150)]. Some DEG such as IFNG, IL18, IL1B, NFKB1, CXCL8, and IL12B were enriched in immune and disease pathways suggesting their possible involvement in the regulation of the host response to S. aureus infection. Four modules (Yellow, Brown, Blue, and Red) were negatively correlated (significantly) with S. aureus subclinical mastitis, and were enriched in functional annotations involved in the regulation of cell migration, cell communication, metabolic process, and blood circulatory system development, respectively. Application of sparse partial least squares discriminant analysis to genes of the Turquoise module identified 5 genes (NR2F6, PDLIM5, RAB11FIP5, ACOT4, and TMEM53) capable of explaining the majority of the differences in the expression patterns between SAP and HC cows. In conclusion, this study has furthered understanding of the genetic changes in the mammary gland and the molecular mechanisms underlying S. aureus mastitis, as well as revealed a list of candidate discriminant genes with potential regulatory roles in response to S. aureus infection.

Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogen responsible for paratuberculosis or Johne’s Disease (JD) in ruminants, which is responsible for substantial economic losses worldwide. MAP transmission primarily occurs through the fecal-oral route, and the introduction of an MAP infected animal into a herd is an important transmission route. In the current study, we characterized MAP isolates from 67 cows identified in 20 herds from the provinces of Quebec and Ontario, Canada. Whole genome sequencing (WGS) was performed and an average genome coverage (relative to K-10) of ∼14.9 fold was achieved. The total number of SNPs present in each isolate varied from 51 to 132 and differed significantly between herds. Isolates with the highest genetic variability were generally present in herds from Quebec. The isolates were broadly separated into two main clades and this distinction was not influenced by the province from which they originated. Analysis of 8 MIRU-VNTR loci and 11 SSR loci was performed on the 67 isolates from the 20 dairy herds and publicly available references, notably major genetic lineages and six isolates from the province of Newfoundland and Labrador. All 67 field isolates were phylogenetically classified as Type II (C-type) and according to MIRU-VNTR, the predominant type was INMV 2 (76.1%) among four distinct patterns. Multilocus SSR typing identified 49 distinct INMV SSR patterns. The discriminatory index of the multilocus SSR typing was 0.9846, which was much higher than MIRU-VNTR typing (0.3740). Although multilocus SSR analysis provides good discriminatory power, the resolution was not informative enough to determine inter-herd transmission. In select cases, SNP-based analysis was the only approach able to document disease transmission between herds, further validated by animal movement data. The presence of SNPs in several virulence genes, notably for PE, PPE, mce and mmpL, is expected to explain differential antigenic or pathogenetic host responses. SNP-based studies will provide insight into how MAP genetic variation may impact host-pathogen interactions. Our study highlights the informative power of WGS which is now recommended for epidemiological studies and to document mixed genotypes infections.

The aim of this study was to evaluate the potential of micronutrients and feed additives to modulate intestinal microbiota and systemic and mucosal immune responses in weaned pigs infected with Salmonella. At weaning, 32 litters of 12 piglets each were allocated to four dietary treatments: 1) control diet (CTRL), 2) CTRL supplemented with chlortetracycline (ATB), 3) CTRL supplemented with a cocktail of feed additives (CKTL); and 4) CKTL diet containing bovine colostrum in replacement of spray-dry animal plasma (CKTL+COL). The CKTL supplement included cranberry extract, encapsulated carvacrol and yeast-derived products and an enriched selenium and vitamin premix. Three weeks after weaning, four pigs per litter were orally inoculated with Salmonella Typhimurium DT104. Half of them were euthanized 3 days post-infection (dpi) and the other half, 7 dpi. The expression of IL6, TNF, IL8, monocyte chemoattractant protein 1 (MCP1), IFNG, cyclooxygenase 2 (COX2), glutathione peroxidase 2 (GPX2) and β-defensin 2 (DEFB2) showed a peaked response at 3 dpi (P < 0.05). Results also revealed that DEFB2 expression was higher at 3 dpi in CTRL and CKTL groups than in ATB (P = 0.01 and 0.06, respectively) while GPX2 gene was markedly increased at 3 and 7 dpi in pigs fed CKTL or CKTL+COL diet compared to CTRL pigs (P < 0.05). In piglets fed CKTL or CKTL+COL diet, intestinal changes in microbial communities were less pronounced after exposure to Salmonella compared to CTRL and progressed faster toward the status before Salmonella challenge (AMOVA P < 0.01). Furthermore, the relative abundance of several families was either up- or down-regulated in pigs fed CKTL or CKTL+COL diet after Salmonella challenge. In conclusion, weaning diet enriched with bovine colostrum, vitamins and mixture of feed additives mitigated the influence of Salmonella infection on intestinal microbial populations and modulate systemic and intestinal immune defences.

