Subclinical mastitis of buffaloes in Asia: prevalence, pathogenesis, risk factors, antimicrobial resistance, and current treatment strategies

SCM in buffalo in Asia can be caused by a variety of pathogens (Fig. 3), including bacteria, fungi, and occasionally, viruses. The prevalence and specific pathogens involved can vary across different regions of Asia.

S. aureus and CNS are frequently identified as common culprits in SCM cases among buffalo in Asia. These bacteria possess the capability to persist within the udder and initiate chronic infections that are notably challenging to treat [6,45]. Presently, they are recognized as significant pathogens responsible primarily for SCM [46]. E. coli is a frequently encountered environmental pathogen that can result in SCM in buffalo. These infections are typically linked to inadequate hygiene and suboptimal management practices [25]. Certain Mycoplasma species, including Mycoplasma bovis and Mycoplasma mycoides, have been documented as responsible agents for SCM in buffalo populations in some Asian countries [15]. Streptococcal species such as Streptococcus agalactiae, S. dysgalactiae, and Streptococcus uberis are additional contributors to instances of SCM in buffalo populations. Streptococcus uberis, in particular, tends to induce a chronic subclinical form of mastitis, although it can also lead to mild to moderate CM [15,45]. Corynebacterium species, such as Corynebacterium bovis, have been identified in cases of SCM in buffalo [15].

Fungal agents specifically yeast species like Candida spp., on occasion, have the potential to trigger SCM in buffalo [47]. Furthermore, SCM has been triggered following a combined intramammary and intranasal administration of bovine herpesvirus 4 to lactating cows, with bovine leukemia virus being identified in mammary tissue of cows experiencing SCM [48].

There are some animal level risk factors (Fig. 3) which are closely related to the occurrence of SCM.

Buffaloes exhibit a higher prevalence of SCM compared to cattle, particularly in high-yield crossbred buffalo such as Murrah, which are at an elevated risk of developing SCM [26]. Teats with a funnel shape were linked to a greater occurrence of SCM compared to those with a cylindrical shape. It was previously reported that cylindrical teats in Murrah buffalo are the most prevalent and exhibit a higher incidence of SCM [49]. Conversely, in some other studies, mastitis is found to be more prevalent in cattle than in buffaloes since buffaloes possess tighter sphincter muscles in comparison to cattle. Additionally, crossbred and exotic cattle exhibit a higher susceptibility compared to zebu cattle [50,51].

Mastitis cases tend to be more frequent in older animals with higher parity, while in buffaloes, a significant proportion of mastitis cases are observed in those that are in their third or fourth parity [27,52]. In addition, it was determined that 90.32% of she-buffaloes aged between 9 to 11 years, 77.27% in the age group of 12 to 14 years, 65.78% among those aged 6 to 8 years, and 41.37% in the age range of 3 to 5 years tested positive for SCM [30]. To corroborate the previous point, a recent study indicated that SCC, a marker for SCM, was notably influenced by factors such as buffalo breed, age, parity, and the time of the year [53].

Dairy animals, especially those in the first two months of milking after giving birth, are highly vulnerable to mastitis [51]. During the peri-parturient period, increased pressure on the teat canal may lead to leakages, providing an opportunity for pathogens to invade the udder. Studies indicate that certain udder and teat characteristics, such as pendulous udder shape, flat or inverted teat ends, and longer/thicker teats, are associated with a higher risk of intra-mammary infection and elevated milk SCCs [54].

Furthermore, teat deformities or injuries, whether caused by chemical injury (ergot, trichothecenes), physical trauma, or heat injury, significantly increases the risk of infection. Uneven milk flow and asymmetrical udder quarters, especially in cases of less pointed and more flattened teats, are associated with higher mastitis rates [55]. Additionally, another study indicates that rear udder quarters are particularly susceptible to mastitis [56].

