Introduction

Mastitis is an infectious disease of the mammary glands characterized by inflammation of the udder tissue in dairy cattle, leading to reduced milk yield and quality [1]. It causes significant economic losses in the global dairy industry [2]. Controlling mastitis remains a global challenge that complicates animal health management [3]. Bovine mastitis is an inflammatory disease classified into clinical and subclinical types based on its characteristics [4]. The etiological agents responsible for the disease may include bacteria, viruses, or fungi, among others. [5]. Milk production often drops as a direct consequence of either the pathogen itself or damage inflicted by the immune response. [6]. The principal bacterial pathogens include Staphylococcus (Gram-positive) and Escherichia coli (Gram-negative) [7]. Mastitis induced by E. coli and other Gram-negative bacteria typically manifests as a clinical condition accompanied by acute and severe inflammation of the mammary gland. The pathogens are subsequently cleared by the host immune system within a few days or following antibiotic therapy [8]. In contrast, mastitis caused by Gram-positive bacteria such as Staphylococcus aureus is often milder; however, antibiotic treatment is frequently ineffective [9], leading to chronic clinical mastitis [10]. Coagulase-negative staphylococci: CoNS (e.g., S. simulans, S. saprophyticus, S. chromogenes, S. epidermidis) are opportunistic pathogens that colonize the teat canal lining and teat skin. Staphylococcal mastitis is one of the most prevalent causes of economic losses due to its impact on reducing milk production [11].

Understanding the mechanisms of the host response to these infectious pathogens and developing appropriate control strategies can play vital roles in mastitis control. The host response to infection has demonstrated significant differences between Gram-negative (E. coli) and Gram-positive (staphylococci) bacteria [12]. Furthermore, these pathogens have been shown to elicit markedly distinct mechanisms within the host innate immune response [13]. Following infection with E. coli or S. aureus, bovine mammary epithelial cells and tissues mount an immune response characterized by the secretion of various cytokines, chemokines, and other specific proteins, which are also detectable in milk [14,15].

The conventional gold-standard methods for diagnosing mastitis including microbial culture, somatic cell count (SCC), and California Mastitis Test (CMT) remain in widespread use. Although these techniques are inexpensive, rapid, and simple to perform, they are consistently associated with limitations in terms of diagnostic accuracy and reliability [16,17]. Currently, mastitis diagnostic methodologies are undergoing modern advancements. Recently, regulatory biomarkers, such as MicroRNAs (miRNAs), have attracted considerable scientific interest [18].

MicroRNAs are endogenous, short non-coding regulatory RNAs (approximately 18–25 nucleotides in length) that regulate gene expression at the post-transcriptional level by binding to fully or partially complementary sites within the 3'UTR of target mRNAs [19]. MicroRNAs modulate the expression of host immune genes which in turn, can substantialy influence host immunity response during mastitis, and a comprehensive understanding of their activity may help in elucidating the etiology of this disease [20].

