Decoding DNA Methylation in Staphylococcus aureus Mastitis: Implications for Immune Regulation and Disease Resistance

Decoding DNA Methylation in Staphylococcus aureus Mastitis: Implications for Immune Regulation and Disease Resistance | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Animal Genetics This is a preprint and has not been peer reviewed. Data may be preliminary. 22 March 2025 V1 Latest version Share on Decoding DNA Methylation in Staphylococcus aureus Mastitis: Implications for Immune Regulation and Disease Resistance Authors : Apeksha , Abhishek Mahendra Todkari 0009-0001-8075-1669 , Ashok Chaudhary , Mir Mehroz Hassan , Diksha Upreti , Shri Ram Saini , Sheikh Firdous Ahmad 0000-0002-7114-0882 , and A. K. Pandey 0009-0000-1390-9983 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174263595.58990203/v1 Published Animal Genetics Version of record Peer review timeline 460 views 224 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Mastitis is a major health and economic threat to the dairy industry, causing massive losses every year worldwide. Staphylococcus aureus is the chief pathogen responsible for most of the subclinical as well as a considerable portion of the clinical cases. Conventional control strategies, including antibiotic treatment and genetic selection for resistance, have limited success due to antimicrobial resistance (AMR) and low heritability of mastitis susceptibility. This calls for the exploration of novel approaches like epigenetics, which offers insights into host-pathogen interactions beyond the genetic variations. This review focuses on DNA methylation changes in the mammary gland that occur during S. aureus mastitis. Recent research works have identified immune suppression and pathogen persistence in relation with DNA methylation during the disease. The microbe has been reported to alter the methylation status of regulatory regions for many immune genes like CXCR1, TNF-α, IL6R, IL10, and C3 , resulting in dysregulation of immune responses in the host, and thereby facilitating pathogen persistence and chronic infection. Along with its own virulence factors, differential DNA methylation status of such genes during infection helps the pathogen to escape host defence, and decreases the intensity of inflammation. Thus, understanding these mechanisms can open new avenues in the field of disease detection, animal selection, and immunotherapy among others. Such an integrative approach offers a revolutionization of mastitis control strategies, ensuring better health and productivity in dairy animals. Introduction The global demand for milk and dairy products has increased significantly over the past few decades, fuelled by factors such as population growth, urbanization, rising incomes, and evolving dietary preferences (Cheng et al ., 2022; Yousefian et al ., 2024). In response, the dairy industry implemented advanced strategies for enhancing per animal milk yield, like nutrition and management, and genetic selection (Cobirka et al ., 2020). The efforts have converged into significant improvements; however, persistent challenges, particularly mastitis, continue to cause substantial losses to the sector. Mastitis is a disease of high concern due to its profound economic, animal welfare, productivity, and public health implications (Detilleux et al ., 2015; Asfaw and Negash, 2017; Dalanezi et al ., 2020; Narayana et al ., 2021). Being a multifactorial condition, it arises from a complex interplay of pathogenic organisms as well as environmental factors, and managemental practices (Jones and Bailey, 2009). A variety of pathogens cause the infection, among which, Staphylococcus aureus has emerged as a major causative agent of both clinical and subclinical mastitis cases, particularly the latter (Reyher et al ., 2011). It is widely accepted as a major threat worldwide owing to its ability to evade host immune responses, produce persistent infections, and develop antimicrobial resistance (AMR). The traditional mastitis control strategies have not completely been effective for eradication of the disease, especially due to increasing cases of AMR, and the low heritability of mastitis susceptibility/ resistance (De Vliegher et al ., 2012; Chandrasekaran et al ., 2014). This has necessitated the exploration of novel mechanisms regulating disease susceptibility and immune responses. Over the past few years, epigenetic mechanisms, specifically DNA methylation, have drawn attention of the scientific community as a contributor to shaping the host-pathogen interactions and associated immune functions. DNA methylation is a heritable modification in the cytosine-phosphate-guanine (CpG) regions of the genome by addition of a methyl group, that influences expression of genes without altering the genetic code itself. There are emerging evidences on the manipulation of host’s DNA methylation levels upon infection by S. aureus within the mammary tissues. Specific changes have been reported in immune-related genes and linked to alterations in the immune responses during course of the disease. Understanding such modifications offers new insights into mechanisms of mastitis susceptibility and resistance, that can be used as novel strategies in control of the disease. This review aims to inspect the various aspects of DNA methylation associated changes in the immune responses to S. aureus induced mastitis. We discuss the mechanisms of DNA methylation, the interaction of S. aureus with the mammary gland methylome, and its impact on the host immune mechanisms. We have highlighted important modifications and the accompanying effects on products of different genes related to the host defence. Finally, possible areas to be explored and probable applications from the understanding of these epigenetic markers have been presented. The integration of epigenetics into mastitis research offers significant potential to uncover novel avenues for a sustainable management of the disease and improving dairy productivity. Mastitis in dairy animals Mastitis is a complex, production-related disease defined as the inflammation of mammary glands. The disease is one of the chief factors that negatively affect quality and quantity of milk in dairy animals, accounting for majority of culling in cattle and buffalo. It is considered as the most critical economic and health challenge in dairy herds (Cobirka et al ., 2020), and is among the most widespread and most costly diseases of dairy animals (Bogni et al ., 2011; Benić et al ., 2018; Jamali et al ., 2018; Narayana et al ., 2021). Reduced milk quantity and discarding of poor-quality milk, increased culling from herd, and additional expenditure on treatment of mastitis lead to huge economic losses for producers (Holmberg et al ., 2012; Detilleux et al ., 2015). Mastitis poses a significant risk to both food safety and environmental health. Apart from this, individual animal’s reproductive performance (Kumar et al ., 2017; Dalanezi et al ., 2020) and overall health may deteriorate due to an increased susceptibility to other diseases (Cha et al ., 2013). Due to the severe pain caused and impact on animal’s health, mastitis poses a challenge for animal welfare as well (Logue and Nolan, 1998; Asfaw and Negash, 2017). Therefore, it has drawn attention of researchers worldwide over decades, and continues to offer newer avenues in animal production yet to be explored. The disease can be triggered by physical injury and/or pathological microorganisms, along with contributors like poor milking practices and environmental factors (Jones and Bailey, 2009). It is classified on the basis of causative agent as environmental or contagious, and as clinical or subclinical based on symptoms manifested in host. Subclinical cases amount for a significantly higher proportion than that of clinical mastitis, respectively being around 25-65% and 5% (Dufour et al ., 2012; Bathla et al ., 2020). Subclinical mastitis is attributed to higher financial losses than clinical mastitis (Azooz et al ., 2020; Morales-Ubaldo et al ., 2023). The severity of mastitis depends on invasive microbes involved and the subsequent immune responses produced (Pighetti and Elliott, 2011; Gogoi-Tiwari et al ., 2017). There is variability in susceptibility to the same pathogen among cows, and lactation stage also influences the incidence of the infection (Burvenich et al ., 2000). The pace, strength, and duration of the immune reaction as well as susceptibility to mastitis are decisively linked to the animal’s genetics (Pighetti and Elliott, 2011; Bobbo et al., 2019). Mastitis agents in bovines and hegemony of Staphylococcus aureus Mastitis is a multi-etiological disease involving bacteria, fungi, mycoplasma, viruses, algae, and yeasts, which may have primary or secondary roles in its occurrence, and may either be environmental or contagious. Bacteria are the most important causal agents among these (Krishnamoorthy et al ., 2021), involving both Gram positive and negative types. In bovine mastitis, S. aureus, Streptococcus uberis, and Escherichia coli are considered as the major pathogens (Wellnitz and Bruckmaier, 2012; Heikkilä et al ., 2018; Saidani et al ., 2018; Krishnamoorthy et al ., 2021). S. aureus and E. coli are zoonotic, with the latter being environmental (Zi et al ., 2018). Gram-negative pathogens, such as E. coli, Klebsiella species, and Pseudomonas species, cause clinical mastitis more often in herds with a low bulk milk SCC; and on the contrary, gram-positive pathogens such as S. aureus, Streptococcus dysgalactiae , and Streptococcus agalactiae cause clinical mastitis more often in herds with a high bulk milk somatic cell count (SCC) (Barkema et al., 1998). S. aureus is the most prevalent contagious mastitis pathogen which causes both clinical and subclinical conditions (Nigam, 2015; Levison et al ., 2016; Singha et al ., 2021; Maddela et al ., 2024) . The microbe colonizes skin of mammary gland and multiplies within the organ, while evading the host’s defence system to mostly cause subclinical or chronic mastitis characterized by a reduced milk yield and an increased SCC in milk (Sutra and Poutrel, 1994; Kibebew, 2017; Cheng and Han, 2020). No other