Genetic polymorphisms in immune- and inflammation-associated genes and their association with bovine mastitis resistance/susceptibility

Bovine mastitis, the inflammation of the mammary gland, is a contagious disease characterized by chemical and physical changes in milk and pathological changes in udder tissues. Depressed immunity and higher expression of inflammatory cytokines with an elevated milk somatic cell count can be observed during mastitis in dairy cattle. The use of somatic cell count (SCC) and somatic cell score (SCS) as correlated traits in the indirect selection of animals against mastitis resistance is in progress globally. Traditional breeding for mastitis resistance seems difficult because of the low heritability (0.10-0.16) of SCC/SCS and clinical mastitis. Thus, genetic-marker-selective breeding to improve host genetics has attracted considerable attention worldwide. Moreover, genomic selection has been found to be an effective and fast method of screening for dairy cattle that are genetically resistant and susceptible to mastitis at a very early age. The current review discusses and summarizes the candidate gene approach using polymorphisms in immune- and inflammation-linked genes (CD4, CD14, CD46, TRAPPC9, JAK2, Tf, Lf, TLRs, CXCL8, CXCR1, CXCR2, C4A, C5, MASP2, MBL1, MBL2, LBP, NCF1, NCF4, MASP2, A2M, and CLU, etc.) and their related signaling pathways (Staphylococcus aureus infection signaling, Toll-like receptor signaling, NF-kappa B signaling pathway, Cytokine-cytokine receptor, and Complement and coagulation cascades, etc.) associated with mastitis resistance and susceptibility phenotypic traits (IL-6, interferon-gamma (IFN-γ), IL17, IL8, SCS, and SCC) in dairy cattle.