Although miRNAs are crucial regulators of the host's response to infectious agents [21], few studies have investigated how bovine miRNA expression levels alter in response to such infections [22]. Currently, efforts have been made to utilize diagnostic biomarkers such as miRNAs, which are present in the microvesicles of milk from dairy cattle. The identification of these biomarkers could provide a more effective alternative method for diagnosis of mastitis [23]. Furthermore, milk contains the highest abundance of miRNAs among various bodily fluids [24]. Studies on miRNA expression changes during bacterial mastitis have demonstrated that stimulating bovine CD14⁺ monocytes with lipopolysaccharide (LPS) or Staphylococcus aureus enterotoxin B (SEB) leads to differential expression of five key inflammation-related miRNAs: miR-9, miR-125b, miR-155, miR-146a, and miR-223 [25]. Moreover, another study reported altered expression of five miRNAs (bta-miR-184, miR-24-3p, miR-148, miR-486, and let-7a-5p) in response to experimental intramammary infection with E. coli [26]. Similarly, in an investigation of mastitis induced by Streptococcus uberis, four out of fourteen studied miRNAs (miR-181a, miR-16, miR-31, and miR-223), which are associated with innate immune regulation and mammary gland function, were found to be differentially regulated [27]. Despite this advantage, the application of milk miRNA expression profiling for the diagnosis of bovine mastitis remains relatively unexplored, with only a limited number of studies addressing this potential [28,29]. The KEGG pathways significantly enriched for differentially expressed miRNA targets are established mediators of the bovine mammary gland's response to bacterial pathogens [22,27]. As an example, a key regulator of innate immunity, miR-146a attenuates the response to bacterial infection by suppressing the expression of TRAF6 and IRAK1 [30]. This molecule acts as a negative feedback regulator by modulating the TLR4/TRAF6/NF-κB signaling pathway during inflammation [31].Current techniques for miRNA detection include conventional methods such as northern blotting [32], microarray [33], and qPCR [34], as well as newer approaches such as digital PCR and small RNA sequencing [35,36]. Nevertheless, the application of miRNAs as biomarkers for the diagnosis and confirmation of bovine mastitis directly from milk has not been extensively studied [37]. The potential of miRNAs in diagnosing or predicting livestock diseases remains incompletely understood [20,38]. Furthermore, investigations into the alterations in miRNA expression profiles in milk from healthy cows and those with naturally acquired subclinical and clinical mastitis remain scarce. Therefore, advancing bovine mastitis research by integrating novel biomarkers with conventional methods may present a promising strategy for enhancing diagnostic and therapeutic outcomes. Accordingly, we selected certain miRNAs that have been most frequently reported in the literature to be associated with inflammatory responses in bovine mastitis. The aim of this study was to employ qPCR to analyze the expression dynamics of these miRNAs in milk samples from naturally infected and healthy cows, comparing three distinct groups: clinical mastitis caused by E. coli, subclinical mastitis associated with coagulase negative staphylococci, and apparently healthy controls.

Materials and methods

Bioinformatic primer design

The primer design was done based on data deposited in MiRBase release 21, a searchable database of published miRNA sequences, and annotation of bovine miRNAs (http://www.mirbase.org; accessed on 27 October 2025). The primers were designed from the stem-loop sequence of the corresponding miRNA by placing the forward and reverse primer flanking to the mature miRNA. The primers for inflammatory miRNA genes (bta-miR-146a, miR-29B-2, miR-148a, miR-223, miR-155, miR-383, miR-200a, miR-205, miR-92a) [25] were designed using the following web server (http://www.srnaprimerdb.com/; accessed on 27 October 2025) and the bovine S18 rRNA was used as a miRNA control [45,46]. Real-time PCR primers were synthesized by TAG Copenhagen, Denmark. The sequences of oligonucleotide primers used in this study are outlined in Table 1.

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Table 1. Sequences of the primers designed in this study.

https://doi.org/10.1371/journal.pone.0352609.t001

Reverse Transcription Quantitative PCR (RT-qPCR) The expression levels of miR-146a, miR-92a, miR-155, miR-29B-2, miR-223, miR-148a, miR-205, miR-200a, and miR-383 were quantified using a StepOnePlus Real-Time PCR System (ThermoFisher Scientific, USA) and a SYBR Green PCR kit (Yekta Tajhiz, Iran). In this study, a total of nine miRNAs, including miR-146a, miR-92a, miR-155, miR-383, miR-29b-2, miR-223, miR-148a, miR-200a, and miR-205, were selected based on their importance in previous studies and their important roles in response to bacterial infections, inflammation, bovine mastitis, modulation of immune responses, and regulation of innate immune responses to infection through the production and release of pro-inflammatory cytokines [30,47–50].