DNA methylation involvement in the regulation of mammary gland inflammatory defense against pathogens, including Staphylococcus aureus(SA) has been documented. However, the mechanisms are not clear. The possible influence of DNA methylation of first introns (DNA-MeFI) on transcriptional activities during subclinical mastitis was studied by profiling the methylome (whole genome methylation sequencing) and transcriptome (RNA-sequencing) of milk somatic cells from cows(n=15) with SA subclinical mastitis (SACs) and healthy cows (HCs)(n=13), followed by bioinformatics processing with standard tools. The DNA-MeFI was calculated, for each of 19411 genes found as expressed in the samples, by averaging the methylation levels of all CpG sites in the first intron. At genome-wide, the DNA-MeFIs was inversely correlated with gene expression (GE) (Pearson’s r=-0.173, p=1.60×10-122)(Figure 1-A). Similarly, the difference in the DNA-MeFI and GE between SACs and HCs was also inversely correlated (Pearson’s r=-0.149, p=4.25×10-97)(Figure 1-B). Application of machine learning by Gaussian Mixed model (GMM) using scikit-learn in python to the changes of DNA-MeFI and GE between SACs and HCs revealed 2866 outliers (GMM’s p< 0.005) that showed significant changes in the DNA-MeFI and/or GE. 644 genes with >10% difference in DNA-MeFI and |log2FC| >1 in GE were selected and referred to as differentially methylated and expressed genes (DME-genes). 644 DME-genes were significantly enriched in 5 GO terms and 11 KEGG pathways related to diseases and immune functions, including Staphylococcus aureus infection and Natural killer cell mediated cytotoxicity (Table 1). Furthermore, 74.84% DME-genes showed inverse changes in DNA-MeFI and GE. Besides, the DNA-MeFI of 264 DME-genes were found to correlate significantly with their GE (|rho| >0.3, FDR<0.05), including 225 DME-genes(85.23%) with inverse correlations(rho< -0.3, FDR< 0.05). 39 DME-genes with positive correlations were significantly enriched in 21 GO terms mainly related to metabolic processes. In conclusion, the DNA-MeFI possibly participate in the regulation of gene expression during bovine subclinical mastitis caused by SA.

Staphylococcus aureus (S. aureus) is one of the most prevalent pathogens of bovine subclinical mastitis and a challenging pathogen in dairy production. DNA methylation involvement in regulating mammary gland inflammatory defense has been documented. This study adopted whole genome bisulfite sequencing and RNA-sequencing technologies to profile the genome-wide DNA methylation and transcriptome landscapes, respectively, of milk somatic cells (MSCs) from 15 cows with S. aureus-induced subclinical mastitis (SACs) and 13 healthy cows (HCs). Bioinformatics processing of data with standard tools indicated that the global DNA methylation patterns of MSCs demonstrated inverse correlations between gene expression and DNA methylation levels at promoter, first exon and first intron regions, suggesting potential regulatory roles of DNA methylation in transcriptional activities. Totally, 26,289 differential methylation regions (DMRs) were identified between SACs and HCs (1,000 bp window, 1,000 bp step, q-value < 0.05, >20% methylation difference and ≥3 differentially methylated cytosines). A total of 887, 334 and 3,414 DMRs were overlapped with promoter, first exon, and first intron regions, respectively, including 363 DMRs harboring transcription start sites. Among them, the methylation levels of 956 DMRs were strongly correlated with the expression levels of their overlapped genes (|Spearman’s correlation coefficient| >0.5, adjusted-p-value< 0.05). Functional annotation of the overlapped genes indicated significant (adjusted-p-value < 0.05) enrichment in 18 GO terms and 1 KEGG pathway related to cellular activities, notably in cell migration, epithelial cell differentiation and tight junction, among others (Figure 1). These results suggest the involvement of DNA methylation in mammary gland defense against S. aureus-invasion. Applying DIABLO method to integrate correlated DMRs and genes, a total of 20 DMR and 10 gene candidate markers were found to discriminate SACs from HCs. The identified candidate markers could serve as reference for enhancing breeding for mastitis resistance and for developing new mastitis management strategies.

Mastitis caused by different pathogens including Streptococcus uberis (S. uberis) is responsible for huge economic losses to the dairy industry. In order to investigate the potential genetic and epigenetic regulatory mechanisms of subclinical mastitis due to S. uberis, the DNA methylome (whole genome DNA methylation sequencing) and transcriptome (RNA sequencing) of milk somatic cells from cows with naturally occurring S. uberis subclinical mastitis and healthy control cows (n = 3/group) were studied. Globally, the DNA methylation levels of CpG sites were low in the promoters and first exons but high in inner exons and introns. The DNA methylation levels at the promoter, first exon and first intron regions were negatively correlated with the expression level of genes at a whole-genome-wide scale. In general, DNA methylation level was lower in S. uberis-positive group (SUG) than in the control group (CTG). A total of 174,342 differentially methylated cytosines (DMCs) (FDR < 0.05) were identified between SUG and CTG, including 132,237, 7412 and 34,693 DMCs in the context of CpG, CHG and CHH (H = A or T or C), respectively. Besides, 101,612 methylation haplotype blocks (MHBs) were identified, including 451 MHBs that were significantly different (dMHB) between the two groups. A total of 2130 differentially expressed (DE) genes (1378 with up-regulated and 752 with down-regulated expression) were found in SUG. Integration of methylome and transcriptome data with MethGET program revealed 1623 genes with significant changes in their methylation levels and/or gene expression changes (MetGDE genes, MethGET P-value < 0.001). Functional enrichment of genes harboring ≥ 15 DMCs, DE genes and MetGDE genes suggest significant involvement of DNA methylation changes in the regulation of the host immune response to S. uberis infection, especially cytokine activities. Furthermore, discriminant correlation analysis with DIABLO method identified 26 candidate biomarkers, including 6 DE genes, 15 CpG-DMCs and 5 dMHBs that discriminated between SUG and CTG. The integration of methylome and transcriptome of milk somatic cells suggests the possible involvement of DNA methylation changes in the regulation of the host immune response to subclinical mastitis due to S. uberis. The presented genetic and epigenetic biomarkers could contribute to the design of management strategies of subclinical mastitis and breeding for mastitis resistance.