Various studies have demonstrated that dairy animals experiencing under or over body condition are more prone to udder health issues [57]. It’s crucial to maintain proper body condition scores (BCS) through methods like regular exercise and feeding management. Managing BCS during the calving period significantly improves productivity and reduces udder health problems [58]. Animals with over BCS can be fed high-fiber, low-concentrate diets, while those with under BCS may benefit from dense feeds, although excessive concentrate levels should be avoided to prevent acidosis [59]. Adding 2%–5% supplemental fat to lactating animal diet can enhance energy balance and improve production status. Nevertheless, it is crucial to ensure that the ratio of n6 to n3 polyunsaturated fatty acids falls within the range of 3.9 to 5.9 to maintain normal immune functions in lactating animals during the transition period [60].

Studies have shown varying dry period lengths (from 0 to 70 days), but recent study suggests an optimal duration of 8 weeks [61]. This period allows time for nutrient replenishment and prepares the animal’s body from a nonproductive to a productive stage [62]. Providing a dry period significantly shorter than optimal can lead to negative energy balance and increase the risk of SCM, especially in young milch animals [62].

SCM is also related to some environmental risk factors as shown in Fig. 3.

Warm and humid weather promote the proliferation of harmful bacteria [63], and the prevalence of mastitis is notably affected by relative humidity and bedding materials [64]. Research suggests that mastitis cases are highest during rainy (16.28%) and summer (75.9%) seasons, whereas winters (8.75%) see fewer instances [30,65]. The variation in resistance to specific climatic conditions among lactating animals may be due to differences in their anatomical structure. Adequate ventilation in animal shelters can help to control humidity and temperature [66].

Studies highlight bedding materials as a significant source of SCM [63]. Select bedding materials that stay dry and clean for extended periods to prevent mastitis and provide comfort to farm animals [67]. Recycled manure solids (RMS) have shown potential but require careful hygiene maintenance and a dry matter content of over 34% [68]. Conversely other materials like rubber mats, concrete floors, and paddy straws have drawbacks. Rubber mats could be expensive investments, while concrete floors may become slippery for animals in wet conditions. Paddy straws are highly susceptible to moisture absorption, which can significantly increase microbial growth. Sand is considered ideal, with a 25 cm layer requiring regular replacement for controlling mastitis [69]. Consistently replacing the top layer of bedding materials and timely raking can aid in maintaining dry and hygienic bedding for animals to move and rest comfortably. This practice may enhance animal comfort levels and contribute to SCM control [70].

SCM leads to oxidative stress and the release of NO-derived free radicals in milk, causing milk loss, reduced antioxidant capacity and vitamin C levels [71]. Vitamin C supplementation in dairy animal diets is recommended to counteract these effects [72]. Prolonged SCM infection negatively affects milk quality and quantity, with increased NO-derived metabolites linked to SCM in milk-producing animals [73].

Milking management is crucial for preventing SCM in dairy animals. Both hand and machine milking methods must be hygienic to avoid SCM [54]. The underhand milking method should be avoided due to its potential for causing teat tissue injuries. Strict hygiene during machine milking is essential to prevent SCM, as unhygienic conditions can promote pathogen growth [54,74]. Automation in milking practices, along with strict hygiene maintenance, can help control SCM. Hygienic practices such as washing hands, udders, utensils, and milking equipment are recommended. Iodine-based teat dips or sprays are advised for preventing SCM. Cleaning milking machines two to three times daily is crucial, and indirect parts should also be cleaned to prevent bacterial contamination. Chlorine-based disinfectants are effective for dairy utensils, while acid-based disinfectants can remove alkaline deposits in machines. Manual cleaning may suffice for small farms, while larger operations may require machine washing systems [75].

Biosecurity measures play a key role in preventing the introduction and spread of pathogens in farms, thereby minimizing the risk of disease transmission and reducing antibiotic usage. Efforts are underway to develop and implement biosecurity tools like the BIOCHECK CATTLE® protocol, which assesses biosecurity levels on dairy farms across different regions. However, studies indicate that many cattle farmers do not fully implement adequate biosecurity measures due to practical and financial constraints [20]. Therefore, while biosecurity is increasingly recognized as a preventive measure, further education of farmers on proper implementation could improve mastitis management strategies, potentially reducing the need for antibiotics in disease prevention and treatment.