Real-Time PCR reactions were performed in a 20 μL reaction volume containing the following components: 10 μL of 2 × SYBR Green qPCR Mix, 0.4 μL of 50 × Passive Reference Dye, 1 μL of specific forward primer, 1 μL of universal reverse primer, and 2.5 μL of cDNA template. The final volume was adjusted to 20 μL with RNase-free ddH₂O. The thermal cycling protocol consisted of an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 62 °C for 30 s, and extension at 72 °C for 30 s. Reaction specificity was confirmed by dissociation curve analysis (melting curve). All reactions were performed in triplicate. Relative gene expression was calculated based on the CT values of the inflammatory miRNA genes and S18 rRNA. The expression levels, represented as fold changes, were reported using the 2^―ΔΔCT method [51].

Results

Quantitative expression profiling of miRNA biomarker candidates

As described in the statistical analysis section, group comparisons were performed using the unpaired t-test or Mann-Whitney U test based on normality assessment. Our findings indicated a significant increase in the expression levels of miR-92a, miR-155, miR-223, and miR-200a in the clinical mastitis group in response to E. coli infection compared to the healthy controls (p < 0.05) with the expression fold change values ranged from 1.23, 2.6, 8.93, and 47.84 (S1 Table 4 in S1 File).

Significant upregulation of miR-223 and miR-200a was observed in both clinical and subclinical mastitis groups compared to healthy controls (p < 0.001). The fold change values in the subclinical mastitis group were 3.89 for miR-223 and 5.81 for miR-200a. miR-200a exhibited the most substantial upregulation among all miRNAs in both disease states.

In clinical mastitis with E. coli infection, a non-significant rise in expression was noted for miR-383 and miR-205 relative to the healthy group, with respective fold changes of 1.8 and 2.5 (p > 0.05). Conversely, in the subclinical mastitis group with CoNS, the observed increase in miR-383 expression (fold change = 1.08) did not reach statistical significance (p > 0.05). Although the expression levels of miR-146a, miR-29B-2, and miR-148 showed a decrease in the clinical mastitis group relative to the healthy controls, with fold changes of 0.46, 0.31, and 0.40, respectively, the differences were not statistically significant (p > 0.05) (S1 Table 5 in S1 File).

In the subclinical mastitis group infected with CoNS, a significant decrease in expression was detected for miR-92a, miR-148a, and miR-205, with respective fold changes of 0.06, 0.23, and 0.43 (p < 0.05). Meanwhile, the expression of miR-146a, miR-29B-2, and miR-155 also exhibited a declining trend, with fold changes of 0.32, 0.90, and 0.19, although these changes did not reach statistical significance (p > 0.05), (Fig 1, Fig 2, Fig 3, Fig 4).

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Fig 1. Relative expression profiles of miR-146a, miR-92a, and miR-155 in milk samples from healthy (H), clinical mastitis (M-E. coli), and subclinical mastitis (S-CoNS) groups.

Data are presented as mean ± SE and were compared using the unpaired Student's t-test and Mann-Whitney U test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

https://doi.org/10.1371/journal.pone.0352609.g001

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Fig 2. Relative expression profiles of miR-383, miR-29B-2, and miR-148a in milk samples from healthy (H), clinical mastitis (M-E. coli), and subclinical mastitis (S-CoNS) groups.

Data are presented as mean ± SE and were compared using the unpaired Student's t-test and Mann-Whitney U test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

https://doi.org/10.1371/journal.pone.0352609.g002

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Fig 3. Relative expression profiles of miR-223, miR-200a, and miR-205 in milk samples from healthy (H), clinical mastitis (M-E. coli), and subclinical mastitis (S-CoNS) groups.

Data are presented as mean ± SE and were compared using the unpaired Student's t-test and Mann-Whitney U test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

https://doi.org/10.1371/journal.pone.0352609.g003

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Fig 4. Heatmap of miRNA expression profiles generated by CIMMiner software (Euclidean distance metric).

