Exploring Probiotics for Subclinical Mastitis Management

Exploring Probiotics for Subclinical Mastitis Management

Nutan Chauhan¹ and Ojal Singh²

¹PhD scholar, Animal nutrition Division

²MVSc scholar, Animal nutrition Division

National Dairy Research Institute, Karnal, Haryana

Email id- nutanc03@gmail.com

Introduction

Mastitis, a mammary gland inflammation, is a significant health issue affecting humans and animals, particularly dairy cows. Subclinical mastitis, characterised by an elevated somatic cell count (SCC) without visible symptoms, poses a challenge in dairy farming due to its impact on milk quality and animal health. Traditional treatments often involve antibiotics, which raise concerns about drug resistance and environmental impact. In recent years, probiotics have emerged as a promising alternative for managing mastitis. Probiotics, live microorganisms that confer health benefits when administered in adequate amounts, have been explored for their potential to prevent and treat mastitis by modulating the microbiota, enhancing immune responses, and inhibiting pathogenic bacteria. In dairy cows, specific probiotic strains such as Lactobacillus lactis and Bifidobacterium breve have shown efficacy in reducing the incidence of subclinical mastitis and lowering SCC in milk. Similarly, in lactating cows, oral probiotics have been found to reduce the incidence of mastitis and alleviate symptoms. Probiotics offer a safer and more sustainable approach compared to antibiotics, potentially mitigating the risks associated with drug resistance and promoting a healthier mammary gland environment.

Types of mastitis and clinical relevance

Mastitis can be epidemiologically categorized into contagious and environmental types and is caused by a wide range of pathogens. An increase in humidity and pollution in the barn environment also raises the load of bacterial pathogens. One study reported a 74.7% prevalence of mastitis at the herd level and a 62.6% prevalence at the cow level. Mastitis can also be classified into three types based on the degree of inflammation: clinical, sub-clinical, and chronic. Clinical bovine mastitis is evident and easily detected by visible abnormalities such as a red and swollen udder, fever (greater than 39.5°C), and loss of appetite in the cow. The milk appears watery and contains flakes and clots. Clinical mastitis can be further divided into per-acute, acute, and sub-acute, depending on the severity of the inflammation. Severe cases of clinical mastitis can be fatal.

In contrast, sub-clinical mastitis shows no visible abnormalities in the udder or milk, but milk production decreases, and there is an increase in the somatic cell count (SCC). Changes in milk composition can be an indicator of sub-clinical mastitis. Figure 1 shows the presence of somatic cells in healthy and infected quarters of a cow. In the milk of a healthy quarter, macrophages are most abundant, followed by smaller percentages of lymphocytes, neutrophils, and epithelial cells. In contrast, an infected quarter, whether with clinical or subclinical mastitis, is predominantly populated with neutrophils, with fewer macrophages, lymphocytes, and epithelial cells.

Fig.1 Healthy and infected quarters of a cow (Adapted from Haxhiaj et al., 2022)

It is recognized and confirmed through laboratory examination of milk or by animal-side tests such as the California mastitis test (CMT) followed by laboratory isolation of the causal agent. The SCC in healthy cow’s milk is between 50,000 to 100,000 cells/ml. Milk with an SCC exceeding 200,000 cells/ml is considered unhealthy for consumers. High SCC in milk reduces milk quality, making the financial losses from sub-clinical mastitis difficult to quantify, but experts agree that these losses are greater than those from clinical cases.

Bovine mastitis risk factors

Several risk factors are known to contribute to the incidence of bovine mastitis, including pathogen, host, and environmental factors. These factors should all be considered in mastitis control programs.

Pathogen factor

Bacterial intra-mammary infection (IMI) is the predominant cause of bovine mastitis, attributed primarily to a few key types such as Staphylococci, Streptococci, and various Gram-negative species like Escherichia coli. While over 150 microorganism species, including viruses, yeasts, mycoplasma, fungi, and bacteria, are capable of infecting the mammary gland, approximately 95% of mastitis cases globally are caused by around 10 major microbial groups (Table 1).