Background
Normally, the mammary gland performs three functions, including the provision of nutrition to offspring in the form of milk, the transfer of immunity from mother to offspring through immunoglobulins in milk, and the provision of protection against microbes (1,2). Bovine mastitis, the inflammation of mammary glands, occurs when the udder is exposed to any physical injury or pathogenic microorganisms (3). Bacterial pathogens, upon entry, compromise the immunity of the mammary gland and trigger the abnormal regulation of the immune system, followed by inflammatory changes, resulting in mastitis (4).
Based on clinical signs, mastitis can be divided into two types, i.e., clinical and sub-clinical mastitis (5)(6)(7)(8). Clinical mastitis is characterized by visible signs of inflammation in the udder and microbiological physical, and chemical changes in the milk (9)(10)(11). Escherichia coli is one of the major bacteria responsible for acute or clinical mastitis, while subclinical mastitis is caused by the grampositive Staphylococcus aureus (12- 14), which is characterized by a marked decrease in milk quantity and quality (15). The other signs of subclinical mastitis are the increase in milk SCC level, which shows the level of leucocytes and epithelial cells in milk (16). Elevated levels of leukocytes are the primary indicator of mammary infection.
An SCC of more than 200,000 cells/mL is an indication of mammary gland infections, while less than 100,000 cells/mL indicates that the cow is uninfected (17). Thus, increased levels of SCC are considered a sign of mastitis. The SCC and/or logtransformed SCC (somatic cell score, SCS) are widely targeted as early indicators of mastitis (3) because of a strong positive genetic correlation (0.6 to 0.90) between mastitis and milk SCC (18)(19)(20). The SCC and SCS have comparatively higher heritability than mastitis (21) and are therefore widely targeted in mastitis control by selecting cows with low SCS/SCC (22). However, increased SCC in early lactation can signify the presence of intra-mammary infection, and in many countries, indirect selection against mastitis using milk SCC is practiced (23). However, in the early phases of infectivity, the neutrophil, including the level of inflammatory cytokines, increases more rapidly than the total SCC (24). Moreover, resistance to the pathogenesis of mastitis is a complicated biological mechanism involving various molecules, cells, and pathways (25). That's why, nowadays, people are more interested in the increasing cells and cytokine levels in milk and blood rather than just the overall SCC, which may show the status of udder health at an earlier stage (26).
The epithelial cells of the inner surface of the mammary gland play a key role in recognizing mastitis-causing pathogens by synthesizing toll-like receptors (TLR2 & TLR4) (27, 28). Upon activation, TLRs further regulate nuclear factor-kB, which translocates into the nucleus and causes the mediation of proinflammatory signaling molecules (tumor necrosis factor-a (TNFa), IL-1b and IL-6, and IL-8) that are essential for the animal's local and systemic immune reactions (27,(29)(30)(31). It has been well-studied that serum cytokines, such as interferon, tumor necrosis factor, IL17, IL6, and IL4, have a key role in inflammatory circumstances, which suggests their possible role in bovine mastitis (32-35). Furthermore, previous studies have also suggested that in addition to SCC and SCS, serum cytokines could also be considered indirect parameters in the control strategies against bovine mastitis (36-38).
The control of mastitis using traditional selection methods is quite challenging due to the low heritability of indirect mastitis resistance phenotypic traits (SCC, SCS, and inflammatory cytokines) and host genetics (39). The use of milk SCC as a surrogate trait for raising mastitis resistance in cattle has achieved limited outcomes (36), thus, the information on molecular markers for mastitis susceptibility/resistance is valuable in identifying genetically mastitis-resistant cattle (40,41). Due to the limited and slow progress in improving udder health using conventional selection procedures with indirect traits, demand has increased for information on molecular markers to enhance host genetics against mastitis in cattle breeding (4,42).
Usually, two types of candidate gene approaches (direct and indirect) are used while looking for genetically mastitis-resistant cattle (43-45). The first approach is the use of linked markers (indirect), which are markers that are closed to the gene or QTL that has a significant role in mastitis. The second is the use of functional or direct genetic markers, in which the polymorphisms in genes associated with variation in mastitis resistance phenotypic traits are targeted (43,46). It is well documented that many genes control mastitis, and all of them contribute considerably to either mastitis resistance or development, so, it is necessary to consider the combination of genes for mastitis resistance because some genes have little effect. It has been documented that the immune and inflammatory response to bacteria is usually regulated by inflammationand immune-associated genes (47).
So far, several genetic polymorphisms in inflammation-and immune-related genes have been identified for their possible association with mastitis resistance phenotypic traits (33, 34, 37, 40, 41, 46, 48-50). In the current review, we discuss all the polymorphisms in immune-and inflammation-associated genes that are linked to mastitis resistance phenotypic traits (SCC, SCS, IL-6, IL8, IL17, and IFN-gamma). Based on the current review, we suggest that all the highlighted SNPs of the genes discussed could be considered potential genetic markers for mastitis resistance in dairy cattle.