Red color intensity corresponds to the level of miRNA upregulation. Groups: M-E (Clinical mastitis, E. coli), M-C (Subclinical mastitis, CoNS), H (Healthy).

https://doi.org/10.1371/journal.pone.0352609.g004

KEGG pathway enrichment analysis of predicted targets for DE miRNAs

The most significant pathways in which these miRNAs are involved include: Metabolic pathways, mTOR signaling pathway, ErbB signaling pathway, Bacterial invasion of epithelial cells, Ras signaling pathway, MAPK signaling pathway, Adherens junction, pathways in cancer, JAK-STAT signaling pathway, Ras signaling pathway, AMPK signaling pathway, Endocytosis, Chemokine signaling pathway, HIF-1 signaling pathway, Calcium signaling pathway, Regulation of actin cytoskeleton, p53 signaling pathway, FoxO signaling pathway, Wnt signaling pathway, AMPK signaling pathway, Cell adhesion moleculesm Platelet activation, and Nucleotide metabolism. Based on the analyses, it can be inferred that miRNAs such as miR-146a, miR-155, miR-92a, miR-29B-2, miR-223, miR-383, miR-148a, miR-200a, and miR-205 play significant roles in numerous crucial biological pathways. The most important of these pathways are involved in inflammatory processes and immune responses, wherein these miRNAs participate by regulating gene expression [54]) S1 Table 6 in S1 File, Fig 5 and Fig 6).

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Fig 5. Network visualization of the regulatory interactions between differentially expressed miRNAs and their target genes in E. coli-induced clinical mastitis and CoNS-induced subclinical mastitis.

In this representation, differentially expressed miRNAs (depicted as red, green, and pink squares and orange triangles) are connected to their respective target genes (white squares). Pink ovals highlight genes that are common targets of two or more miRNAs.

https://doi.org/10.1371/journal.pone.0352609.g005

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Fig 6. A network of differentially expressed miRNAs, along with a subset of target genes involved in pivotal pathways such as immunity, metabolic, cancer, inflammation, and bacterial invasion.

In this representation, differentially expressed miRNAs (depicted as red, green, and pink squares and orange triangles) are connected to their respective target genes (yellow ovals). Blue ovals highlight genes that are common targets of two or more miRNAs.

https://doi.org/10.1371/journal.pone.0352609.g006

Discussion

Evidence from the literature reveals a pattern of miRNA dysregulation in bovine mastitis. An analysis of blood by Chen et al. revealed a significant decrease in the expression of miR-148a, miR-146a, and miR-155, and an increase in the expression of miR-223 and miR-29b in mastitis cows [55]. Corroborating these findings in milk, studies by Srikok et al. and Ngo et al. confirmed the downregulation of miR-146a, miR-155, miR-148a, and miR-29b in clinical and subclinical mastitis, with miR-29b exhibiting the most pronounced decrease [37,56]. In a different model, Chuammitri et al. reported that miR-146a expression was suppressed in E. coli LPS-stimulated bovine neutrophils treated with quercetin [57]. In the study by Dilda et al., the expression of inflammation-related miRNAs, such as miR-155, miR-146a, and miR-223, was upregulated in bovine monocytes stimulated with E. coli lipopolysaccharide [25]. Similarly, Wang et al. found that the expression of miR-146a and miR-146b was increased in the mammary gland tissue of cows with clinical and subclinical mastitis [29]. Furthermore, Lai et al. reported that the expression of miR-146a, miR-383, and miR-155 was upregulated in the milk of cattle with clinical mastitis [38]. Our findings on miRNA expression are consistent with previous studies by Chen, Chuammitri, Srikok, and Ngo [37,55,57]. In our study, the expression levels of miR-146a, miR-148a, and miR-29B-2 were reduced in the milk of cows with clinical mastitis naturally infected with E. coli and in subclinical mastitis caused by CoNS. Among these, the downregulation of miR-148a in subclinical mastitis was significant. However, the decreases in miR-146a and miR-29B-2 expression were not significant in either clinical or subclinical mastitis. The expression pattern of miR-146a in the present study contradicted those previously reported by Lai, Wang, and Dilda. Furthermore, Srikok et al. found the downregulation of miR-29B-2 to be significant and proposed it as a potential biomarker candidate [37]. Lawless et al. also reported that the expression of miR-29B-2 was downregulated in mammary epithelial cells infected with Streptococcus uberis [22]. The miR-29 family plays a role in the epigenetic regulation of lactation-related genes in bovine mammary epithelial cells [48]. miR-29B acts by inhibiting the NF-κB pathway and TNFAIP3 (a negative regulator of the NF-κB pathway) [49]. In our bioinformatic analysis, CXCR1 was identified as a predicted target of miR-29B-2. This gene plays a pivotal role in the chemokine signaling pathway, suggesting a potential mechanism for immune regulation in mastitis.