signs of disease appear in such cases apart from this; however, the udder tissue is damaged irreversibly, which may ultimately lead to AMR in addition to decreased milk production (Cheng and Han, 2020). Economic losses related with S. aureus clinical and subclinical mastitis result from the decrease in milk production and quality, the expenses on antibiotics and veterinary fees, and the withdrawal period of cows treated with antibiotics (Halasa et al ., 2007; Aghamohammadi et al ., 2018). Horizontal transfer of antibiotic resistance genes (Lerminiaux and Cameron, 2019), the emergence of livestock-associated methicillin-resistant S. aureus (MRSA) strains (García-Álvarez et al ., 2011; Holmes and Zadoks, 2011), and the public pressure to reduce antibiotic use in animal production are the few important reasons to find efficient non-antibiotic control measures for this disease. Therefore, breeding for mastitis resistant cattle becomes even more important. Immunity to S. aureus in mastitis Mastitis resistance depends on the mammary gland immune system. Physical barriers constitute the first line of defence in the gland against infection. It comprises of udder and teat morphology, milking speed, innate immunity, and the mechanical and antimicrobial teat canal barrier, through which bacteria must penetrate to cause intramammary infection (Burvenich et al ., 2000; Rainard and Riolett, 2006; Günther and Seyfert, 2018). After establishment of infection, innate and adaptive immunities play their role. Strain specific immune response has also been reported for S. aureus. Some strains cause rapid and intense immune response leading to clearance of organism from milk, and hence associated with mild clinical mastitis, while others cause persistent mastitis due to milder immune responses (Engler et al ., 2022). Strain diversity (Sivakumar et al., 2023), multiple virulence factors (Soares et al., 2017), persistent infection (Campos et al., 2022), immune evasion (De Jong et al., 2019), and low cure rates of anti-biotherapies (Erskine et al., 2002) are the critical factors making S. aureus an important pathogen in context of mastitis. Chronic and subclinical mastitis caused by S. aureus has low response to antibiotics due to colonization in intracellular epithelium (Erskine et al., 2002). Role of epigenetics in breeding for mastitis resistance Mastitis can be controlled by management practices and antibiotic treatment of affected animals, though these approaches alone have not been able to eradicate this rampant disease (Wang et al ., 2020). Further, antimicrobial resistance (AMR) has been reported time and again, causing low cure rates, along with posing a public health hazard (Barkema et al ., 2006; Chandrasekaran et al ., 2014). Mastitis control in dairy animals consumes a massive 80% of the total antibiotics used in this industry (Ashraf et al ., 2020), indicating predicament of the issue. Underscoring the effective, but short-term and limited gains from the above approaches, another important strategy, involving the use of genetic selection of animals, offers a parallel and permanent solution. Identification of animals’ susceptibility or resilience for the disease can help in early intervention through preventive measures and also aid in efficient selection (Bouzeraa et al ., 2024). Thousands of genes are involved in the immune response against different pathogens, but animals with better responses can be identified using conventional quantitative genetic principles based on phenotype (Mallard et al ., 2015). Heritability of immune responses, both cell-mediated (CMIR) and antibody-mediated (AMIR), is satisfactorily high to enable selection of animals for disease resistance (Hernandez et al ., 2006; Thompson-Crispi et al ., 2012). However, susceptibility to mastitis has been established as a trait with low heritability (Lush, 1950; Walawski, 1999; de Haas et al ., 2002; De Vliegher et al ., 2012), which necessitates the use of modern breeding strategies involving genomic selection aided by genome wide association studies (GWAS), transcriptomic, post-transcriptomic and epigenetic studies (Wang et al ., 2020). Gene transcription is regulated by both genetic mutations and epigenetic modifications (Zhang et al ., 2018). Genetic markers like singly nucleotide polymorphisms (SNPs) are frequently being used to assist genomic selection for livestock species like cattle (Bouquet and Juga, 2013; Obšteter and Gorjanc, 2021). Epigenetics, defined as the study of heritable changes in gene expression that do not involve alterations to the underlying DNA sequence (Ho et al ., 2010), has emerged as a powerful tool in recent years for understanding complex traits like mastitis in livestock. The epigenome encompassing the entire range of these potentially inheritable modifications across the genome, is contingent on the genetic, environmental, and other non-genetic factors (Bernstein et al., 2007; Ibeagha-Awemu and Khatib, 2017). Therefore, unlike genome, the epigenome is inordinately dynamic through lifetime of an individual, and also, within an individual, different cells/tissues express unique epigenetic profiles despite harbouring the same genetic instruction. Mastitis involves a triadic interaction between the host, pathogen, and environment, wherein the host factors include both its genetics and epigenetics components (Dego, 2020). The epigenetic component is affected by environmental cues which may be external, like temperature, chemicals, and pathogens, or internal, like nutrition status, hormones, and oxidative stress (James et al., 2004; Petronis, 2010; Tando and Matsui, 2023). The epigenetic alterations can result through several mechanisms, all of which work in concert to regulate gene expression without changing the genetic code itself. These mechanisms or epigenetic marks include DNA methylation, histone modifications, non-coding RNAs (ncRNAs), and chromatin remodelling. Among these, DNA methylation is the most studied and characterized epigenetic mechanism that plays a significant role in regulating mammary gland health and function in livestock (Ibeagha-Awemu and Zhao, 2015; Ivanova et al ., 2021). Pathogenic microbes including S. aureus have well been reported to alter the expression of various genes through the host’s epigenetic machinery (Van Nhieu and Arbibe, 2009; Heimer et al ., 2010; Gómez-Díaz et al ., 2012; Niller et al ., 2012). DNA methylation DNA methylation involves the covalent addition of methyl groups to the 5’ position of cytosine nucleotides in the DNA, usually at cytosine-phosphate-guanine (CpG) sites (Ibeagha-Awemu and Zhao, 2015; Triantaphyllopoulos et al., 2016). CpG sites or islands (CGIs) are genomic regions with an unusually high frequency of cytosine and guanine residues, present in most of the gene promoters, and are typically devoid of methylation (Illingworth and Bird, 2009). Many CpG islands (CGIs) are present in the bovine genome. DNA methylation at a single nucleotide position is considered equivalent to a polymorphism, and is termed as methylation variable position (MVP) (González-Recio, 2012). DNA methyltransferase enzymes like DNMT1, DNMT3a, and DNMT3b catalyse DNA methylation, using S-adenosyl-methionine (SAM) as methyl donor (Miranda and Jones, 2007). Here, DNMT1 functions as a ”maintenance” methyltransferase, copying existing methylation patterns after DNA replication, while DNMT3A and DNMT3B act as ”de novo” methyltransferases, establishing new methylation patterns during development and differentiation, essentially creating new methylation marks on previously unmethylated DNA (Tajima et al ., 2022). The process typically leads to gene silencing by blocking transcription factors from binding to the DNA. Hence, in general, hypermethylation in the regulatory regions of DNA is associated with inactivation of genes, whereas the reverse happens at lower levels of methylation. Conversely, gene expression is said to be increased if the hypermethylation occurs within the body of genes (Langevin and Kelsey, 2013). Insights into DNA methylation patterns can help explore the “black box” of phenotypic variation that has not possibly been explained by the genetic variations (Ibeagha-Awemu and Yu, 2021). Modern day assessment of DNA methylation can be carried out using techniques which are (1) bisulphite-based, like bisulphite sequencing (BS-Seq) (Stirzaker et al., 2014), methylation-specific PCR (MSP) (Herman et al., 1996), combined bisulphite restriction analysis (COBRA) (Xiong and Laird, 1997), and pyrosequencing (Tost and Gut, 2007), (2) enzyme-based, like methylation-sensitive restriction enzyme PCR (MSRE-PCR) (Zhang et al., 2009), HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) (Khulan et al., 2006), (3) affinity-based, like methylated DNA immunoprecipitation (MeDIP) (Weber et al., 2005), and methyl-CpG binding domain capture (MBD-Seq) (Serre et al., 2010), and (4) sequencing-based, like whole-genome bisulfite sequencing (WGBS) (Lister et al ., 2009), reduced representation bisulfite sequencing (RRBS) (Meissner et al., 2005), oxidative bisulfite sequencing (oxBS-Seq) (De Borre and Branco, 2021), and single-molecule real-time sequencing (SMRT-Seq) (Flusberg et al., 2010). In dairy cattle, the role of DNA methylation in milk production has been well documented. Studies show that methylation differences between lactating and non-lactating stages influence the synthesis of milk components. DNA methylation at specific genomic loci has been associated with milk yield, composition, and mammary gland function (Wang and Ibeagha-Awemu, 2021). This understanding is crucial for addressing and utilizing the information on mastitis-related alterations in the DNA. In general, the global CpG methylation is higher in mastitis-positive animals, and more significant in the regulatory regions of DNA like promoters, first exons, and first introns (e.g. Wang et al., 2023; Wang et al., 2024). DNA methylation within the first intron during S. aureus -induced subclinical mastitis in dairy cows was found to influence immune-related pathways, potentially affecting the cow’s ability to respond to the infection (Wang et al ., 2022). However, some studies have reported