Methods for collection of literature studies
For this review, we used Google Scholar, Web of Science, and PubMed. Furthermore, we used NCBI to verify the information regarding the genes, such as the number of exons and chromosome location. We only considered data published in the English language and in SCI journals. In addition, we used data that was published between 2000 and 2022. Only the polymorphisms located in exonic regions, promoters, or 5 or 3 untranslated regions of genes were utilized in the current study. Finally, we used the online software, DAVID for biological signaling pathways. The keywords, such as SCS, SCC, inflammatory cytokines, immune-and inflammation-associated genes, and polymorphisms were considered while collecting literature for the current review.
3 Genetic polymorphisms of genes associated with mastitis resistance/ susceptibility in dairy cattle The detection of single nucleotide polymorphisms (SNPs) in genes regulating the mammary gland's innate immunity in response to pathogens has attracted considerable attention within the field of genetic markers in mastitis control research (51-55). Several immuneand inflammation-associated genes and their polymorphisms have been discovered for their association with bovine mastitis susceptibility/ resistance (33, 56-60). All the reported polymorphisms in immuneand inflammation-associated genes are summarized in Table 1. 3.1 Innate immune-compartmentassociated genes 3.1.1 Bovine peptidoglycan recognition protein 1 (PGLYRP1) Bovine peptidoglycan recognition protein 1 located on chromosome 18, having 3 exons, has a significant role in regulating inflammation, response to infection, and post-infection healing (97). Because PGLYRP1 works as a receptor for murein peptidoglycans (PGN) of gram-positive and gram-negative bacteria, it is considered a key player in the activation of innate immunity (92). Considering its important role in immunity and inflammation, PGLYRP1 has been widely targeted in bovine mastitis research (92,97). A higher expression of PGLYRP1 has been documented in cows with mastitis (36, 97-99). Consequently, the polymorphisms (G + 102C, T -12G) in PGLYRP1 and SNP-C+4867T in PGLYRP2 in the exonic region showed an association with milk SCS in dairy cattle (100,101). Moreover, a study reported that the polymorphism rs68268284 at exon 1 of PGLYRP1 correlated with milk SCC (92), as mentioned in Table 1. 3.1.2 Calcium channel, voltage-dependent, alpha-2/delta subunit 1 (CACNA2D1) Calcium channel, voltage-dependent, alpha-2/delta subunit 1 residing on chromosome 4, containing 42 exons, is another key genetic signature with an important role in clinical mastitis. It has been documented that SNP-G519663A at exon 18 (80), SNP-G519663A, and SNP-A526745G at exon 24 in the bovine CACNA2D1 gene were significantly linked with lower milk SCS levels and mastitis resistance in dairy cattle (82, 102). Moreover, it has also been noticed that SNP G38819398A at exon 18 (81), variant-T38826986G at exon 19 (103), and SNP-C367284A of CACNA2D1 (104) were positively linked with milk SCS in Sahiwal and Karan Fries. The above findings show that CACNA2D1 could be a potential genetic marker against mastitis resistance in dairy cattle.

Mannose-binding lectin (MBL)
Mannose-binding lectin is a critical player belonging to the collectin protein family that attaches to a variety of microorganisms and regulates the innate immunity lectin-complement pathway (69). MBL-A and MBL-C proteins are encoded by the MBL1 and MBL2 genes respectively. The reduced level of MBL may expose dairy cattle to various infections, including mastitis (66). Similarly, a study reported a strong correlation of SNP g.2651G>A in the MBL gene with milk SCS, suggesting its possible role in mastitis resistance (65). In addition, other studies have also reported the significant association of SNPs in the MBL with milk SCS, an essential phenotypic indicator of bovine mastitis (66, 70).

Mannose-binding lectin-associated serine protease 2 (MASP2)
Mannose-binding lectin-associated serine protease 2 which is located on chromosome 16 and contains 11 exons, is considered the key protease of the complement system. The MASP2 gene plays an important role in innate immunity and creates resistance to infections of the mammary gland (105) and is therefore widely studied for its link with mastitis in dairy cattle (83,105). The polymorphism G553A in MASP2 was found to be associated with mastitis resistance and lower milk SCC in Chinese Holsteins (83). Similarly, another study also reported that the polymorphisms (g.14047A > C, g.14248T > C, and g.14391C > T) in MASP2 had a significant correlation with lower milk SCC and mastitis resistance in dairy cattle (105), as shown in Table 1.

Lactoferrin (Lf)
Lactoferrin is an important, iron-binding glycoprotein member of the serum-transferring protein family, which is produced by the mammary gland and immune cells and distributed in external secretions, such as milk, tears, and polymorphonuclear neutrophil (PMN) cells and plays a crucial role in eliminating bacterial load in certain organs. The Lf has several biological functions, including immunity and bacteriostatic activities, which protect the udder from pathogenic infections (106). A review article comprehensively highlighted the role of lactoferrin in bovine mastitis (107). The increased mRNA expression of Lf and the elevated level of SCC in the mammary gland with mastitis indicate their role in acute phase response in the mammary gland during mastitis (108). Similarly, another study reported a higher Lf expression during mammary gland infection (109). Recently, several reports have proven the association of genetic polymorphisms in the bovine Lf gene with mastitis susceptibility (53, [110][111][112][113][114][115][116]. One study documented that SNPs (SNP −190 G>A, −270 T > C, and −190 G >A) in the bovine lactoferrin gene were associated with milk SCS. Furthermore, it has been demonstrated that a higher expression of this gene may render dairy cows susceptible to mastitis (78).

