Mastitis, an inflammation of the mammary gland, is characterized by changes in udder tissue, along with physical, chemical, bacterial, and sensory abnormalities in the milk (Kibebew, 2017). This is the most prevalent and substantial barrier to dairy productivity in Sri Lanka and an economically significant illness affecting dairy cows worldwide (Ranasinghe et al., 2021).

Reduced milk quality and quantity, milk discarded during treatment, veterinary and pharmaceutical costs, increased labor requirements, early culling, and cow mortality in extreme circumstances are contributing factors to the financial losses connected with mastitis (Kibebew, 2017); (Kerro Dego, 2021)). Subclinical mastitis (SCM), which is much more prevalent than clinical mastitis in dairy cattle, is the most insidious type. However, it does not display obvious clinical symptoms in lactating cows. As a result, SCM often remains unnoticed for prolonged periods, causing significant hidden financial losses and acting as a recurring source of infection in dairy herds (Silva et al., 2021). According to (Puerto et al., 2021), SCM alone accounts for 20%-60% of the economic losses associated with clinical mastitis, mostly due to decreased milk production and rejection. It has a complex multifactorial etiology that includes environmental and infectious pathogens. Long-lasting subclinical mastitis infections are often caused by contagious microorganisms such as Staphylococcus aureus and Streptococcus agalactiae, which are well adapted to survive within the mammary gland and spread during milking practices.

On the other hand, housing and floor materials are the primary sources of environmental pathogens, including coagulase-negative staphylococci, streptococci, and coliforms (M.A. et al., 2022); (Shoaib et al., 2023). Additionally, factors associated with the host and management, such as parity, lactation stage, milk yield, hygienic procedures, and prior mastitis history, have a critical impact on the prevalence of SCM.

Numerous studies have shown significant occurrences of SCM in Sri Lanka, although estimates of its occurrence vary markedly by area. Notably, S. aureus is consistently detected as the predominant pathogen, with cow-level prevalence ranging from roughly 12% to over 55% in several districts (Ranasinghe et al., 2021). Previous studies have reported the contribution of Escherichia coli, Klebsiella spp., and Streptococcus spp., suggesting the involvement of both environmental and contagious pathogens in the prevalence of SCM in Sri Lankan dairy systems (Sanotharan et al., 2016); (Rahularaj et al., 2019). These results collectively demonstrate that SCM is a widespread problem across the country’s many agroecological regions.

The extensive use of antimicrobials for mastitis treatment and prevention has heightened concerns about antimicrobial resistance (AMR) in dairy production systems. In Sri Lanka, increasing resistance levels among mastitis-related pathogens, especially S. aureus, to frequently utilized antimicrobials have been documented, potentially undermining treatment effectiveness and posing substantial risks to both animal and public health (Jayaweera et al., 2018); (Priyantha et al., 2021). Nevertheless, region-specific data on antimicrobial susceptibility patterns and the frequency of multidrug resistance among SCM-associated pathogens are scarce, fragmented, and insufficient to inform evidence-based therapeutic decision-making (Bari et al., 2022).

The Monaragala District in the Uva Province is distinguished by primarily small- to medium-sized dairy farms managed by widespread, semi-intensive, and intensive systems. Despite the ongoing growth of the dairy sector in this region, issues such as inadequate milking hygiene, insufficient veterinary assistance, and low farmer awareness persist, thus hindering proper mastitis management. Many studies have reported the prevalence of SCM and patterns of antimicrobial resistance in Sri Lanka; however, there are still few region-specific studies that use robust multivariable analytical techniques to integrate epidemiology, hierarchical risk factors, bacterial etiology, and antimicrobial resistance.

Using a population-averaged multivariable modeling methodology, the present study integrates SCM prevalence, risk-factor analysis, bacterial characterization, and antibiotic resistance profiling to provide the first comprehensive assessment in Monaragala District. Therefore, the objectives of the current study were to: (i) detect the prevalence of SCM; (ii) identify the risk factors of SCM; (iii) identify the bacterial pathogens causing SCM; and (iv) evaluate their antimicrobial resistance profile. The findings of this study will support evidence-based mastitis control strategies and promote prudent antimicrobial use in Sri Lanka’s dairy sector.