lower overall methylation levels in infected animals compared to healthy ones (e.g. Ju et al ., 2020; Nayan et al., 2022). Staphylococcus aureus Mastitis: Pathogenesis and DNA Methylation S. aureus infection is established in three phases. The first phase involves adhesion of the organism to host cell and extracellular matrix. This is mediated by adhesion molecules like ClfA and ClfB. In the second phase, invasion of tissues occurs with the help of fibronectin and fibronectin-binding protein (FnBP), wherein an overexpression of FnBP leads to higher invasion. Finally in the third phase, evasion of host immune response occurs (Campos et al., 2022; Middleton, 2008; Almeida et al., 1996). A differential expression of various virulence factors takes place in the different stages of infection. In S. aureus , expression of virulence factors is under the control of global regulators like the accessory gene regulator (Agr) which allows transition from colonisation phase to invasive phase, and the alternative transcriptional sigma factor (SigB) which promotes expression of adhesions and biofilm (Novick and Geisinger, 2008). Concomitantly, the pathogen manipulates host immune responses through epigenetic alterations including DNA methylation. Studies indicate that S. aureus infection induces hypermethylation in pro-inflammatory cytokine genes such as TNF-α and IL6R genes, potentially dampening the immune response and allowing persistent infections (Zhang et al., 2018; Wang 2020; Dong 2021; Wang et al., 2024). In buffalo, hypermethylation of promoters for PZP (pregnancy zone protein) and CPAMD8 (complement 3 and pregnancy zone protein-like, a2-macroglobulin domain-containing protein 8) genes has been observed (Nayan et al ., 2022). Both the genes are broad spectrum protease inhibitors and involved in opsonization and phagocytosis of pathogens. Thus, their downregulation during mastitis supports persistence of S. aureus for longer periods inside the host. Host Immune Response and DNA Methylation Alterations The mammary gland relies on both innate and adaptive immunity to combat infections. The innate immune system provides first line of defence through physical barriers, pattern recognition receptors (PRRs), inflammation, complement activation, phagocytosis, lactoferrins, cytokines etc. Bacterial penetration into the udder is physically blocked by the tight sphincters at teat ends in between milkings (Jones and Bailey, 2009). However, these sphincters are relaxed in early dry period and immediately after milking, increasing the susceptibility to mastitis during these periods (Rainard and Riolett, 2006). Secondly, keratin and calcium binding protein layer of the teat canal has bacteriostatic and bactericidal properties (Smolenski et al., 2015; Notcovich, 2021). Mammary epithelial cells have PRRs for the recognition of pathogen associated molecular patterns (PAMPs) (Aitken et al ., 2011). PRR-PAMP binding results in release of various cytokines and chemokines depending on the PPRs involved, and leads to recruitment of the cells of immune system. Cytokines are signalling chemicals which may either promote inflammation upon encounter of pathogen (proinflammatory cytokines), or restrict the activity of proinflammatory cytokines (anti-inflammatory cytokines). The proinflammatory cytokines are released early in infection, as soon as the mammary epithelial cells identify the components of different mastitic bacteria (Lahouassa et al., 2007; Cheng et al., 2020; Vitenberga-Verza et al., 2022). The CXCR1 (C-X-C motif chemokine receptor 1) is a G-protein coupled receptor specifically involved in triggering of neutrophil chemotaxis and activation via its binding to ligands like IL-8 (CXCL8) and CXCL6 (Turner et al ., 2014). Neutrophils being the primary immune cells in udder infections, make this mechanism an essential part of immune responses to mastitis. However, S. aureus has evolved sophisticated mechanisms to modulate these immune mechanisms through DNA methylation changes. For example, increased CpG site methylation in the CXCR1 gene has been observed in S. aureus -infected mammary epithelial cells, impairing the host immune defence, and facilitating bacterial persistence (Dinarello, 2000; Mao et al., 2015; Wang et al., 2020). CSF2RB , which encodes for the common receptor β chain of cytokines like GM-CSF, IL3 and IL5, has been reported as downregulated under promoter hypermethylation after S. aureus mastitis in buffalo (Nayan et al ., 2022). This suggests that subclinical mastitis reduces immune function by downregulation of these cytokines. Similarly, altered DNA methylation profiles were reported for other genes involved in S. aureus induced inflammation, like IL6R, TNF, IL10, IL17 etc. (Zhang et al ., 2018; Wang et al ., 2020). However, a cell culture study revealed contrasting results, i.e. DNA hypomethylation-led increase in expression of cytokines TNF-α, IL-1β, IL-6, IL-8 , and chemokines CXCL1 and CXCL6 , in response to bacterial peptidoglycan (PGN) and lipoteichoic acid (LTA) (Wu et al., 2020). By complex interactions of such mechanisms, S. aureus cell wall components promote subclinical and chronic mastitis due to their weak but persistent inflammatory activity (Eckel and Ametaj, 2016). Methylation and gene expression changes are dependent on the type of pathogen as well as the duration of exposure to the pathogen. Pathogen-dependent gene expression mRNA expression of inflammatory cytokines and chemokines like TNF-α, IL1β , and IL8 is more pronounced after stimulation from lipopolysaccharide (LPS), the cell wall component of Gram-negative bacteria, as compared to that from lipoteichoic acid (LTA), the constituent of Gram-positive bacterial cell wall. Therefore, S. aureus is frequently isolated from subclinical and chronic cases, in contrast to gram negative bacteria like E. coli (Yang et al., 2008; Wellnitz et al., 2011). TNF-α , an important cytokine for inflammation and immunity, was reported as downregulated due to promoter hypermethylation in cases of LTA-bearing S. aureus mastitis (Zhang et al., 2018; Wang et al., 2020; Dong et al., 2021; Wang et al., 2024), whereas mammary epithelial cells showed its hypomethylation and resultant increased expression upon LPS stimulation (Dong et al ., 2021). The STAT5-binding lactational enhancer present upstream to the αS1-casein gene’s promoter is normally hypomethylated in lactating mammary glands. A pathogen-specific epigenetic response occurs in this region, where remethylation leads to suppression of αS1-casein synthesis in case of E. coli acute mastitis, contributing to a metabolic shift toward immune defence. In contrast, subclinical S. aureus infections do not induce any such changes, allowing continued αS1-casein synthesis, which may contribute to the pathogen’s long-term survival in the mammary gland (Vanselow et al., 2006). Exposure-dependent gene expression Different durations of mammary epithelium stimulation with LTA have shown variable mRNA concentrations of immune mediators like IL-1β, IL-8, TNF-α, CXCL6 and β-defensin. A rapid increase was observed within 2-4 hours of stimulation, followed by a return to baseline levels after 8-16 hours, and negligible concentrations by 24 hours (Strandberg et al., 2005). Acute mastitis, where exposure time to S. aureus is relatively short, is characterized by higher concentrations of IL-12 and IFN-γ for significantly longer time periods, reflecting its clinical nature (Bannerman et al., 2004). A dose dependent effect was demonstrated for LPS, wherein milk genes were supressed at its lower doses and immune genes were expressed at higher doses, owing to changes in methylation patterns at the corresponding promoter regions (Chen et al., 2019). Examples of the lactation-related genes reported were ACACA, ACSS2 and S6K1 (Chen et al., 2019). Leucocytes and DNA methylation Macrophages, neutrophils, and lymphocytes provide the next layer of protection after pathogen entry through physical barriers. They harbour PRRs on their surface and activate both innate and acquired immune responses (Sordillo et al., 1997; Soltys et al., 1999). Healthy mammary tissue mainly contains macrophages, and after recognition of pathogens, these macrophages release pro-inflammatory cytokines leading to recruitment of neutrophils (Alhussien et al., 2021; Rainard, 2003). Neutrophils are important for defence in acute as well as chronic mastitis (Rainard, and Riollet, 2003), as they act as the first line of defence and eliminate pathogens by phagocytosis (Paape et al., 2003). Level of neutrophils in the blood of cows in the puerperal period is highly heritable and is associated with susceptibility to clinical mastitis (Burvenich et al ., 2000; König and May, 2019). Therefore, a decrease in neutrophil count after parturition is associated with higher incidences of mastitis (Yamaguchi et al., 1999). Also, a major constituent of neutrophil secondary granules, lactoferrin (an iron binding protein), exhibits bacteriostatic activity against S . aureus (Bennett and Kokocinski, 1978; Diarra et al., 2002). DNA methylation has been associated with lactoferrin expression (Grant et al., 1999), which further opens the perspectives of studying the possible immune context vis-à-vis mastitis. Unlike neutrophils, the lymphocytes are recruited later in infection, and their interaction with S. aureus becomes crucial in determining the severity and outcome of the disease (Ziegler et al ., 2011; Rainard et al., 2022 ). These include T cells, B cells, and natural killer (NK) cells. Differential DNA methylation has been observed in peripheral blood lymphocytes for associated genes involved in inflammation, namely NRG1 (hypomethylated), MST1 (hypomethylated) and NAT9 (hypermethylated) during S. aureus subclinical mastitis, making them suitable epigenetic candidate genes (Song et al., 2016). NRG1 (neuregulin 1) , an epidermal growth factor, interacts with ErbB receptors in mammary gland and promotes anti-inflammatory macrophage (M2) responses (Zhao et al ., 2011). In phagocytes, the MST1 (mammalian sterile 20-like kinase 1) enhances the production of bactericidal reactive oxygen species (ROS) (Geng et al ., 2015), whereas NAT9 (N-acetyltransferase 9) is known to exhibit antiviral effects. Complement and DNA methylation The complement system is a part of humoral innate immune system, which comprises of a group of proteins that destroy the membranes of invading microorganisms, and also enhance phagocytosis through opsonization (Janeway et al., 2001). S. aureus is susceptible to the opsonising activity of complement proteins, but resistant to their lytic action (Rooijakkers et al., 2007; Laarman et al., 2011). This resistance is achieved by the interaction of bacterial ClfA with host complement regulator factor 1, resulting into cleavage of C3b (Hair et al., 2010). C3b is a critical component in the complement cascade. In absence of a functional C3b, formation of membrane attack complex is therefore inhibited, leading to suppressed lytic action of the complement. This mechanism of immune evasion by S. aureus was reported coinciding with promoter hypermethylation in the C3 gene of Indian buffalo, revealing contributions from epigenetic regulation (Nayan et al., 2022). Immune Evasion and Epigenetic Modifications by S. aureus S. aureus employs multiple mechanisms to evade immune responses in host: (1) secretion of toxins and immune modulators that impair host white blood cells, (2) production of protein A, which interferes with opsonization and phagocytosis, and (3) biofilm formation, which protects the bacteria from phagocytosis and antibiotics. Toxins and immune modulators: The organism secretes toxins and immune modulators that impair host white blood cells (WBCs). Exotoxins such as TSST-1, staphylococcal enterotoxins (SE), and staphylococcal enterotoxin-like proteins (SEl) play an important role in pathogenesis within the mammary gland by acting as superantigens leading to a massive cytokine release (Fitzgerald et al., 2001; Tollersrud et al., 2006). Enterotoxins M and H cause inflammation, necrosis, and apoptosis of mammary epithelium (Liu et al., 2014; Zhao et al., 2020). Leucocidins help S. aureus escape the immune system by disrupting WBC function (Alonzo and Torres, 2014). Tissue necrosis is caused by the α and β hemolysins (von Hoven et al., 2016; Cifrian et al., 1996). All these chemicals also increase cell permeability, exert lymphotoxic effects and lyse RBCs for iron acquisition (Huseby et al., 2007). Research on S. aureus induced nasal polyps has shown enterotoxin B to influence DNA methylation patterns in the nasal tissue (Pérez-Novo et al., 2013), and its impact on methylation status in udder tissue warrants further investigation. The TERT (telomerase reverse transcriptase) gene, despite remaining hypermethylated during both coagulase-negative (CNS) and coagulase-positive (CPS) staphylococcal infections, showed downregulation in the latter case only (Ząbek et al., 2020). This suggests a direct role of bacterial exotoxins on the host signalling pathways, and that these bacterial factors can override methylation-based repression under certain conditions. Protein A: Staphylococcal protein A (SpA) is a surface protein found in the bacterial cell wall, and exhibits anti-opsonic properties by preventing FcR-mediated phagocytosis (Forsgren and Sjöquist, 1966; Becker et al., 2014). Biofilm formation: In a biofilm, many layers of bacterial cells are embedded in the extracellular matrix, which adheres to biological surfaces and is made up of polysaccharide intracellular adhesions (PIA) mainly (Arciola et al., 2015; Formosa-Dague et al., 2016). Attachment, microcolony formation, maturation and detachment are the stages involved in development of biofilm (Pereya et al., 2016). Extracellular matrix of this stratified structure acts as a barrier, providing resistance to antibiotics, and also helps in evasion of immune response, making it significant for pathogenicity of S. aureus . Presence of biofilm has been described in udder of cows suffering from intramammary infection (Schönborn et al., 2016). The microbe also uses several other immune evasion mechanisms in addition to the abovementioned. For example, hypomethylation of the anti-inflammatory cytokine IL10 contributes to immune suppression, leading to chronic and subclinical mastitis (Wang et al ., 2020). Although direct evidence linking various bacterial factors to DNA methylation changes in host is limited, studies on other bacterial components (Qin et al., 2021) point to the possibility of elucidating specific effects of these elements as well. Involvement of non-genic regions Interestingly, in a study, DNA hypermethylation was observed in repeat elements (REs) LINE-1 and tRNA-derived SINE, which correlated with positive gene expression changes in immune-related genes PADI4 and CCAR1 respectively (Wang et al., 2024). Transposon MTD , member of the long terminal repeat (LTR) retrotransposon family showed hypomethylation in S. aureus subclinical mastitis cases in the same study (Wang et al., 2024). These observations suggest that certain non-genic elements can also influence gene transcription in unexpected ways, possibly by acting as alternative regulatory elements, and can be explored in future studies. Table 1: Genes with differential expression associated with DNA methylation changes in S. aureus mastitis ACTN1 Controls the organization of the actin cytoskeleton Hypermethylation Downregulated Wang et al., 2024 BOLA-DOB Antigen presentation and heat resistance Hypomethylation Upregulated Wang et al., 2024 BTK B cell development and maturation Hypomethylation Upregulated Wang et al ., 2020 C3 * Complement component Hypermethylation Downregulated Nayan et al ., 2022 CD4 Co-receptor for TCR during antigen recognition Hypermethylation Downregulated Wang et al., 2024 CLDN3 Forms tight junctions between cells Hypermethylation Downregulated Wang et al., 2024 CLDN4 Increases the complexity of tight junctions Hypermethylation Downregulated Wang et al., 2024 CPAMD8* Protease Inhibitor Hypermethylation Downregulated Nayan et al ., 2022 CSF2RB * Common receptor β chain of cytokines and chemokines like GMCSF, IL3, IL5 Hypermethylation Downregulated Nayan et al ., 2022 CSN1S1 Encodes αS1-casein milk protein Hypermethylation Downregulated Wang et al., 2024 CXADR Cell adhesion, viral reception, and signaling Hypermethylation Downregulated Wang et al., 2024 CXCL1 Neutrophil chemotaxis, angiogenesis Hypomethylation Upregulated Wu et al., 2020 CXCL17 Angiogenesis, neutrophil traffiking, antimicrobial activity Hypermethylation Downregulated Wang et al., 2024 CXCL6 Neutrophil chemotaxis, wound healing, tissue repair Hypomethylation Upregulated Wu et al., 2020 CXCR1 Induces inflammation Hypermethylation Downregulated Wang et al ., 2020 ; Wang et al ., 2024 EGR1 Regulates cell proliferation and apoptosis Hypermethylation Downregulated Wang et al., 2024 FGF1 Wound healing and cell proliferation Hypermethylation Downregulated Wang et al., 2024 FGF2 Tissue repair, wound healing Hypermethylation Downregulated Wang et al., 2024 IL10 Anti-inflammatory cytokine Hypomethylation Upregulated Wang et al ., 2020 IL17 Neutrophil mobilization and activation Hypomethylation Upregulated Wang et al ., 2020 IL1R2 IL1 receptor, anti-inflammatory effect Hypermethylation Downregulated Wang et al ., 2020 IL-1β Inflammation, fever Hypomethylation Upregulated Wu et al., 2020 ; Wang et al., 2024 IL2RA Regulates T cell function Hypomethylation Upregulated Wang et al., 2024 IL-6 Inflammation, haematopoiesis Hypomethylation Upregulated Wu et al., 2020; IL6R IL6 receptor. Induces proinflammatory response Hypermethylation Downregulated Zhang et al ., 2018 ; Wang et al ., 2024 IL-8 Neutrophil chemotaxis, angiogenesis Hypomethylation Upregulated Wu et al., 2020 IRF2 Development and maturation of NK cells Hypermethylation Downregulated Wang et al., 2024 KLF4 Regulates cell proliferation, differentiation, and apoptosis Hypermethylation Downregulated Wang et al., 2024 LINE-1 # Retrotransposon Hypermethylation PADI4 upregulated Wang et al., 2024 MEF2A T-cell activation, muscle development, cardiovascular function, and neuronal activity Hypomethylation Upregulated Wang et al., 2024 MST1 Anti-inflammatory molecule (Reduces IL1β, IL6, TNFα) Hypomethylation Upregulated Song et al., 2016 NAT9 N-Acetyltransferase- Antiviral effect Hypermethylation Downregulated Song et al., 2016 NCF1 Promotes neutrophil activity and phagocytosis Hypomethylation Upregulated Wang et al., 2024 Nckap5 $ Immune cell apoptosis Hypomethylation Upregulated Wang et al., 2019 ; Wang et al., 2024 NFKBIA Negative regulator of inflammation Hypermethylation Downregulated Wang et al ., 2020 NRG1 Anti-inflammatory molecule Hypermethylation Downregulated Song et al., 2016 PZP * Protease inhibition; Th-1 immune response inhibition Hypermethylation Downregulated Nayan et al ., 2022 RAC2 Regulates secretion, phagocytosis, and cell polarization Hypomethylation Upregulated Wang et al., 2024 SLC9A3R1 Sodium/hydrogen exchanger regulatory cofactor (NHE-RF1 Hypermethylation Downregulated Wang et al., 2024 SP4 Enables transcription factor activity Hypermethylation Downregulated Wang et al., 2024 SPRY2 Regulates cell proliferation, differentiation, and survival Hypermethylation Downregulated Wang et al., 2024 SYK Encodes tyrosine kinase- B cell development, cellular adhesion, innate immune recognition Hypomethylation Upregulated Wang et al., 2024 TERT Maintains telomerase, promotes cell division Hypermethylation Upregulated/ Downregulated Ząbek et al., 2020 TGFB2 Regulates cell growth, differentiation, role in wound healing Hypermethylation Downregulated Wang et al., 2024 TNF-a Pro-inflammatory cytokine Hypermethylation Downregulated Wang et al ., 2020; TNFSF8 Activation and differentiation of T and B cells Hypomethylation Upregulated Wang et al ., 2020 transposon MTD $# Acts as LTR-retrotransposons; Hypomethylation Upregulated Wang et al., 2019 tRNA-derived SINE # Non coding Hypermethylation CCAR1 upregulated Wang et al., 2024 * Study on water buffalo $ Study in mouse model # Non-genic regions Gaps in research While most of the current research has focused on bulk tissue analysis, cell-specific studies on macrophages, neutrophils etc. in addition to mammary epithelial cells, can provide more precise insights into immune regulation through