Transferrin (Tf)
Transferrin is a b-globulin protein involved in iron ion transportation and plays a significant role in the regulation of innate immunity against microbial pathogens and blocked pathogenic access to iron (117, 118). Being a key player in immunity, Tf has been studied for its association with mastitis resistance in dairy cattle (79). In addition, it has been documented that cows with SNP g.13942T>C in Tf show the lowest milk SCS level. Moreover, they noticed a higher expression of Tf in the mammary glands of cows with mastitis, which suggests its crucial role in mastitis resistance (79).

Neutrophil cytosolic factor 4 (NCF4)
Calcium channel, voltage-dependent, alpha-2/delta subunit 1 is located on bovine chromosome 5 and contains 9 exons. Being a key component of the nicotinamide dinucleotide phosphate (NADPH) oxidase complex, NCF4 plays an important role in the regulation of biochemical pathways and innate immune responses against microbial infection (119). The role of the NCF4 has been well studied in bovine mastitis research (76,77). Ju et al. reported that the polymorphism g.18174 A>G on exon 9 of NCF4 showed a noteworthy relationship with high milk SCC levels and is responsible for the mastitis susceptibility of Chinese Holsteins (77). Another study found an SNP on g.18475 A>G in the 3′ UTR of NCF4, which was linked with higher levels of milk SCC, suggesting its critical role in mastitis susceptibility (76). It has been well established that, upon challenge with bacteria, the microRNAs (miRNAs) were significantly expressed in the mammary gland, which shows their role in the regulation of host immunity (120). Several studies have documented the role of miRNAs in bovine mastitis and host immune  regulation against pathogenic infections (76,96,(121)(122)(123)(124)(125). Consequently, a study reported that the miRNAs-mRNA interaction significantly regulates the expression of NCF4, which is linked with mastitis susceptibility and host immunity (76). Similarly, the SNP g.10766 T>C in NCF1 caused the aberrant splice variant NCF1-TV1 production, which showed an association with high milk SCS in dairy cattle (126).

Bovine Alpha-2-macroglobulin (A2M) gene
Bovine Alpha-2-macroglobulin gene has 38 exons and is located on chromosome 5. By binding with proteases, alpha-2macroglobulin (A2M) in the plasma and tissues of vertebrates and invertebrates acts as a defense barrier against pathogens (127). Recently, a study briefly explored the role of the A2M gene in immunity, inflammation, and infectious diseases (128). As mastitis is characterized by immune depression followed by inflammatory changes, making it a key player in immunity and inflammation, A2M has been widely studied in mastitis susceptibility in dairy cattle research (127,129). The SNP c.3535A> T at exon 29 of the A2M gene caused the aberrant splice variant A2M-AS4 production, which exposes dairy cattle mastitis (127). The Bta-miR-2898 was found to be up-regulated in cows with mastitis when compared with healthy cows. It was experimentally proven that polymorphism at point c.4659_4661delC of the A2M gene significantly influences the target bta-miR-2898 binding affinity. This shows that it might be possible that A2M has a significant association with mastitis susceptibility (129).