Antibiotics are extensively used in intensive livestock production, accounting for over 50% of global antimicrobial use in veterinary medicine [94]. By 2030, antimicrobial use in food-producing animals is projected to increase significantly, with estimates ranging from 11.5% to 67% [95,96]. In dairy production (cattle an buffalo), mastitis is the primary reason for antibiotic use, mainly administered via intramammary preparations or systemic applications. Common antibiotics for mastitis include penicillins, sulfonamides, ampicillin, cloxacillin, and aminoglycosides. Additionally, symptomatic and supportive therapies help reduce inflammation, improve antibiotic efficacy, and accelerate recovery and milk production [11,94]. However, controlling mastitis requires the cautious administration of antibiotics, often given preventively during the dry period. When treating CM, antibiotics should be selected based on the results of culture and sensitivity tests [16], considering the diverse antibiotic susceptibility patterns observed in mastitis-causing pathogens. Resistance issues highlight the importance of careful antibiotic selection to avoid ineffective treatments [97]. However, antibiotic usage can lead to residue concerns in milk and poses a risk of antibiotic resistance. Alternative strategies, such as non-steroidal anti-inflammatory drugs (NSAIDs), are explored for supportive therapy [98]. For challenging pathogens like S.aureus, traditional antibiotic therapies have limited effectiveness due to biofilm formation and unique host adaptations, driving the search for innovative solutions like vaccines and novel peptides [16]. Effective mastitis management also requires continuous monitoring of antibiotic resistance, awareness campaigns, and legal frameworks promoting judicious antibiotic use [99,100]. In some cases, extended antibiotic treatments or combination therapies are explored to improve cure rates, but the overall efficacy varies based on factors such as pathogen type and udder environment. For challenging pathogens like Prototheca spp. no effective therapies exist, leading to investigations into alternative disinfectants like guanidine [101]. Additionally, nano therapy has garnered a significant attention for delivering antimicrobial agents as drug delivery vehicle in the treatment of SCM. Different types of nanoparticles, such as liposomes, nanogels, polymeric nanoparticles, inorganic nanoparticles, and solid lipid nanoparticles, have demonstrated potential in managing SCM caused by bacteria like S. aureus [102]. Studies have demonstrated their effectiveness against multi-drug resistant strains, enhancing antibacterial activity and reducing dosing intervals for antibiotics. Specifically, chitosan nanoparticles [103] and metal nanoparticles like silver, copper, and zinc oxide [104] have exhibited antibacterial properties without harming mammary glands. Plant-derived nanoparticles, such as silver-nanoparticle-decorated quercetin and curcuminnano-formulations, have also displayed anti-biofilm activity against multi-drug resistant bacteria causing SCM [105]. Combining chitosan with antibiotics like cloxacillin has proven effective in inhibiting biofilm formation, improving clearance, and reducing intracellular bacteria viability, offering a novel, contamination-free method for mastitis prevention, particularly against multi-drug resistant strains [106]. However, further research is needed to validate these findings in vivo.