DNA methylation alterations. Since the variable nature of epigenetics through time is well known, longitudinal studies can be designed to assess the long-term effects. Differences in DNA methylation responses among dairy breeds remain unexplored till date, and an understanding of these differences can be crucial for selective breeding strategies in future. A multitude of staphylococcal metabolites interact with the host’s immune system to influence the outcome of infection, and more of these can be inspected in the epigenetic aspect. A comprehensive understanding of immune regulation can be developed through large-scale epigenome-wide association studies (EWAS) which link DNA methylation profiles with mastitis susceptibility in dairy animals. Applications and potential future research areas Based on findings from different studies, important DNA methylation markers can be selected and used as epigenetic biomarkers for early detection of S. aureus mastitis. Information on methylation patterns in immune-related genomic regions can assist in selection of animals with natural resistance to S. aureus infections. Targeted epigenetic immunotherapy to alter the immune system epigenome has been used successfully for diseases like cancer in humans, and it can be applied for mastitis management on similar lines. Site-specific epigenetic modifications in DNA methylation patterns can be carried out using systems like CRISPR to enhance mastitis resistance. Conclusion Over years, the dairy industry has continued to face persistent challenge to production in the form of mastitis, accounting for substantial financial losses, and compromised animal welfare, product quality and quantity, and public health concerns. Staphylococcus aureus -induced mastitis is particularly a menace for the farmer due to concerns of immune evasion, antimicrobial resistance (AMR), and persistent infections. Traditional control strategies of improving management and genetic selection have been met with limited success due to the aforementioned factors as well as the low heritability of mastitis susceptibility trait. Therefore, supplementing these with information from epigenetic marks like DNA methylation, becomes important in disease control. S. aureus modulates host immune responses function through DNA methylation of many immune genes complementing its virulence factors, leading to persistence of the infection. Hypomethylation of promoter regions in immunosuppressive genes like IL10 , leading to their upregulation and in contrast, hypermethylation in pro-inflammatory CXR1, TNF-α, IL6R, C3 etc. causing their downregulation explains the lowering of immune responses caused during S. aureus mastitis . Hence, these genes can potentially act as epigenetic biomarkers for the disease. These biomarkers can enable early disease detection as well as selection of mastitis resistant animals. Application of EWAS, and use of epigenetic immunotherapy and targeted interventions through CRISPR have prospective of developing novel mastitis control strategies. Integrating epigenetic insights in genomic selection programmes can assure enhancement of the resilience of dairy animals against S. aureus mastitis in the future . Author contributions Apeksha: Conceptualization; writing – original draft. Abhishek Mahendra Todkari: Conceptualization; writing – original draft. Mir Mehroz Hassan: Writing – review and editing. Ashok Chaudhary: Writing – review and editing. Diksha Upreti: Writing – review and editing. Shri Ram Saini: Writing – review and editing. Sheikh Firdous Ahmad: Writing – review and editing. A. K. Pandey: Conceptualization; writing – review and editing Acknowledgements The authors sincerely acknowledge the Director and Joint Director (Research), ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar for providing their invaluable support and necessary infrastructural facilities for this review. Conflict of interest statement The authors declare that there are no conflicts of interest associated with this study. Data availability statement No new data were generated or analysed as part of this review. References Aghamohammadi, M., Haine, D., Kelton, D.F., Barkema, H.W., Hogeveen, H., Keefe, G.P. and Dufour, S., 2018. Herd-level mastitis-associated costs on Canadian dairy farms. Frontiers in veterinary science , 5 , p.355975. Aitken, S.L., Corl, C.M. and Sordillo, L.M., 2011. Immunopathology of mastitis: insights into disease recognition and resolution. Journal of mammary gland biology and neoplasia , 16 , pp.291-304. Alhussien, M.N., Panda, B.S. and Dang, A.K., 2021. A comparative study on changes in total and differential milk cell counts, activity, and expression of milk phagocytes of healthy and mastitic indigenous sahiwal cows. Frontiers in Veterinary Science , 8 , p.670811. Almeida, R.A., Matthews, K.R., Cifrian, E., Guidry, A.J. and Oliver, S.P., 1996. Staphylococcus aureus invasion of bovine mammary epithelial cells. Journal of dairy science , 79 (6), pp.1021-1026. Alonzo III, F. and Torres, V.J., 2014. The bicomponent pore-forming leucocidins of Staphylococcus aureus. Microbiology and Molecular Biology Reviews , 78 (2), pp.199-230. Arciola, C.R., Campoccia, D., Ravaioli, S. and Montanaro, L., 2015. Polysaccharide intercellular adhesin in biofilm: structural and regulatory aspects. Frontiers in cellular and infection microbiology , 5 , p.7. Asfaw, M. and Negash, A., 2017. Review on impact of bovine mastitis in dairy production. Adv Biol Res , 11 (3), pp.126-131. Ashraf, A. and Imran, M., 2020. Causes, types, etiological agents, prevalence, diagnosis, treatment, prevention, effects on human health and future aspects of bovine mastitis. Animal health research reviews , 21 (1), pp.36-49. Azooz, M.F., El-Wakeel, S.A. and Yousef, H.M., 2020. Financial and economic analyses of the impact of cattle mastitis on the profitability of Egyptian dairy farms. Veterinary world , 13 (9), p.1750. Bannerman, D.D., Paape, M.J., Lee, J.W., Zhao, X., Hope, J.C. and Rainard, P., 2004. Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection. Clinical and Vaccine Immunology , 11 (3), pp.463-472. Barkema, H.W., Schukken, Y.H. and Zadoks, R.N., 2006. Invited review: The role of cow, pathogen, and treatment regimen in the therapeutic success of bovine Staphylococcus aureus mastitis. Journal of dairy science , 89 (6), pp.1877-1895. Barkema, H.W., Schukken, Y.H., Lam, T.J.G.M., Beiboer, M.L., Wilmink, H., Benedictus, G. and Brand, A., 1998. Incidence of clinical mastitis in dairy herds grouped in three categories by bulk milk somatic cell counts. Journal of dairy science , 81 (2), pp.411-419. Bathla, S., Sindhu, A., Kumar, S., Dubey, S.K., Pattnaik, S., Rawat, P., Chopra, A., Dang, A., Kaushik, J.K. and Mohanty, A.K., 2020. Tandem Mass Tag (TMT)-based quantitative proteomics reveals potential targets associated with onset of Sub-clinical Mastitis in cows. Scientific Reports , 10 (1), p.9321. Becker, S., Frankel, M.B., Schneewind, O. and Missiakas, D., 2014. Release of protein A from the cell wall of Staphylococcus aureus. Proceedings of the National Academy of Sciences , 111 (4), pp.1574-1579. Benić, M., Maćešić, N., Cvetnić, L., Habrun, B., Cvetnić, Ž., Turk, R., Đuričić, D., Lojkić, M., Dobranić, V., Valpotić, H. and Grizelj, J., 2018. Bovine mastitis: a persistent and evolving problem requiring novel approaches for its control-a review. Veterinarski arhiv , 88 (4), pp.535-557. Bennett, R.M. and Kokocinski, T., 1978. Lactoferrin content of peripheral blood cells. British Journal of Haematology , 39 (4), pp.509-521. Bernstein, B.E., Meissner, A. and Lander, E.S., 2007. The mammalian epigenome. Cell , 128 (4), pp.669-681. Bobbo, T., Penasa, M. and Cassandro, M., 2019. Genetic aspects of milk differential somatic cell count in Holstein cows: A preliminary analysis. Journal of dairy science , 102 (5), pp.4275-4279. Bogni, C., Odierno, L., Raspanti, C., Giraudo, J., Larriestra, A., Reinoso, E., Lasagno, M., Ferrari, M., Ducrós, E., Frigerio, C. and Bettera, S., 2011. War against mastitis: Current concepts on controlling bovine mastitis pathogens. Science against microbial pathogens: Communicafing current research and technological advances , pp.483-494. Bouquet, A. and Juga, J., 2013. Integrating genomic selection into dairy cattle breeding programmes: a review. Animal , 7 (5), pp.705-713. Bouzeraa, L., Martin, H., Plessis, C., Dufour, P., Marques, J.C., Moore, S., Cerri, R. and Sirard, M.A., 2024. Decoding epigenetic markers: implications of traits and genes through DNA methylation in resilience and susceptibility to mastitis in dairy cows. Epigenetics , 19 (1), p.2391602. Burvenich, C., Detilleux, J., Paape, M.J. and Massart-Leën, A.M. 2000. Physiological and genetic factors that influence the cow resistance to mastitis, especially during early lactation. Flem Vet J , pp.9-20. Campos, B., Pickering, A.C., Rocha, L.S., Aguilar, A.P., Fabres-Klein, M.H., de Oliveira Mendes, T.A., Fitzgerald, J.R. and de Oliveira Barros Ribon, A., 2022. Diversity and pathogenesis of Staphylococcus aureus from bovine mastitis: Current understanding and future perspectives. BMC Veterinary Research , 18 (1), p.115. Chandrasekaran, D., Venkatesan, P., Tirumurugaan, K.G., Nambi, A.P., Thirunavukkarasu, P.S., Kumanan, K., Vairamuthu, S. and Ramesh, S., 2014. Pattern of antibiotic resistant mastitis in dairy cows. Veterinary World , 7 (6). Chen, J., Wu, Y., Sun, Y., Dong, X., Wang, Z., Zhang, Z., Xiao, Y. and Dong, G., 2019. Bacterial lipopolysaccharide induced alterations of genome-wide DNA methylation and promoter methylation of lactation-related genes in bovine mammary epithelial cells. Toxins , 11 (5), p.298. Cheng, J., Zhang, J., Han, B., Barkema, H.W., Cobo, E.R., Kastelic, J.P., Zhou, M., Shi, Y., Wang, J., Yang, R. and Gao, J., 2020. Klebsiella pneumoniae isolated from bovine mastitis is cytopathogenic for bovine mammary epithelial cells. Journal of dairy science , 103 (4), pp.3493-3504. Cheng, W.N. and Han, S.G., 2020. Bovine mastitis: Risk factors, therapeutic strategies, and alternative treatments—A review. Asian-Australasian journal of animal sciences , 33 (11), p.1699. Cheng, R., Wang, Q. and Wei, L., 2022. Income growth, employment structure