In contrast to the studies by Chen and Srikok, and consistent with the findings of Dilda and, the expression level of miR-155 in our study was upregulated in clinical mastitis caused by E. coli, but downregulated in subclinical mastitis. This suggests that miR-155 expression in Gram-negative bacterial infections may differ from that observed in Gram-positive infections. As previously established, bovine mastitis caused by Gram-positive and -negative bacterial pathogens triggers distinct host response patterns, which consequently lead to varying degrees of mastitis severity [26]. Different miRNA response patterns to Gram-positive and -negative bacteria have also been demonstrated in previous studies [25,50]. Although there has been limited overlap among the miRNAs identified across studies, changes in the expression of a large number of miRNAs have been reported [58]. miR-155 acts as a pro-inflammatory agent, and its early upregulation during innate immunity may amplify inflammatory signaling, a response that correlates with acute inflammation following E. coli infection [59]. Furthermore, bioinformatics analysis demonstrated that miR-155 targets key genes, including CBL and RAC1, which are involved in important biological pathways such as pathways such as the Ras signaling pathway, the MAPK signaling pathway, and bacterial invasion of epithelial cells.

One possible reason for the difference in miR-155 expression levels between our study and that of Srikok may be the use of skim milk instead of whole milk in the present study, which could explain the observed discrepancies in our data. This also highlights the importance of sample type when examining miRNA expression in milk or other body fluids for disease diagnosis. Moreover, previous studies have shown that miRNA expression profiles vary between lactating and non-lactating mammary glands [50], between mature milk and colostrum, and even across different organs (lung, brain, liver, and spleen) and whole blood [60]. Several other factors may account for the differences in miRNA expression observed in our study compared to previous research: the use of different sample types, such as milk, blood, monocytes, or mammary epithelial cells. The application of various laboratory techniques for profiling expression, such as NGS and real-time PCR, which can influence the specificity and sensitivity of the results as well [29,55]. In some studies, bacterial components were used to induce infection, and miRNA expression was examined at different time points [25,29]. In contrast, the present study and some similar investigations did not impose time constraints on the infection process [37]. Additionally, many studies did not specify the bacterial species responsible for mastitis, which may explain the differential expression of certain miRNAs depending on the specific pathogen involved.

Consistent with the studies conductedby Chen, Dilda, and Srikok, miR-223 expression was elevated in both clinical and subclinical mastitis in our study. Notably, the upregulation of miR-223 was significantly more pronounced in clinical mastitis caused by E. coli compared to subclinical mastitis associated with CoNS. Similarly, Pu et al. reported increased miR-223 expression in the mammary gland tissue of cows infected with Streptococcus agalactiae [61]. In contrast, Bagnicka reported a decrease in miR-223 expression in the mammary gland tissue of cattle infected with CoNS [62]. Moreover, Tzelos et al. found no statistically significant difference in miR-223 expression between mastitis and healthy cattle when comparing whole milk and skim milk samples [58]. Nevertheless, previous studies have suggested that miR-223 may serve as a novel biomarker for the diagnosis of bacterial mastitis [55, 63].