The cow-level prevalence of SCM in Monaragala District was 18.7%, which was substantially lower than the prevalence reported in Kurunegala (57.5%) and Ampara (45.5%), but higher than that reported in selected areas of Nuwara Eliya (11.8%) (Sanotharan et al., 2016); (Rahularaj et al., 2019); (Ranasinghe et al., 2021). This moderate prevalence may be attributed to differences in agroecological conditions, herd structure, housing systems, hygiene management practices, and production intensity in Monaragala District, where small- to medium-scale herds are predominantly managed under semi-intensive and extensive systems (Sanotharan et al., 2016).

Other South Asian smallholder dairy systems have reported comparable levels of SCM prevalence. For example, reports reveal that the cow-level SCM prevalence ranges from 20% to 75% in Bangladesh, 18% to 87% in India, and 17% to 81% in Pakistan(Senthilkumar et al., 2020); (Bari et al., 2022).

The quarter-wise prevalence was relatively uniform in the present study, indicating limited evidence of quarter-specific predisposition under the prevailing management conditions. This, in turn, supports the view that SCM transmission is influenced more by management- and hygiene-related factors. The observed farm-level prevalence results further suggest that SCM is widely distributed across herds and does not concentrate in a few, highly affected farms. This finding emphasizes the importance of implementing herd-level rather than cow-level control strategies.

Dairy herds in Bagmati Province, Nepal, have reported a comparable prevalence pattern (Tiwari et al., 2022) In a similar context, (Hussein et al., 2022) observed a relatively higher quarter-level SCM prevalence (30.2%) among dairy farms in Qena Governorate, Egypt. In contrast, other studies have reported a substantially higher SCM prevalence in forequarters than in hindquarters. It suggests that potential anatomical or exposure-related differences exist across specific production systems (El-Kholy et al., 2018); (Mourya et al., 2020), possibly contributing to the observed differences in SCM prevalence between forequarters and hindquarters in dairy farms.

Animal-level factors significantly influenced SCM occurrence in the study area. Pure exotic breeds were more susceptible than crossbred cows, probably due to their higher production potential, increased metabolic stress, and reduced adaptability to local environments. These factors have been consistently associated with immune suppression and an increased likelihood of SCM occurrence (Taponen et al., 2017); (Curone et al., 2018). As parity increases, enlargement of the teat size, relaxation of the teat sphincter, and pathogen exposure may further facilitate bacterial entry into a cow’s udder (Yimam et al., 2020).

The lactation stage also influenced the risk of SCM, with lower odds in early lactation compared with late lactation. This pattern likely reflects prolonged exposure to mastitis pathogens and a gradual decrease in teat sphincter competence toward the end of lactation (Qayyum et al., 2016)(Bari et al., 2022).

Similarly, lower-yielding cows exhibited reduced odds of SCM than high-yielding animals. High milk production is associated with prolonged opening of the teat canal before and after milking and increased physiological stress. Both these factors enhance susceptibility to intramammary infections (Bari et al., 2022). These findings are consistent with those of (Biswas & Sarker, 2017), who reported a substantially lower prevalence of SCM in low-yielding cows (33.3%) than in high-yielding cows (87.5%).

In addition, a history of mastitis emerged as a strong predictor of current SCM, highlighting the persistence of intramammary infections and the importance of effective post-treatment monitoring. Cows previously affected by mastitis are at a higher risk of reinfection. It suggests that repeated microbial challenges to mammary tissues, coupled with other stressors, may increase susceptibility to infections(Bronzo et al., 2020). This association may also result from incomplete elimination of infective agents during treatment. Clinical signs are suppressed, but infection persists in a subclinical form.

At the farm level, production system intensity was a significant determinant of SCM risk. Cows managed under intensive and semi-intensive systems exhibited higher odds of SCM, due to intensification without corresponding improvements in hygiene and management. Pathogen exposure is increased through higher stocking densities and greater environmental contamination. Moreover, in intensive systems, cows tend to remain in confined areas for prolonged periods, which may be sanitized inadequately. Overcrowding may also exacerbate physiological stress. Similar associations between intensive systems and increased SCM prevalence have been widely reported in South Asia and other regions (Tedla et al., 2018); (Bihon et al., 2019); (Pugliese et al., 2021). Implementing appropriate housing interventions, such as the use of clean, comfortable bedding materials, has enhanced cow comfort and reduced SCM prevalence (Singh et al., 2020).