transition and the rise of modern markets: The impact of urbanization on residents’ consumption of dairy products in China. Plos one , 17 (4), p.e0267006. Cifrian, E., Guidry, A.J., Bramley, A.J., Norcross, N.L., Bastida-Corcuera, F.D. and Marquardt, W.W., 1996. Effect of staphylococcal β toxin on the cytotoxicity, proliferation and adherence of Staphylococcus aureus to bovine mammary epithelial cells. Veterinary microbiology , 48 (3-4), pp.187-198. Cobirka, M., Tancin, V. and Slama, P., 2020. Epidemiology and classification of mastitis. Animals , 10 (12), p.2212. Dalanezi, F.M., Joaquim, S.F., Guimarães, F.F., Guerra, S.T., Lopes, B.C., Schmidt, E.M.S., Cerri, R.L.A. and Langoni, H., 2020. Influence of pathogens causing clinical mastitis on reproductive variables of dairy cows. Journal of dairy science , 103 (4), pp.3648-3655. De Borre, M. and Branco, M.R., 2021. Oxidative bisulfite sequencing: an experimental and computational protocol. DNA Modifications: Methods and Protocols , pp.333-348. de Haas, Y., Barkema, H.W. and Veerkamp, R.F., 2002. Genetic parameters of pathogen-specific incidence of clinical mastitis in dairy cows. Animal Science , 74 (2), pp.233-242. De Jong, N.W., Van Kessel, K.P. and Van Strijp, J.A., 2019. Immune evasion by Staphylococcus aureus. Microbiology spectrum , 7 (2), pp.10-1128. De Vliegher, S., Fox, L.K., Piepers, S., McDougall, S. and Barkema, H.W., 2012. Invited review: Mastitis in dairy heifers: Nature of the disease, potential impact, prevention, and control. Journal of dairy science , 95 (3), pp.1025-1040. Dego, O.K., 2020. Bovine mastitis: part I. Animal reproduction in veterinary medicine , p.149. Detilleux, J., Kastelic, J.P. and Barkema, H.W., 2015. Mediation analysis to estimate direct and indirect milk losses due to clinical mastitis in dairy cattle. Preventive veterinary medicine , 118 (4), pp.449-456. Diarra, M.S., Petitclerc, D. and Lacasse, P., 2002. Effect of lactoferrin in combination with penicillin on the morphology and the physiology of Staphylococcus aureus isolated from bovine mastitis. Journal of dairy science , 85 (5), pp.1141-1149. Dong, Y., An, D., Wang, J., Liu, H., Zhang, Q., Zhao, J. and Lu, W., 2021. Effect of DNA methylation on LPS-induced expression of tumour necrosis factor alpha (TNF-α) in bovine mammary epithelial cells. Indian Journal of Animal Research , 55 (9), pp.1079-1084. Dufour, S., Dohoo, I.R., Barkema, H.W., Descôteaux, L., Devries, T.J., Reyher, K.K., Roy, J.P. and Scholl, D.T., 2012. Epidemiology of coagulase-negative staphylococci intramammary infection in dairy cattle and the effect of bacteriological culture misclassification. Journal of dairy science , 95 (6), pp.3110-3124. Eckel, E.F. and Ametaj, B.N., 2016. Invited review: Role of bacterial endotoxins in the etiopathogenesis of periparturient diseases of transition dairy cows. Journal of dairy science , 99 (8), pp.5967-5990. Engler, C., Renna, M.S., Beccaria, C., Silvestrini, P., Pirola, S.I., Pereyra, E.A., Baravalle, C., Camussone, C.M., Monecke, S., Calvinho, L.F. and Dallard, B.E., 2022. Differential immune response to two Staphylococcus aureus strains with distinct adaptation genotypes after experimental intramammary infection of dairy cows. Microbial Pathogenesis , 172 , p.105789. Erskine, R.J., Walker, R.D., Bolin, C.A., Bartlett, P.C. and White, D.G., 2002. Trends in antibacterial susceptibility of mastitis pathogens during a seven-year period. Journal of Dairy Science , 85 (5), pp.1111-1118. Fitzgerald, J.R., Monday, S.R., Foster, T.J., Bohach, G.A., Hartigan, P.J., Meaney, W.J. and Smyth, C.J., 2001. Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens. Journal of bacteriology , 183 (1), pp.63-70. Flusberg, B.A., Webster, D.R., Lee, J.H., Travers, K.J., Olivares, E.C., Clark, T.A., Korlach, J. and Turner, S.W., 2010. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nature methods , 7 (6), pp.461-465. Formosa-Dague, C., Feuillie, C., Beaussart, A., Derclaye, S., Kucharikova, S., Lasa, I., Van Dijck, P. and Dufrene, Y.F., 2016. Sticky matrix: adhesion mechanism of the staphylococcal polysaccharide intercellular adhesin. ACS nano , 10 (3), pp.3443-3452. Forsgren, A. and Sjöquist, J., 1966. “Protein a” from S. aureus: I. Pseudo-immune reaction with human γ-globulin. The Journal of Immunology , 97 (6), pp.822-827. García-Álvarez, L., Holden, M.T., Lindsay, H., Webb, C.R., Brown, D.F., Curran, M.D., Walpole, E., Brooks, K., Pickard, D.J., Teale, C. and Parkhill, J., 2011. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study. The Lancet infectious diseases , 11 (8), pp.595-603. Geng, J., Sun, X., Wang, P., Zhang, S., Wang, X., Wu, H., Hong, L., Xie, C., Li, X., Zhao, H. and Liu, Q., 2015. Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity. Nature immunology , 16 (11), pp.1142-1152. Gilbert, F.B., Cunha, P., Jensen, K., Glass, E.J., Foucras, G., Robert-Granié, C., Rupp, R. and Rainard, P., 2013. Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system. Veterinary research , 44 , pp.1-23. Gogoi-Tiwari, J., Williams, V., Waryah, C.B., Costantino, P., Al-Salami, H., Mathavan, S., Wells, K., Tiwari, H.K., Hegde, N., Isloor, S. and Al-Sallami, H., 2017. Mammary gland pathology subsequent to acute infection with strong versus weak biofilm forming Staphylococcus aureus bovine mastitis isolates: a pilot study using non-invasive mouse mastitis model. PLoS One , 12 (1), p.e0170668. Gómez-Díaz, E., Jorda, M., Peinado, M.A. and Rivero, A., 2012. Epigenetics of host–pathogen interactions: the road ahead and the road behind. PLoS pathogens , 8 (11), p.e1003007. González-Recio, O., 2012. Epigenetics: a new challenge in the post-genomic era of livestock. Frontiers in genetics , 2 , p.106. Grant, D.J., Shi, H. and Teng, C.T., 1999. Tissue and site-specific methylation correlates with expression of the mouse lactoferrin gene. Journal of molecular endocrinology , 23 (1), pp.45-55. Günther, J. and Seyfert, H.M., 2018, November. The first line of defence: insights into mechanisms and relevance of phagocytosis in epithelial cells. In Seminars in immunopathology (Vol. 40, No. 6, pp. 555-565). Berlin/Heidelberg: Springer Berlin Heidelberg. Hair, P.S., Echague, C.G., Sholl, A.M., Watkins, J.A., Geoghegan, J.A., Foster, T.J. and Cunnion, K.M., 2010. Clumping factor A interaction with complement factor I increases C3b cleavage on the bacterial surface of Staphylococcus aureus and decreases complement-mediated phagocytosis. Infection and immunity , 78 (4), pp.1717-1727. Halasa, T., Huijps, K., Østerås, O. and Hogeveen, H., 2007. Economic effects of bovine mastitis and mastitis management: A review. Veterinary quarterly , 29 (1), pp.18-31. Heikkilä, A.M., Liski, E., Pyörälä, S. and Taponen, S., 2018. Pathogen-specific production losses in bovine mastitis. Journal of dairy science , 101 (10), pp.9493-9504. Heimer, S.R., Yamada, A., Russell, H. and Gilmore, M., 2010. Response of corneal epithelial cells to Staphylococcus aureus. Virulence , 1 (4), pp.223-235. Herman, J.G., Graff, J.R., Myöhänen, S.B.D.N., Nelkin, B.D. and Baylin, S.B., 1996. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proceedings of the national academy of sciences , 93 (18), pp.9821-9826. Hernandez, A., Quinton, M., Miglior, F. and Mallard, B.A., 2006. Genetic parameters of dairy cattle immune response traits. Ho, D.H. and Burggren, W.W., 2010. Epigenetics and transgenerational transfer: a physiological perspective. Journal of Experimental Biology , 213 (1), pp.3-16. Holmberg, M., Fikse, W.F., Andersson‐Eklund, L., Artursson, K. and Lundén, A., 2012. Genetic analyses of pathogen‐specific mastitis. Journal of Animal Breeding and Genetics , 129 (2), pp.129-137. Holmes, M.A. and Zadoks, R.N., 2011. Methicillin resistant S. aureus in human and bovine mastitis. J Mammary Gland Biol Neoplasia , 16 , pp.373-382. Huseby, M., Shi, K., Brown, C.K., Digre, J., Mengistu, F., Seo, K.S., Bohach, G.A., Schlievert, P.M., Ohlendorf, D.H. and Earhart, C.A., 2007. Structure and biological activities of beta toxin from Staphylococcus aureus. Journal of bacteriology , 189 (23), pp.8719-8726. Ibeagha-Awemu, E.M. and Khatib, H., 2017. Epigenetics of livestock breeding. In Handbook of epigenetics (pp. 441-463). Academic Press. Ibeagha-Awemu, E.M. and Yu, Y., 2021. Consequence of epigenetic processes on animal health and productivity: is additional level of regulation of relevance?. Animal Frontiers , 11 (6), pp.7-18. Illingworth, R.S. and Bird, A.P., 2009. CpG islands–‘a rough guide’. FEBS letters , 583 (11), pp.1713-1720. Ivanova, E., Le Guillou, S., Hue-Beauvais, C. and Le Provost, F., 2021. Epigenetics: new insights into mammary gland biology. Genes , 12 (2), p.231. Jamali, H., Barkema, H.W., Jacques, M., Lavallée-Bourget, E.M., Malouin, F., Saini, V., Stryhn, H. and Dufour, S., 2018. Invited review: Incidence, risk factors, and effects of clinical mastitis recurrence in dairy cows. Journal of dairy science , 101 (6), pp.4729-4746. James, S.J., Cutler, P., Melnyk, S., Jernigan, S., Janak, L., Gaylor, D.W. and Neubrander, J.A., 2004. Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism. The American journal of clinical nutrition , 80 (6), pp.1611-1617. Janeway Jr, C.A., Travers, P., Walport, M. and Shlomchik, M.J., 2001. The complement system and innate immunity. In Immunobiology: The Immune System in Health and Disease. 