Consequently, our findings support the potential of miR-223 as a candidate biomarker for diagnosing clinical mastitis, given its expression in all cases of both clinical and subclinical mastitis, with significantly elevated levels in clinical mastitis. miR-223 is known to play a key role in the immune response during bovine mastitis [64], as well as in pulmonary and bacterial peritonitis infections [65]. According to Fang et al., bta-miR-223 acts as a key post-transcriptional regulator in the bovine mammary gland during S. aureus infection, where it controls the expression of pivotal innate immune genes like CXCL14 [66]. Similarly, Tucker et al. emphasized the importance of miR-223 in regulating immune responses during mastitis infection [67]. Moreover, in humans, miR-223 has been identified as a specific and sensitive biomarker for the diagnosis of sepsis [68]. miR-223 also plays a critical role in the innate immune response during myeloid differentiation and in the function and activation of granulocytes, making it a key miRNA in infection and inflammation [62], and our bioinformatic screening suggests that it may also influence the Ras and HIF-1 pathways through an unexpected association with PLCG1 expression.

Bta-miR-223 is highly conserved across 15 different species without any changes in base pairing, and this evolutionary conservation underscores its importance and benefits to the host. Therefore, a therapeutic agent designed to target this miRNA could potentially be applicable across multiple species [67]. Similar to the findings of Lai et al. and Jadhav et al., we also observed an upregulation of miR-383 in the milk of cows with both clinical and subclinical mastitis; however, this increase was not statistically significant. Correspondingly, Jadhav et al. reported elevated expression of miR-383 in the milk of buffaloes with clinical mastitis [69]. Furthermore, it has been shown that the expression level of miR-383 increases in the RAW264.7 macrophage cell line stimulated with LPS [70]. The upregulation of miR-383 modulates the immune response by regulating genes such as IL-1, TNF, COX-2, TLR4, and CXCL-1, and the resulting tissue reaction to mammary gland bacterial infection in mastitis leads to inflammation [70]. Consequently, investigating the altered expression of miR-383 in milk and mammary tissue during mastitis provides insight into the mechanisms by which miRNAs regulate immune pathways [61]. Immune-related signaling pathways, such as JAK-STAT, Ras, and AMPK, are regulated by miR-383.

Other miRNAs investigated in our study were miR-200a and miR-205 in both clinical and subclinical mastitis. Compared to the healthy control group, miR-200a was significantly upregulated in clinical mastitis caused by E. coli. In subclinical mastitis caused by CoNS, miR-200a also showed upregulation relative to healthy controls.

Furthermore, miR-205 exhibited a non-significant upregulation in clinical mastitis compared to the healthy group, whereas it showed a significant downregulation in subclinical mastitis. In a study, Luoreng et al. demonstrated that miR-200a and miR-205 play important roles in the late immune stage of E. coli mastitis, and their expression levels were significantly elevated [71]. Our results are consistent with the findings of Luoreng et al.. In contrast, Li et al. reported a downregulation of miR-205 and miR-200b in mammary epithelial tissue infected with staphylococcus aureus [72]. This finding aligns with our observation of miR-205 expression in subclinical mastitis infected with CoNS, suggesting that the downregulation of miR-205 may be a common response to Gram-positive bacteria, independent of the specific species. The miR-200 family, which has been increasingly studied in recent years, is known to be upregulated in a wide range of diseases, a pattern that corroborates our results. Furthermore, Luoreng et al. demonstrated that bovine miR-200a regulates the development of E. coli-induced mastitis by targeting the bovine ZEB1 gene [71]. One study demonstrated that miR-200a-3p expression is elevated in alcoholic hepatocytes and induces apoptosis by targeting the ZEB2 gene [73]. Our bioinformatic analysis indicated that miR-200 is also involved in two key functional clusters: in cancer pathways by targeting genes such as CDK6, ERBB2, and TGFBR2, and in endocytosis by targeting genes including PSD, GIT2, PDGFRA, WWP1, and TGFBR2.