Probiotics are indeed live microorganisms which are administered in adequate amounts to provide health benefits to the host [107]. These benefits are achieved through various mechanisms, including the modulation of the gut microbiota, enhancement of the gut barrier function and mucosal integrity, modulation of the immune system, and inhibition of pathogens [101,102]. Lactic acid bacteria, a type of probiotic, have become popular in the treatment and prevention of mastitis, an inflammatory condition common in dairy animals. Strains of Lactobacillus have strong immunomodulatory properties and can protect against mastitis when used as feed supplements, teat dips, or through intramammary inoculations [108]. These bacteria form protective biofilms in the udder, inhibiting the growth of mastitis-causing pathogens. Studies have identified specific strains like Lactobacillus brevis 1595, L. brevis 1597, L. plantarum 1610, Lactobacillus lactis subsp. lactis, and Lactobacillus perolens among the total of 165 isolates obtained from sampling of the teat canal exhibited, high colonization capacities in bovine mammary epithelial cells, preventing invasion by pathogenic bacteria [109]. The two strains of lactic acid bacteria, L. lactis subsp. lactis CRL 1655 and L. perolens CRL 1724, isolated from bovine milk, demonstrated inhibitory activity against bovine mastitis pathogens. This effect was achieved through co-aggregation and adherence to the epithelial cells of the bovine teat canal during in vitro evaluation [110]. Incorporating lactic acid bacteria into animal feed is considered effective in preventing bovine mastitis. These bacteria adhere to mammary gland surfaces, hindering pathogenic bacterial invasion. Lactobacillus strains eg. L. casei BL23 also modulate the innate immune response, reducing pro-inflammatory cytokine expression in infected mammary epithelial cells and inhibiting bacterial adhesion and internalization [111]. Intramammary infusion of lactic acid bacteria (L. lactis subsp. lactis CRL 1655 and L. perolens CRL 1724) induces pro-inflammatory activity, promoting neutrophil influx into milk during lactation and drying-off periods [112]. Probiotic-based teat disinfectants have proven superior to commercial disinfectants, reducing mastitis-associated bacteria by altering the teat apex microbiota and preventing colonization by pathogens. Although probiotics hold potential, using them alone or in conjunction with repeated milk-out is not advised for the management of CM in lactating dairy animals. Moreover, microbial extracts derived from actinomycetes have exhibited antimicrobial properties against a range of bacteria responsible for mastitis, indicating their potential as viable options for mastitis treatment [113]. These extracts effectively hinder bacterial growth and are utilized for the development of efficient mastitis treatments.

Antimicrobial peptides (AMPs) are potent antimicrobial agents that play a crucial role in the innate immune system [114]. These peptides are widely available in nature and can provide antimicrobial activity against different organisms including bacteria, viruses, fungi, and parasites. They function through various mechanisms, such as disrupting bacterial membranes-leading to cell lysis and death or by modulating the immune system [108]. AMPs are known to exhibit broad-spectrum activity against various bacteria, including drug-resistant strains, and can work synergistically with conventional antibiotics. AMPs like beta defensins and cathelicidins are crucial in the innate immunity of vertebrates. Beta defensins act as the first line of defense against intramammary infections (IMIs) in dairy animals [115], while cathelicidins, released by infiltrating neutrophils during mastitis, have broad-spectrum antimicrobial activity [116]. Bovine cathelicidins, such as BMAP-27 and BMAP-28, show potential for mastitis treatment [117]. Bacteriocins, another type of AMP, are synthesized and secreted by various bacteria and are considered alternatives to antibiotics. Examples like Nisin and Bovicin HC5 have demonstrated antimicrobial efficacy against mastitis-causing bacteria [118]. However, bacterial ability to develop resistance against these compounds through different mechanisms, including protease production, surface charge modification, efflux pump activation, and potential toxicity and low bioavailability poses significant challenges in widespread use of AMPs as antimicrobial agents [105,109–119].