Cluster of differentiation 4 (CD4) gene
During mastitis, the inflammatory cells are recruited to the site of infection, where T cells, particularly CD4 cells, were predominantly observed (130). Polymorphisms in CD4 and STAT5B genes and their link with mastitis resistance phenotypic traits have been well studied (49). The polymorphism in CD4 at loci g.13598C>T has been identified for its significant association with SCS, which is a crucial indicator of mastitis. Additionally, the study reported that although polymorphisms in STAT5B did not show any link with mastitis, when a combination analysis was conducted with CD4 gene polymorphisms, it was noticed that the combination of both genes showed a considerable effect on SCS (49). In addition, a study has reported a significant association of polymorphisms at point T104010752C and C104028410T in CD4 and LAG3 genes, respectively, with milk SCC (131). Based on the published studies, it can be concluded that CD4 might be a valuable addition to the genetic markers for mastitis resistance in dairy cattle.

Toll-like receptors
Toll-like receptors, the family of recognition patterns, are associated with the regulation of innate immunity (132, 133). Recently, various reports have been published on the role of TLRs in mastitis (134-141). The toll-like receptors on mammary epithelial cells show early expression during mammary infection because of interaction with microbial pathogens (142). Upon activation, TLR regulates the expression of several chemokines and pro-inflammatory cytokines, which further facilitate the recruitment of neutrophils and activate innate and acquired immune responses (53). The SNPs within the pattern recognition receptors (PRR) may alter the host response to pathogens and lead to either mastitis resistance or susceptibility (71).
Bovine TLR4, which is located on chromosome 8, contains 4 exons, and is a type I transmembrane protein and member of the TLR family, has been identified as a key pattern recognition receptor (PRR) (143). The TLR4 regulates innate immune system cells when stimulated by pathogen-associated molecular patterns (PAMPs) of foreign microorganisms, including viruses, fungi, and bacteria (144). Furthermore, Wang et al. (143) reported that the LPS, which is considered an essential PAMPS, could be found in most gram-negative and some gram-positive bacteria that interact with TLR4 in mammary epithelial cells, which is why the higher expression of TLR4 has been noticed during mastitis in dairy cattle (143). A study reported that SNP-rs8193069 in the TLR4 gene was significantly associated with higher milk SCC (145, 146). Similarly, another study found that the SNP-1,397-C-T at exon 3 of TLR4 correlated with milk SCS in Chinese Holstein, Sanhe cattle, and Chinese Simmental (145, 147). Furthermore, Mesquita et al. (71) documented that SNP at point rs8193060 G>A in TLR4 was significantly associated with the lowest milk SCS level in Brazilian Holsteins. From the above studies, we concluded that TLR4 has a critical role in mastitis development, and it might, therefore, be considered a potential genetic marker for mastitis resistance.
The polymorphism (SNP -79 T > G) in bovine TLR1 predisposes dairy cattle to mammary infection due to the excessive activation of NF-kB signaling and poor immune response to pathogens (148). Furthermore, it has been revealed that mutation in TLR1 also influences the expression of CXCL8 (IL-8) (149), TLR2, and IL-6 (34), and high expression of these genes has been reported in the mammary epithelial cells of cows with mastitis. Besides, TLR1 can establish heterodimers with TLR2 (150) to widen the recognized ligands. Pant et al. revealed that mutations in TLR2 and caspase recruitment domain 15 (CARD15) are associated with milk SCS and increase the susceptibility of cattle to mastitis. TLR2 and CARD15 are key pattern recognition receptors that play an essential role in the stimulation of inflammatory and immune response (151, 152). A study documented that SNP T385G at exon 2 in the TLR2 gene is linked with high milk SCS and mastitis susceptibility (72).

Lipopolysaccharide-binding protein (LBP)
Lipopolysaccharide-binding protein is another critical gene that has a vital role to play in the innate immune recognition of gram-negative bacteria in dairy cattle. The gram-negative bacteria has been documented to have an essential role in mastitis (87). LBP is the critical protein that combines with LPS, a primary plotter from gramnegative bacteria with the ability to activate inflammation in animals. Furthermore, by binding with LPS, the LBP is presented to CD14 + cells, resulting in the regulation of the TLR4 pathway. The TLR4 pathway further activates the pro-inflammatory response caused by tumor necrosis factor-a (TNF-a), interleukin (IL) -1, or IL-6. In addition, with the help of the soluble form of CD14, the LPS-LBP complex also activates the CD14 cell inflammatory response, which shows that LBP could significantly contribute to blocking the inflammatory cascade before the release of inflammatory cytokines. Cheng et al. (87) reported that mutations in the LBP gene have a significant association with mastitis in dairy cattle, as shown in Table 1.