However, the association between miR-200a and mastitis in dairy cattle remains poorly understood, as current research in this area is limited and calls for more comprehensive and in-depth investigations. Likewise, the role of miR-205 in regulating the immune system has only been examined in a handful of studies [71]. Additional research indicates that miR-205 expression is elevated in individuals with allergic rhinitis [74], and that it modulates erbB2/erbB3 expression in breast cancer cells, thereby facilitating apoptosis [75]. Notably, a miRNAomic analysis identified miR-205 as a candidate miRNA linked to mastitis resistance [64]. The miR-205 COMMD1-NF-κB signaling axis promotes the amplification of the inflammatory response [76]. Our bioinformatic findings indicated that by targeting key genes in major signaling pathways such as Wnt, MAPK, FoxO, and p53, miR-205 plays a significant role in modulating inflammatory responses during bovine mastitis.

Based on limited previous studies and our results, miR-200a could also serve as a biomarker for clinical mastitis caused by E. coli infection and be involved in the immune response in cattle, given that its expression changes were highly significant. However, elucidating its precise function in bovine mastitis will require further investigation. Another miRNA examined in our study was miR-92a. Its expression was significantly elevated in the milk of cows with clinical mastitis, whereas it was markedly reduced in the subclinical mastitis group. miR-92a plays a vital role in the host’s bacterial defense mechanisms in dairy cattle [77]. Additionally, miR-92a ranks among the most abundantly expressed miRNAs across different fractions of bovine milk, including milk fat, whey, and cellular components [78]. In a study, Casas et al. reported that miR-92a was downregulated in the serum of beef cattle infected with Mycoplasma bovis and suggested it as a potential biomarker [77]. Similarly, we observed downregulation of miR-92a in subclinical mastitis cases caused by coagulase-negative staphylococci (CoNS), despite differences in sample types and bacterial pathogens between the studies. Lia et al. further demonstrated that miR-92a is downregulated in Toll-like receptor (TLR)-expressing macrophages. Moreover, miR-92a modulates the production of pro-inflammatory cytokines such as IL-6 and TNF-α; its overexpression suppresses these cytokines, while its inhibition leads to increased IL-6 and TNF-α levels [79]. TLRs play a critical role in initiating immune responses and recognizing pathogens in the host [79]. Dysregulation of miR-92a is a recurrent feature in a spectrum of human malignancies, with documented aberrant expression in cancers of the lung, breast, stomach, prostate, colon, pancreas, liver, and kidney [80]. Lai et al. proposed miR-92a as a housekeeping gene for analysis of milk samples from mastitis cows, as its expression was stable in both healthy and mastitis animals [81]. However, research on miR-92a in cattle remains limited. Contrary to their findings, our analysis revealed that miR-92a expression differed significantly among the healthy, subclinical, and clinical mastitis groups. Our study encountered several limitations. A primary constraint was the single time-point sampling and the challenge of determining the precise onset of infection within the dairy herds. Most miRNAs are expressed differentially at various stages post-infection, reflecting their rapid temporal dynamics. Furthermore, many miRNAs induce relatively subtle changes in gene expression in response to infection, as they function as fine-tuners of gene regulation [82, 83]. Additionally, our sampling was conducted on cattle with natural infections, which may explain the discrepancies with studies employing in vivo experimental models. This inconsistency could be attributed to the potentially vastly different infectious agent doses in a natural setting. Consequently, future validation through a large-scale cohort study is essential to definitively assess the suitability of miRNAs as early diagnostic biomarkers for bovine mastitis.

Conclusion

Analysis of miRNA expression patterns in mastitis not only holds great promise for early detection even before clinical symptoms appear, but also offers valuable insights into disease status and underlying causes. Moreover, the accessibility of non-invasive samples such as milk makes miRNAs especially attractive as valuable diagnostic biomarkers. The significant changes observed in miRNA expression levels in response to infections highlight their potential as early indicators of mastitis. This suggests that miRNAs could open a new frontier in mastitis diagnosis and/or prognosis. That said, validating these miRNAs as reliable diagnostic tools will require further investigation with larger sample sizes and diverse populations.

Supporting information

Acknowledgments

The authors are grateful for the support of the Department of Pathobiology, Faculty of Veterinary Medicine, Bu-Ali Sina University.