Hygiene-related practices have been demonstrated to be strongly protective against SCM. For instance, frequent floor cleaning and good overall housing hygiene are associated with reduced SCM occurrence. Additionally, proper disposal of urine and manure, together with routine cleaning and disinfection of barns, lowers the risk of environmental mastitis caused by coliform bacteria. It limits the transmission of contagious mastitis pathogens such as Staphylococcus aureus(Klaas & Zadoks, 2018). Differences in how hygiene-related variables are defined and measured across the studies have led to variability in the identification of specific risk factors. Nevertheless, the lack of separation of infected cows, poor hygienic practices, and the absence of teat dipping have been consistently reported as significant risk factors for SCM (Sanotharan et al., 2016).

S. aureus was the predominant pathogen isolated from CMT-positive milk samples, indicating that SCM in Monaragala District is primarily driven by contagious transmission. A similar predominance of S. aureus has been reported in smallholder dairy systems operating under comparable management conditions in Bangladesh (Abebe et al., 2016).

The high prevalence observed in the present study likely reflects prevailing smallholder management practices, suboptimal milking hygiene, widespread reliance on hand-milking, and limited implementation of dry cow therapy. Consistent with this interpretation, previous studies from Sri Lanka have also documented poor adoption of post-milking hygienic practices, which may facilitate transmission and persistence of S. aureus infections (Ranasinghe et al., 2021).

The persistence of S. aureus is further enhanced by its ability to invade deep mammary tissues and establish chronic intramammary infections that are difficult to eliminate during lactation using conventional antimicrobial therapy (Calvinho & Dallard, 2023). In comparison, the relatively low prevalence of Gram-negative and other environmental pathogens may suggest limited environmental mastitis pressure in the study area. The dominance of contagious pathogens stresses the need to prioritize improvements in milking hygiene and herd-level control measures rather than relying solely on environmental interventions.

The antimicrobial resistance patterns observed among mastitis pathogens raise important concerns for mastitis management and antimicrobial stewardship. Resistance to commonly used antimicrobials, along with the presence of multidrug-resistant isolates, underscores the potential consequences of prolonged, unregulated antimicrobial use in dairy herds. The resistance patterns should be interpreted with caution due to differences in antimicrobial testing across isolates; however, the findings are consistent with regional reports and indicate increasing resistance among mastitis pathogens (Jayaweera et al., 2018); (Priyantha et al., 2021).

These results emphasize the need to prioritize preventive mastitis control strategies, reduce reliance on antimicrobial therapy, and promote rational drug use. Improving hygiene and management practices may substantially reduce the burden of SCM in Monaragala District. Interventions focused on milking hygiene, regular floor cleaning, and udder sanitation may lead to meaningful reductions in SCM prevalence and, consequently, lower dependence on antimicrobial treatments. The observed antimicrobial resistance patterns underscore the urgency of strengthening mastitis prevention programs and antimicrobial stewardship in the Sri Lankan dairy sector.

SCM prevalence among dairy cows in Monaragala District was 18.7%. This was primarily related to contagious pathogens, particularly Staphylococcus aureus. Furthermore, SCM occurrence was significantly associated with some animal-level factors, including breed, parity, lactation stage, milk yield, and previous mastitis history, as well as farm management intensity and hygiene practices. Importantly, the detection of antimicrobial resistance highlights concerns regarding treatment efficacy in smallholder dairy systems. Given these findings, strengthening farm hygiene, mastitis prevention strategies, and prudent antimicrobial use is therefore essential to improve udder health, milk quality, and the sustainability of dairy production in Sri Lanka.

We certify that there is no conflict of interest with any financial, personal, or other relationships with other people or organizations related to the material discussed in the manuscript.

We are grateful to the farm owners for their consent to participate in data collection and to the lab assistants for their assistance in the laboratory process.

During the preparation of this work, the authors used ChatGPT to improve readability and language. The authors reviewed and edited the content after using this tool as needed and take full responsibility for the publication’s content.