Cytokines encoded genes and their receptors associated with mastitis
The inflammatory cytokines and their associated genes have attracted considerable attention in mastitis research (38). Interleukin-8 receptor a (IL-8RA), coded by the chemokine receptor CXCR1, is located on the surface of neutrophil and connects the pro-inflammatory IL-8 with high affinity and is, therefore, targeted widely as a potential marker in mastitis control research (153). Due to the immunosuppressive nature of S. aureusmastitis, IL8RA plays an important role in the control of bovine mastitis by enhancing immunity (154). IL8, also called CXC chemokine ligand 8 (CXCL8), is located on chromosome 6 and has 4 exons. IL8 is considered a potent mediator of inflammation and is also involved in the recruitment of leukocytes to sites of infection (155). The polymorphisms in the IL8 gene have been the focus of some mastitis research (156,157). A polymorphism at +472 A>G in IL8 was reported to be associated with high milk SCC in S. aureus mastitis-infected dairy cattle (153). Moreover, the SNP -105G>A in IL8 has been found to be associated with high milk SCS, immunity enhancement, and mastitis resistance (85). It has been documented that the mutation at point +735 G>C could change the amino acid glutamine to histidine in the amino chain of CXCR2, which is linked to calcium signaling and G-protein interaction and has a key role in mastitis (158).
The CXCR1 gene, which coded interleukin 8 receptor a (IL8RA) and has the potential to bind IL8 with high affinity, is located on the surface of neutrophil surface (159,160). The role of CXCR1 has been well-studied in mastitis resistance in dairy cattle research (158). Bacteria activate the CXCR1 after interaction with TLR4, which further regulates the NF-kB signaling. The NF-kB translocates to the nucleus, binds with DNA, and causes the expression of the CXCR1 gene. Furthermore, the interaction of CXCR1 or CXCR2 with IL-8 brings changes in neutrophils, which allow their chemotaxis toward the infection in the mammary gland (161), mediates the cell survival and migration, and increases the activity of phagocytosis (162,163). Several polymorphisms in CXCR genes have been identified so far for their possible role in mastitis resistance. Polymorphisms (SNPs -1830AA, -1768TT, and -344TT) (164) and mutation at point (SNP -1768T>A) (165) in the CXCR1 gene were linked with high milk SCC in dairy cattle. Similarly, another study also reported the significant association of SNP c.337A>G and c.365C>T in CXCR1 with the milk SCC, suggesting their role in the host response against mastitis (166). A study found that the CXCR1+472 variant was significantly linked to milk SCS and increases the susceptibility to S. aureus-mastitis in dairy cattle (153). Similarly, other studies have also reported a strong link between CXCR1 mutations c.980AG, c.735C>G, CXCR1+472, CXCR1+777, and CXCR1−1768 at position 5′ upstream region with milk SCC in dairy cattle (153,165,(167)(168)(169). Based on published research, it can be concluded that CXCR1 and its reported polymorphisms might be considered potential markers for mastitis resistance/susceptibility in dairy cattle.
The bovine IL-17 located on chromosome 23, containing 3 exons, is another critical gene studied for its important role in immunity and inflammation pathology (170). Comprehensive reviews have been published on the role of IL-17 in the mediation of immunity and inflammation (171,172). Furthermore, it has been demonstrated that the members of the IL-17 family have a key function in acute and chronic inflammation and have been associated with enhancing the host's defense against microbial organisms (173, 174). Hu et al. reported that the expression of IL-17 was significantly up-regulated upon challenging the mammary glands of mice with lipopolysaccharide (LPS). The elevated level of IL-17 is also associated with the regulation of nuclear factor-kB (NF-kB) signaling, which is crucial in mastitis susceptibility. The blockage of IL-17 with the anti-IL-17A antibody has been shown to protect dairy cows from LPS-induced mastitis by suppressing the pro-inflammatory cytokine levels, myeloperoxidase activity, and neutrophil infiltration and NF-kB signaling pathway (175). In 2017, Usman and his co-workers found polymorphism (24392436C/T) in IL-17F and mutation (24345410 A > G) in IL-17A, which shows an association between the regulation of immunity and inflammation signaling and high milk SCS in both Chinese Holstein and Sanhe cattle (38). Furthermore, the polymorphism (1578A>G) in IL17A regulated the milk SCC and, thus, their expression might be a potential marker for mastitis susceptibility (91). The IL-17A production was documented during S. uberis mastitis (176), and slightly increased expression was also noticed in S. aureus-infected cows' somatic cells (177). Furthermore, an invitro study illustrated that the IL-17A reinforces the ability of mammary epithelial cells (MEC) to resist the consequences of S. aureus (178). Additionally, it has been found that IL-17F and IL-17A are significantly regulated in mammary tissue in response to E.coli (179). Moreover, Roussel and his co-workers experimentally proved that IL-17 F and IL-17A could play an essential role in regulating host-pathogen relations during mastitis development (179). In addition, a study reported that IL-17 positively regulates CD4+ T cells to facilitate the immune system against pathogenic infection (180).