Immunotherapy provides an alternative approach to mastitis treatment, utilizing immunological techniques. It was previously reported that, microbeads carrying specific antibodies and an enhancer of phagocytosis, known as Y-complex, were used to treat mastitic cows infected with E. coli,S. dysgalactiae, or CNS [120]. This treatment was as effective as antibiotics and superior to NSAIDs in eliminating bacteria. Meloxicam, an NSAID, when used alongside antibiotics, improved cow fertility [121]. Additionally, interleukin-2 (IL-2) injected into the skin region after calving enhanced milk markers (SCC, serum amyloid A, lactoferin, NAGase) related to immune responses [122]. Immuno-stimulants such as Saccharomyces cerevisae yeast extracts and egg yolk immunoglobulins (IgYs) have demonstrated potential. The infusion of yeast extract into the mammary gland increased immune cell activity, reducing the risk of new infections. Specific egg yolk immunoglobiulin IgYs exhibited inhibitory and phagocytic activity against E. coli and S. aureus isolated from mastitic animals in vitro, suggesting their potentials for therapeutic treatment against mastitis in dairy cows [123]. Furthermore, antibodies targeting S. uberis adhesion molecule (SUAM) provided better protection against S. uberis infection in dairy cows, reducing clinical symptoms and bacterial counts, indicating improved clearance of pathogens and reduced IMIs [124]. A summary of the use and functioning of immunotherapy in treating mastitis is depicted in Fig. 6. In addition, a native secretory factor is also a naturally occurring substance released by cells or tissues into the bloodstream or surrounding environment, playing vital roles in biological processes such as cell signaling and immune response modulation. These factors encompass a range of molecules including hormones, cytokines, growth factors, and enzymes [125]. Lactoferrin (Lf), a natural whey protein derived from mammary glands and classified as a native secretory factor, exhibits notable antibacterial and anti-inflammatory properties (Fig. 6). It enhances penicillin’s inhibitory activity against bacteria, especially in penicillin-resistant strains [126], by blocking beta-lactamase activity [127]. Bovine lactoferricin gene (LFcinB) transfected into mammary cells increases LFcinB secretion, exhibiting strong antibacterial activity against S. aureus and E. coli [128]. Phospholipases A2 reduce inflammation and improve cell viability in vitro, and a single PLA2G1B application in chronic S. dysgalactiae cases clears inflammation and bacteria [129]. Homeopathy shows limited effectiveness compared to antibiotics, with suboptimal bacteriological and cytological cure rates [130]. In CM of dairy lactating cows, non-antimicrobial treatments like homeopathy are not recommended.

Fig. 6. Immuno-therapeutic approaches for the treatment of sub-clinical and clinical mastitis. Method 1: microbeats and formation of Y-complex, method 2: injecting interleukin-2 (IL-2) and Cytokine, method 3: mammary infusion of Saccharomyces cerevisae, method 4: egg yolk antibody (Ig-Y) therapy, method 5: using anti-recombinant Streptococcus uberis adhesion molecules.

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Bacteriophages, or phages, are viruses that specifically infect bacteria. They can be used therapeutically to target and destroy pathogenic bacteria without harming the host’s cells or beneficial microbiota [131]. Treating bacteria that form biofilms is challenging due to their resistance to conventional antibiotics. Various bacteriophages have been studied for their effectiveness against mastitis-causing bacteria like S. aureus, S agalactiae, S uberis, Klebsiella pneumonia, Klebsiella oxytoca, and E. coli [20,132]. While promising, these evaluations have been primarily in vitro, requiring further in vivo studies to confirm their efficacy in clinical cases. One approach involves phage cocktails, which have shown superiority over individual phages in mouse models of mastitis [133]. However, phages can induce specific immune responses, potentially affecting their therapeutic success [134]. Thermostable phages, resistant to high temperatures, have been identified, and the lytic effectiveness of a bacteriophage mixture comprising three phages, STA1.ST29, EB1.ST11, and EB1.ST27, was assessed against S. aureus isolates. The marked reduction in S. aureus bacterial density highlighted the therapeutic potential of bacteriophage therapy [135]. Despite these advancements, further research is essential to validate the in vivo efficacy of bacteriophage therapy for managing bovine mastitis. Additionally, bacteriophage-derived endolysins have proven effective against Gram-positive pathogens by breaking down the peptidoglycan layer of bacterial cell walls, facilitating phage release during the lytic cycle [136]. A novel bacteriophage-derived peptidase, CHAPK, demonstrated efficacy in disrupting biofilm-forming staphylococci, making it a potential candidate for preventing S. aureus colonization on udder skin when included in teat-dip solutions [137]. Other anti-staphylococcal peptidoglycan hydrolases like lysostaphin, LasA, ALE-1, broth lysate, CsCl, LytM, AtlA, AtlE, LysK, SAL-1, MV-L, ClyS, and LysH5 have also been identified for controlling and treating staphylococcal infections [138].