5th edition . Garland Science. Jones, G.M. and Bailey, T.L., 2009. Understanding the basics of mastitis. Ju, Z., Jiang, Q., Wang, J., Wang, X., Yang, C., Sun, Y., Zhang, Y., Wang, C., Gao, Y., Wei, X. and Hou, M., 2020. Genome-wide methylation and transcriptome of blood neutrophils reveal the roles of DNA methylation in affecting transcription of protein-coding genes and miRNAs in E. coli-infected mastitis cows. BMC genomics , 21 , pp.1-14. Khulan, B., Thompson, R.F., Ye, K., Fazzari, M.J., Suzuki, M., Stasiek, E., Figueroa, M.E., Glass, J.L., Chen, Q., Montagna, C. and Hatchwell, E., 2006. Comparative isoschizomer profiling of cytosine methylation: the HELP assay. Genome research , 16 (8), pp.1046-1055. Kibebew, K., 2017. Bovine mastitis: A review of causes and epidemiological point of view. Journal of Biology, Agriculture and Healthcare , 7 (2), pp.1-14. König, S. and May, K., 2019. Invited review: Phenotyping strategies and quantitative-genetic background of resistance, tolerance and resilience associated traits in dairy cattle. Animal , 13 (5), pp.897-908. Krishnamoorthy, P., Suresh, K.P., Jayamma, K.S., Shome, B.R., Patil, S.S. and Amachawadi, R.G., 2021. An understanding of the global status of major bacterial pathogens of milk concerning bovine mastitis: a systematic review and meta-analysis (Scientometrics). Pathogens , 10 (5), p.545. Kumar, N., Manimaran, A., Kumaresan, A., Jeyakumar, S., Sreela, L., Mooventhan, P. and Sivaram, M., 2017. Mastitis effects on reproductive performance in dairy cattle: a review. Tropical animal health and production , 49 , pp.663-673. Laarman, A.J., Ruyken, M., Malone, C.L., van Strijp, J.A., Horswill, A.R. and Rooijakkers, S.H., 2011. Staphylococcus aureus metalloprotease aureolysin cleaves complement C3 to mediate immune evasion. The Journal of Immunology , 186 (11), pp.6445-6453. Lahouassa, H., Moussay, E., Rainard, P. and Riollet, C., 2007. Differential cytokine and chemokine responses of bovine mammary epithelial cells to Staphylococcus aureus and Escherichia coli. Cytokine , 38 (1), pp.12-21. Langevin, S.M. and Kelsey, K.T., 2013. The fate is not always written in the genes: epigenomics in epidemiologic studies. Environmental and molecular mutagenesis , 54 (7), pp.533-541. Lerminiaux, N.A. and Cameron, A.D., 2019. Horizontal transfer of antibiotic resistance genes in clinical environments. Canadian journal of microbiology , 65 (1), pp.34-44. Levison, L.J., Miller-Cushon, E.K., Tucker, A.L., Bergeron, R., Leslie, K.E., Barkema, H.W. and DeVries, T.J., 2016. Incidence rate of pathogen-specific clinical mastitis on conventional and organic Canadian dairy farms. Journal of dairy science , 99 (2), pp.1341-1350. Lister, R., Pelizzola, M., Dowen, R.H., Hawkins, R.D., Hon, G., Tonti-Filippini, J., Nery, J.R., Lee, L., Ye, Z., Ngo, Q.M. and Edsall, L., 2009. Human DNA methylomes at base resolution show widespread epigenomic differences. nature , 462 (7271), pp.315-322. Liu, Y., Chen, W., Ali, T., Alkasir, R., Yin, J., Liu, G. and Han, B., 2014. Staphylococcal enterotoxin H induced apoptosis of bovine mammary epithelial cells in vitro. Toxins , 6 (12), pp.3552-3567. LOGUE, C.J.K. and NOLAN, A., RECOGNISING AND CONTROLLING PAIN AND INFLAMMATION IN MASTITIS. Lush, J.L., 1950. Inheritance of susceptibility to mastitis. Journal of Dairy Science , 33 (2), pp.121-125. Maddela, P., Makwana, P.M., Parmar, S.M., Kalyani, I.H., Patel, D.R. and Parasana, D.K., 2024. Cultural and Biochemical Characterization of Staphylococcus aureus Isolates from Bovine Clinical and Subclinical Mastitis. Indian Journal of Veterinary Sciences & Biotechnology , 20 (3). Mallard, B.A., Emam, M., Paibomesai, M., Thompson-Crispi, K. and Wagter-Lesperance, L., 2015. Genetic selection of cattle for improved immunity and health. Japanese Journal of Veterinary Research , 63 (Supplement 1), pp.S37-S44. Mao, Y.J., Zhu, X.R., Li, R., Chen, D., Xin, S.Y., Zhu, Y.H., Liao, X.X., Wang, X.L., Zhang, H.M., Yang, Z.P. and Yang, L.G., 2015. Methylation analysis of CXCR1 in mammary gland tissue of cows with mastitis induced by Staphylococcus aureus. Genet. Mol. Res , 14 , pp.12606-12615. Meissner, A., Gnirke, A., Bell, G.W., Ramsahoye, B., Lander, E.S. and Jaenisch, R., 2005. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic acids research , 33 (18), pp.5868-5877. Middleton, J.R., 2008. Staphylococcus aureus antigens and challenges in vaccine development. Expert review of vaccines , 7 (6), pp.805-815. Miranda, T.B. and Jones, P.A., 2007. DNA methylation: the nuts and bolts of repression. Journal of cellular physiology , 213 (2), pp.384-390. Morales-Ubaldo, A.L., Rivero-Perez, N., Valladares-Carranza, B., Velázquez-Ordoñez, V., Delgadillo-Ruiz, L. and Zaragoza-Bastida, A., 2023. Bovine mastitis, a worldwide impact disease: prevalence, antimicrobial resistance, and viable alternative approaches. Veterinary and animal science , 21 , p.100306. Narayana, S.G., Schenkel, F., Miglior, F., Chud, T., Abdalla, E.A., Naqvi, S.A., Malchiodi, F. and Barkema, H.W., 2021. Genetic analysis of pathogen-specific intramammary infections in dairy cows. Journal of Dairy Science , 104 (2), pp.1982-1992. Nayan, V., Singh, K., Iquebal, M.A., Jaiswal, S., Bhardwaj, A., Singh, C., Bhatia, T., Kumar, S., Singh, R., Swaroop, M.N. and Kumar, R., 2022. Genome-wide DNA methylation and its effect on gene expression during subclinical mastitis in water buffalo. Frontiers in Genetics , 13 , p.828292. Nigam, R., 2015. Incidence and pattern of antibiotic resistance of Staphylococcus aureus isolated from clinical and subclinical mastitis in cattle and buffaloes. Asian Journal of Animal Sciences , 9 (3), pp.100-109. Niller, H.H., Banati, F., Ay, E. and Minarovits, J., 2012. Microbe-induced epigenetic alterations. In Patho-epigenetics of disease (pp. 419-455). New York, NY: Springer New York. Notcovich, S., 2021. The physiology of the keratin plug formation in the teat canal of dairy cattle and its interaction with current and novel methods for prevention of intramammary infections: a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science, Massey University, Turitea, Palmerston North, New Zealand (Doctoral dissertation, Massey University). Novick, R.P. and Geisinger, E., 2008. Quorum sensing in staphylococci. Annual review of genetics , 42 (1), pp.541-564. Obšteter, J., Jenko, J. and Gorjanc, G., 2021. Genomic selection for any dairy breeding program via optimized investment in phenotyping and genotyping. Frontiers in genetics , 12 , p.637017. Paape, M., Bannerman, D., Zhao, X. and Lee, J.W., 2003. The bovine neutrophil: Structure and function in blood and milk. Veterinary research , 34 (5), pp.597-627. Pereyra, E.A., Picech, F., Renna, M.S., Baravalle, C., Andreotti, C.S., Russi, R., Calvinho, L.F., Diez, C. and Dallard, B.E., 2016. Detection of Staphylococcus aureus adhesion and biofilm-producing genes and their expression during internalization in bovine mammary epithelial cells. Veterinary Microbiology, 183, pp.69-77. Pérez-Novo, C.A., Zhang, Y., Denil, S., Trooskens, G., De Meyer, T., Van Criekinge, W., Van Cauwenberge, P., Zhang, L. and Bachert, C., 2013. Staphylococcal enterotoxin B influences the DNA methylation pattern in nasal polyp tissue: a preliminary study. Allergy, Asthma & Clinical Immunology, 9, pp.1-5. Petronis, A., 2010. Epigenetics as a unifying principle in the aetiology of complex traits and diseases. Nature , 465 (7299), pp.721-727. Pighetti, G. M. and Elliott, A. A. 2011 Gene polymorphisms: the keys for marker assisted selection and unraveling core regulatory pathways for mastitis resistance, J. Mammary. Gland Biol ., 16, 421– 432. Qin, W., Scicluna, B.P. and van der Poll, T., 2021. The role of host cell DNA methylation in the immune response to bacterial infection. Frontiers in immunology , 12 , p.696280. Rainard, P. and Riollet, C., 2003. Mobilization of neutrophils and defense of the bovine mammary gland. Reproduction nutrition development , 43 (5), pp.439-457. Rainard, P. and Riollet, C., 2006. Innate immunity of the bovine mammary gland. Veterinary research , 37 (3), pp.369-400. Rainard, P., 2003. The complement in milk and defense of the bovine mammary gland against infections. Veterinary research , 34 (5), pp.647-670. Rainard, P., Foucras, G. and Martins, R.P., 2022. Adaptive cell-mediated immunity in the mammary gland of dairy ruminants. Frontiers in Veterinary Science , 9 , p.854890. Reyher, K. K., S. Dufour, H. W. Barkema, L. Des Côteaux, T. J. Devries, I. R. Dohoo, G. P. Keefe, J.-P. Roy, and D. T. Scholl. 2011. The National Cohort of Dairy Farms—A data collection platform for mastitis research in Canada. J. Dairy Sci. 94:1616– 1626. Rooijakkers, S.H., Milder, F.J., Bardoel, B.W., Ruyken, M., van Strijp, J.A. and Gros, P., 2007. Staphylococcal complement inhibitor: structure and active sites. The Journal of Immunology , 179 (5), pp.2989-2998. Saidani, M., Messadi, L., Soudani, A., Daaloul-Jedidi, M., Châtre, P., Ben Chehida, F., Mamlouk, A., Mahjoub, W., Madec, J.Y. and Haenni, M., 2018. Epidemiology, antimicrobial resistance, and extended-spectrum beta-lactamase-producing Enterobacteriaceae in clinical bovine mastitis in Tunisia. Microbial Drug Resistance , 24 (8), pp.1242-1248. Schönborn, S. and Krömker, V., 2016. Detection of the biofilm component polysaccharide intercellular adhesin in Staphylococcus aureus infected cow udders. Veterinary microbiology , 196 , pp.126-128. Serre, D., Lee, B.H. and Ting, A.H., 2010. MBD-isolated Genome Sequencing provides a high-throughput and comprehensive survey of DNA methylation in the human genome. Nucleic acids research , 38 (2), pp.391-399. Singha, S., Koop, G., Persson, Y., Hossain, D., Scanlon, L., Derks, M., Hoque, M.A. and Rahman, M.M., 2021. Incidence, etiology, and risk factors of clinical mastitis in dairy cows under semi-tropical circumstances in Chattogram, Bangladesh. Animals , 11 (8), p.2255. Sivakumar, R., Pranav, P.S., Annamanedi, M., Chandrapriya, S., Isloor, S., Rajendhran, J. and Hegde, N.R., 2023. Genome sequencing and comparative genomic analysis of bovine mastitis-associated Staphylococcus aureus strains from India. BMC genomics , 24 (1), p.44. Smolenski, G.A., Cursons, R.T., Hine, B.C. and Wheeler, T.T., 2015. Keratin and S100 calcium-binding proteins are major constituents of the bovine teat canal lining. Veterinary research , 46 , pp.1-10. Soares, B.S., Melo, D.A., Motta, C.C., Marques, V.F., Barreto, N.B., Coelho, S.M.O., Coelho, I.S. and Souza, M.M.S., 2017. Characterization of virulence and antibiotic profile and agr typing of Staphylococcus aureus from milk of subclinical mastitis bovine in State of Rio de Janeiro. Arquivo Brasileiro de Medicina Veterinária e Zootecnia , 69 , pp.843-850. Soltys, J. and Quinn, M.T., 1999. Selective recruitment of T-cell subsets to the udder during staphylococcal and streptococcal mastitis: analysis of lymphocyte subsets and adhesion molecule expression. Infection and Immunity , 67 (12), pp.6293-6302. Song, M., He, Y., Zhou, H., Zhang, Y., Li, X. and Yu, Y., 2016. Combined analysis of DNA methylome and transcriptome reveal novel candidate genes with susceptibility to bovine Staphylococcus aureus subclinical mastitis. Scientific reports , 6 (1), p.29390. Sordillo, L.M., Shafer-Weaver, K. and DeRosa, D., 1997. Immunobiology of the mammary gland. Journal of dairy science , 80 (8), pp.1851-1865. Stirzaker, C., Taberlay, P.C., Statham, A.L. and Clark, S.J., 2014. Mining cancer methylomes: prospects and challenges. Trends in Genetics , 30 (2), pp.75-84. Strandberg, Y., Gray, C., Vuocolo, T., Donaldson, L., Broadway, M. and Tellam, R., 2005. Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells. Cytokine , 31 (1), pp.72-86. Sutra, L. and Poutrel, B., 1994. Virulence factors involved in the pathogenesis of bovine intramammary infections due to Staphylococcus aureus. Journal of Medical Microbiology , 40 (2), pp.79-89. Tajima, S., Suetake, I., Takeshita, K., Nakagawa, A., Kimura, H. and Song, J., 2022. Domain structure of the Dnmt1, Dnmt3a, and Dnmt3b DNA methyltransferases. DNA Methyltransferases-Role and Function , pp.45-68. Tando, Y. and Matsui, Y., 2023. Inheritance of environment-induced phenotypic changes through epigenetic mechanisms. Environmental Epigenetics , 9 (1), p.dvad008. Thompson-Crispi, K.A., Sewalem, A., Miglior, F. and Mallard, B.A., 2012. Genetic parameters of adaptive immune response traits in Canadian Holsteins. Journal of dairy science , 95 (1), pp.401-409. Tollersrud, T., Kampen, A.H. and Kenny, K., 2006. Staphylococcus aureus enterotoxin D is secreted in milk and stimulates specific antibody responses in cows in the course of experimental intramammary infection. Infection and immunity , 74 (6), pp.3507-3512. Tost, J. and Gut, I.G., 2007. DNA methylation analysis by pyrosequencing. Nature protocols , 2 (9), pp.2265-2275. Triantaphyllopoulos, K.A., Ikonomopoulos, I. and Bannister, A.J., 2016. Epigenetics and inheritance of phenotype variation in livestock. Epigenetics & chromatin , 9 , pp.1-18. Turner, M.D., Nedjai, B., Hurst, T. and Pennington, D.J., 2014. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research , 1843 (11), pp.2563-2582. Van Nhieu, G.T. and Arbibe, L., 2009. Genetic reprogramming of host cells by bacterial pathogens. F1000 biology reports , 1 . Vanselow, J., Yang, W., Herrmann, J., Zerbe, H., Schuberth, H.J., Petzl, W., Tomek, W. and Seyfert, H.M., 2006. DNA-remethylation around a STAT5-binding enhancer in the αS1-casein promoter is associated with abrupt shutdown of αS1-casein synthesis during acute mastitis. Journal of molecular endocrinology , 37 (3), pp.463-477. Vitenberga-Verza, Z., Pilmane, M., Šerstņova, K., Melderis, I., Gontar, Ł., Kochański, M., Drutowska, A., Maróti, G. and Prieto-Simón, B., 2022. Identification of inflammatory and regulatory cytokines IL-1α-, IL-4-, IL-6-, IL-12-, IL-13-, IL-17A-, TNF-α-, and IFN-γ-producing cells in the milk of dairy cows with subclinical and clinical mastitis. Pathogens , 11 (3), p.372. von Hoven, G., Rivas, A.J., Neukirch, C., Klein, S., Hamm, C., Qin, Q., Meyenburg, M., Füser, S., Saftig, P., Hellmann, N. and Postina, R., 2016. Dissecting the role of ADAM10 as a mediator of Staphylococcus aureus α-toxin action. Biochemical Journal , 473 (13), pp.1929-1940. Walawski, K., 1999. Genetic aspects of mastitis resistance in cattle. Journal of Applied Genetics , 40 (2), pp.117-128. Wang, D., Wei, Y., Shi, L., Khan, M.Z., Fan, L., Wang, Y. and Yu, Y., 2019. Genome-wide DNA methylation pattern in a mouse model reveals two novel genes associated with Staphylococcus aureus mastitis. Asian-Australasian journal of animal sciences , 33 (2), p.203. Wang, M., Liang, Y., Ibeagha-Awemu, E.M., Li, M., Zhang, H., Chen, Z., Sun, Y., Karrow, N.A., Yang, Z. and Mao, Y., 2020. Genome-wide DNA methylation analysis of mammary gland tissues from Chinese Holstein cows with Staphylococcus aureus induced mastitis. Frontiers in Genetics , 11 , p.550515. Wang, M. and Ibeagha-Awemu, E.M., 2021. Impacts of epigenetic processes on the health and productivity of livestock. Frontiers in Genetics , 11 , p.613636. Wang, M., Laterrière, M., Dudemaine, P.L., Bissonnette, N., Gagné, D. and Ibeagha-Awemu, E.M., 2022. PSVIII-B-15 Potential Involvement of DNA Methylation of First Intron in Transcriptional Regulation During Bovine Subclinical Mastitis Caused by Staphylococcus Aureus. Journal of Animal Science , 100 (Supplement_3), pp.310-311. Wang, M., Bissonnette, N., Laterrière, M., Gagné, D., Dudemaine, P.L., Roy, J.P., Sirard, M.A. and Ibeagha-Awemu, E.M., 2023. Genome-wide DNA methylation and transcriptome integration associates DNA methylation changes with bovine subclinical mastitis caused by Staphylococcus chromogenes. International Journal of Molecular Sciences , 24 (12), p.10369. Wang, M., Bissonnette, N., Laterrière, M., Dudemaine, P.L., Gagné, D., Roy, J.P., Sirard, M.A. and Ibeagha-Awemu, E.M., 2024. DNA methylation haplotype block signatures responding to Staphylococcus aureus subclinical mastitis and association with production and health traits. BMC biology , 22 (1), p.65. Weber, M., Davies, J.J., Wittig, D., Oakeley, E.J., Haase, M., Lam, W.L. and Schuebeler, D., 2005. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nature genetics , 37 (8), pp.853-862. Wellnitz, O. and Bruckmaier, R.M., 2012. The innate immune response of the bovine mammary gland to bacterial infection. The veterinary journal , 192 (2), pp.148-152. Wellnitz, O., Arnold, E.T. and Bruckmaier, R.M., 2011. Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland. Journal of Dairy Science , 94 (11), pp.5405-5412. Wu, Y., Chen, J., Sun, Y., Dong, X., Wang, Z., Chen, J. and Dong, G., 2020. PGN and LTA from Staphylococcus aureus induced inflammation and decreased lactation through regulating DNA methylation and histone H3 acetylation in bovine mammary epithelial cells. Toxins , 12 (4), p.238. Xiong, Z. and Laird, P.W., 1997. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic acids research , 25 (12), pp.2532-2534. Yamaguchi, T., Hiratsuka, M., Asai, K., Kai, K. and Kumagai, K., 1999. Differential distribution of T lymphocyte subpopulations in the bovine mammary gland during lactation. Journal of dairy science , 82 (7), pp.1459-1464. Yang, W., Zerbe, H., Petzl, W., Brunner, R.M., Günther, J., Draing, C., von Aulock, S., Schuberth, H.J. and Seyfert, H.M., 2008. Bovine TLR2 and TLR4 properly transduce signals from Staphylococcus aureus and E. coli, but S. aureus fails to both activate NF-κB in mammary epithelial cells and to quickly induce TNFα and interleukin-8 (CXCL8) expression in the udder. Molecular immunology , 45 (5), pp.1385-1397. Yousefian, N., Alam, S., Ramappa, K.B., Schlecht, E. and Dittrich, C., 2024. Cows in the city: How consumer demands sustain urban dairying in the IT capital of India. Urban Agriculture & Regional Food Systems , 9 (1), p.e20070. Ząbek, T., Semik-Gurgul, E., Ropka-Molik, K., Szmatoła, T., Kawecka-Grochocka, E., Zalewska, M., Kościuczuk, E., Wnuk, M. and Bagnicka, E., 2020. Locus-specific interrelations between gene expression and DNA methylation patterns in bovine mammary gland infected by coagulase-positive and coagulase-negative staphylococci. Journal of dairy science , 103 (11), pp.10689-10695. Zhang, Y., Rohde, C., Reinhardt, R., Voelcker-Rehage, C. and Jeltsch, A., 2009. Non-imprinted allele-specific DNA methylation on human autosomes. Genome biology , 10 , pp.1-11. Zhang, Y., Wang, X., Jiang, Q., Hao, H., Ju, Z., Yang, C., Sun, Y., Wang, C., Zhong, J., Huang, J. and Zhu, H., 2018. DNA methylation rather than single nucleotide polymorphisms regulates the production of an aberrant splice variant of IL6R in mastitic cows. Cell Stress and Chaperones , 23 (4), pp.617-628. Zhao, W., Shen, Y. and Ren, S., 2011. Endogenous expression of Neuregulin-1 (Nrg1) as a potential modulator of prolactin (PRL) secretion in GH3 cells. Cell and Tissue Research , 344 , pp.313-320. Zhao, Y., Tang, J., Yang, D., Tang, C. and Chen, J., 2020. Staphylococcal enterotoxin M induced inflammation and impairment of bovine mammary epithelial cells. Journal of dairy science , 103 (9), pp.8350-8359. Zi, C., Zeng, D., Ling, N., Dai, J., Xue, F., Jiang, Y. and Li, B., 2018. An improved assay for rapid detection of viable Staphylococcus aureus cells by incorporating surfactant and PMA treatments in qPCR. BMC microbiology , 18 , pp.1-8. Ziegler, C., Goldmann, O., Hobeika, E., Geffers, R., Peters, G. and Medina, E., 2011. The dynamics of T cells during persistent Staphylococcus aureus infection: from antigen‐reactivity to in vivo anergy. EMBO molecular medicine , 3 (11), pp.652-666. Information & Authors Information Version history V1 Version 1 22 March 2025 Peer review timeline Published Animal Genetics Version of Record 25 Sep 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Animal Genetics Keywords staphylococcus aureus dna methylation epigenetics immune response mastitis Authors Affiliations Apeksha ICAR - Indian Veterinary Research Institute View all articles by this author Abhishek Mahendra Todkari 0009-0001-8075-1669 Government of Maharashtra View all articles by this author Ashok Chaudhary ICAR - Indian Veterinary Research Institute View all articles by this author Mir Mehroz Hassan ICAR - Indian Veterinary Research Institute View all articles by this author Diksha Upreti ICAR - Indian Veterinary Research Institute View all articles by this author Shri Ram Saini ICAR - Indian Veterinary Research Institute View all articles by this author Sheikh Firdous Ahmad 0000-0002-7114-0882 ICAR - Indian Veterinary Research Institute View all articles by this author A. K. Pandey 0009-0000-1390-9983 [email protected] ICAR - Indian Veterinary Research Institute View all articles by this author Metrics & Citations Metrics Article Usage 460 views 224 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Apeksha, Abhishek Mahendra Todkari, Ashok Chaudhary, et al. Decoding DNA Methylation in Staphylococcus aureus Mastitis: Implications for Immune Regulation and Disease Resistance. Authorea . 22 March 2025. DOI: https://doi.org/10.22541/au.174263595.58990203/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.174263595.58990203/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'a02556435952c13d',t:'MTc3OTg4ODIzNg=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();