The association of genetic polymorphisms in JAK-STAT pathway genes with mastitis
The JAK-STAT has been widely studied for its critical role in immunity and inflammation (181,182), and evidence indicates that  (187,196); current analysis bta04620:Toll-like receptor signaling pathway CXCL8, LBP, CD14, TLR4, TLR2 (187,195); current analysis bta04613:Neutrophil extracellular trap formation C5, NCF1, NCF4, HMGB1, TLR4, TLR2 current analysis; (197,198 Staphylococcus aureus can cause multiple forms of infections ranging from superficial skin infections to food poisoning and life-threatening infections. The organism has several ways to divert the effectiveness of the immune system: secreting immune-modulating proteins that inhibit complement activation and neutrophil chemotaxis or lysis, modulating sensitivity to cationic antimicrobial peptides (such as defensin) by increasing the positive net charge of its cytoplasmic membrane, and the expression of superantigens that prevent the development of a normal immune response or cause an emetic response when ingested. persistent activation of this pathway might lead to many immunity- (183) and inflammation-related diseases (184,185). Due to its significant role in immunity, cell proliferation, cell differentiation, and inflammation, the JAK-STAT pathway has been widely targeted for therapeutic purposes in several diseases (186). Furthermore, playing a critical role in mammary gland development, any abnormal regulation of the JAK-STAT pathway may disturb normal function, resulting in impairment of mammary gland development and exposure to mammary infections. As mastitis is an immunity and inflammatory-related disease, the JAK-STAT pathway should be explored in mastitis control research (187). JAK2 and STAT5A & B are the key parts of JAK-STAT signaling, which have been recently studied for their association with mastitis resistance (32, 34, 37, 48, 95). Furthermore, it has been noticed that the polymorphism at point 39630048C/T in JAK2 was associated with interleukin-17 (IL-17) (34), IL-6 and IFN-g (32). In addition, the SNPs (39652267A/G, 39631175T/C) in the JAK2 gene were documented for their significant links with milk SCC, IL-6, and IFN-g (32, 34). The mutation (39631044G/A) in the Jak2 gene was noticed to be significantly associated with milk SCS in Chinese Holstein (34).
Moreover, the variation at point 39645396C/T in the JAK2 gene was linked to milk SCC, IL-6, and IFN-g (37), while SNP-39631044G/A in JAK2 was associated with milk SCS (34). Consequently, it is predicted that the SNP (39645396C/T), which regulates the production of inflammatory cytokines, is responsible for changing the amino acid from lysine to asparagines and can, therefore, be targeted as a functional candidate marker for mastitis resistance (37). Other studies also reported a higher expression of IL-6 in Plasma cell mastitis (PCM), which indicates that the IL-6/ STAT3 pathway could play a vital role in the pathogenesis of PCM (188, 189). A variety of cytokines and growth factors activate STATs, which are a family of latent transcription factors. Members of the STATs family are involved in growth, differentiation, survival, and apoptosis. Among the seven members of the STATs family (STAT1-4, 5a, 5b, and 6) in mammalian cells, STAT5A and STAT5B are the most closely linked and are the result of duplication (190). STAT5, a primary gene of the JAK/STAT inflammation signaling pathway, has an essential role to play in prolactin-induced mammary gland development and is associated with mammary gland development in transgenic mice (191). A