Herbal therapy is a promising approach for mastitis treatment due to its lack of adverse effects. Ethno-veterinary medicine, focusing on herbal remedies offers alternatives to manage bovine mastitis, demonstrating antibacterial, anti-inflammatory, and immune-modulatory properties (Table 2) [139–166]. Chinese herbs, such as Diploclisiaglaucescens and Curcuma longa, exhibit analgesic and anti-inflammatory effects comparable to standard medications [167]. Various administration methods, including topical, oral, and intramammary routes, are utilized. Herbal therapies such as plant extracts, like moringa, possess anti-inflammatory and antioxidant properties, aiding in udder inflammation and oxidative stress reduction. It also inhibited the expression of pro inflammatory cytokines (tumor necrosis factor [TNF]-α, IL-1β, and IL-6), cyclooxygenase-2 expression, downregulated NF-κβ as well as upregulated heme- oxygenase-1 and NADPH [168]. In southern Brazil, plants like Achilleamillefolium and Baccharistrimera are used orally and topically for their anti-inflammatory and immunomodulatory effects. Oxytropisglabra inhibits biofilm formation in bacteria (S. epidermidis) associated with mastitis [169]. Integrating herbal extracts with conventional treatments improves mastitis management. Some herbal preparations, like PHYTO-MASTVR, containing FDA-recognized safe ingredients, show potential for mastitis treatment, although effectiveness may vary [170]. Animal-derived compounds like bee venom, lactic acid bacteria from honeybees, and propolis possess anti-inflammatory properties and demonstrate antibacterial activity against major mastitis-causing pathogens in laboratory settings. They also exhibit inhibitory effects on S. aureus and E. coli [140–142]. Essential oils derived from Allium sativum, Cinnamon cassia, lemongrass, and M. verticillata exhibit inhibitory properties against various species of Staphylococcus and E. coli, while also enhancing the phagocytic activities of immune cells. Furthermore, they demonstrate immunomodulatory effects and inhibit Streptococcus uberis strains [155–161]. A depiction of the role and mechanism of action of herbal therapy in treating mastitis is provided in Fig. 7.

Fig. 7. A promising approach for the use of herbal and animal derived medicines in the treatment of mastitis. Antibacterial, analgesic and anti-inflammatory properties of medicinal herbs. Plant sources (Moringa oleifera, Diploclisia glaucescens, Curcuma longa, Achillea millefolium), plant extract (Limonene, Thymol, Lactoferrin, Eucalyptus extract), animal source (Lactic acid bacteria from honey bee, Propolis etc.), essential oils from (Allium sativum, Cinnamon cassia, Lemongrass oil, M. verticillata).

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Bovine mammary stem cells play a crucial role in maintaining udder health and can be utilized to treat mastitis-induced structural/cytological defects [171]. Mesenchymal stem cells derived from fetal bone marrow and adipose tissue exhibit antibacterial activity, enhancing bacterial clearance by promoting innate immune responses and AMP expression which is mediated by b-defensin 4 A and NK-lysine 1 activity [172]. Human mesenchymal stem cells show broad-spectrum antimicrobial activity, mediated by the enzyme indoleamine 2,3-dioxygenase (IDO), while murine stem cells lack this activity, indicating species-specific differences [173]. Allogeneic adipose tissue mesenchymal stem cell (ATMSC) therapy reduced bacterial count in mastitis-affected cows without adverse effects [174]. Intramammary inoculation with allogeneic ATMSCs (2.5 × 10⁷) lowered the bacterial count in the milk of cows with CM compared to untreated cows. Bovine mammary stem cell therapy can be applied to regenerate mammary tissues by either repairing or replacing damaged tissue. These stem cells have the capacity to differentiate into epithelial, myoepithelial, and/or cuboidal/columnar cells of the udder tissue. Utilizing bovine mammary stem cells helps mitigate the risk of rejection and potential side effects. Given that mammary stem cells are essential for the growth, renewal, and turnover of mammary epithelial cells, they can be employed for tissue repair and enhancing milk production [169]. Additional research on the isolation and characterization of mammary stem cells is crucial for gaining a deeper understanding of normal epithelial cell development in mammary tissue [171]. Despite various established and emerging treatment techniques, mastitis remains a challenge due to its diverse causes and clinical manifestations. Farmer’s knowledge and skills in mastitis management are crucial, as highlighted in a survey completed by Swedish dairy farmers. Overall, while stem cell therapy holds promise, further advancements and research are necessary before its widespread application in mastitis treatment [174].