few FIGURE 2 Inflammatory immune response requires the recruitment of leukocytes to the site of inflammation upon foreign insult. Chemokines are small chemoattractant peptides that provide directional cues for cell trafficking and are therefore vital for protective host response. In addition, chemokines regulate a plethora of biological processes of hematopoietic cells, leading to cellular activation, differentiation, and survival. The chemokine signal is transduced by chemokine receptors (G-protein coupled receptors) expressed on the immune cells. After receptor activation, the alpha-and betagamma-subunits of G protein dissociate to activate diverse downstream pathways, resulting in cellular polarization and actin reorganization. Various members of small GTPases are involved in this process. The induction of nitric oxide and the production of reactive oxygen species are also regulated by the chemokine signal via calcium mobilization and diacylglycerol production. studies noticed a significant association of polymorphism in the STAT5A and STAT5B genes with mastitis resistance phenotypic traits (34, 37). They documented that polymorphism in STAT5A (43046497A/C) was associated with IL-6 and also changed the amino acid isoleucine to valine (34). Similarly, mutation at point 43673888A>G in the STAT5B gene was significantly linked to mastitis resistance phenotypic traits (IL-4 and SCC) (37). Bochniarz et al. reported the elevated level of IL-6 and the reduced level of IL-4 in the milk and serum of cows infected with S. aureus (192). Khatib et al. noticed that variant 12195T/C in STAT5A was significantly linked to a decrease in milk fat and protein percentages as well as levels of SCS in dairy cattle (193). Based on the above-published data, it can be concluded that JAK-STAT signaling plays a key role in immunity and inflammation; thus, the polymorphisms in the genes of JAK-STAT signaling might be valuable additions to the genetic markers for increasing genetic mastitis resistance in dairy cattle.
4 Bioinformatic analysis to find the biological signaling pathways for the above-mentioned genes Bioinformatics analysis was performed to discover the significantly regulated biological signaling pathways of the above-mentioned genes. For this purpose, we collected all the genes (mentioned in Supplementary Table 1) with their Ensembl IDs (already discussed in the current review article) and uploaded them to the online DAVID software (https://david.ncifcrf.gov/tools.jsp) (194). Through bioinformatics analysis, we reported several immunity and inflammatory signaling pathways (Supplementary Table 2), of which, staphylococcus aureus infection, chemokine signaling pathway, toll-like receptor signaling pathway, complement and coagulation cascades, cytokine-cytokine receptor interaction, and NF-kappa B signaling pathway were found for their involvement in mastitis (Table 2). In addition, the regulation mechanisms of staphylococcus aureus infection, chemokine signaling pathway, and toll-like receptors signaling pathways are shown in Figures 1-3 respectively.

Conclusion
Altogether, we concluded that an appropriate approach to identifying genetically resistant animals would be to study the association of host genetics with mastitis susceptibility and resistance, thus, genetic marker selection was determined as the best approach to screening genetically mastitis-resistant cattle. In the current review, we have highlighted potential genetic polymorphisms in inflammation-and immune-associated gene markers that are significantly associated with mastitis resistance/