Table 1 Pathogens causing mastitis

These bacterial infections can be classified into two categories based on their origin: contagious and environmental. Contagious mastitis refers to infections that can be transmitted from cow to cow, particularly during milking. Pathogens such as Staphylococcus aureus and Streptococcus agalactiae, along with less common species like Mycoplasma bovis and Corynebacterium, colonize the cow’s udder and teat skin, invading and proliferating within the teat canal. These pathogens are capable of establishing sub-clinical infections, typically characterized by an elevated somatic cell count (SCC). Environmental pathogens such as Escherichia coli and Streptococcus uberis invade and multiply within the cow’s udder, eliciting a host immune response, and are often rapidly eliminated. A wide array of bacterial species has been reported to cause environmental mastitis, including Streptococcus spp. (e.g., S. uberis), coliform species (e.g., E. coli, Klebsiella spp., Enterobacter spp.), and Pseudomonas spp.

Host factor

Breeding and genetic

Significant influences on susceptibility or resistance to mastitis can be attributed to genetic factors and dairy cow breeding. Breeds of cattle with high yields, especially Holstein-Friesian cattle, have a higher genetic susceptibility to mastitis than breeds with medium yields. There is a decreased incidence of mastitis in Jersey cattle as opposed to Holstein-Friesian cattle. Multiparous cows exhibit immunological incompetence, which makes them more vulnerable to intra-mammary infections (IMI) than primiparous cows.

Udder structure

The structure of the udder also impacts susceptibility to infection. Cattle with large, funnel-shaped teats, pendulous udders, or blind quarters after calving are at higher risk of sub-clinical mastitis. Additionally, teat size and the distance from teat to floor can reduce the in vitro activity of leukocytes in milk, thereby increasing the occurrence of intra-mammary infections (IMIs).

Age

Age is another factor influencing infections. Older cows are more susceptible to infections, likely due to the wider or permanently partially open teat canal resulting from frequent milking. Additionally, the mammary epithelium of older cows has increased permeability, primarily due to the irreversible damage caused by previous inflammations.

Transition period

The period from three weeks before to three weeks after parturition, known as the transition or periparturient period, is a high-risk time for dairy cows to develop diseases like mastitis. Research indicates that intra-mammary infections (IMIs) are more common during parturition and the first month of lactation. This increased incidence of mastitis is attributed to immunosuppression, which is linked to heightened oxidative stress and reduced antioxidant defenses.

Host nutritional stress and the immune system

Dairy cattle require a substantial increase in energy and nutrients during lactation in order to synthesize milk and colostrum. Cattle encounter a negative energy balance when their feed intake isn’t sufficient to fulfil these lactation requirements. Deficits in trace metals (iron, copper, zinc, cobalt, and chromium), amino acids (lysine and L-histidine), and vitamins (A, C, E, β-carotene, and lycopene) are associated with this negative energy balance.

Environment factor

Environmental conditions and herd management practices significantly influence animal health and welfare. Maintaining cleanliness and ensuring comfort for the herd can effectively reduce the incidence and severity of mastitis. Factors such as high stocking density, contaminated floors, wet bedding, inadequate ventilation, and hot, humid climates create conditions that promote the growth of mastitis pathogens.

Economic significance

Clinical and subclinical mastitis cause milk loss, which means that the milk must be disposed of. Treatment expenditures and related expenses are also incurred. 60–70% of the total losses related to mastitis infections are attributable to clinical mastitis, which results in three times more production losses than subclinical mastitis. Clinical mastitis also generates large economic losses. Mastitis was shown to be responsible for roughly 49% of losses resulting from milk devaluation and 37% of losses resulting from veterinary expenses  on dairy animals in central India.

Advancement in the treatment of mastitis

Intra-mammary administration of antibiotics is the conventional therapy widely used for treating clinical mastitis during lactation and for preventing new infections during dry-off. However, concerns about antimicrobial resistance due to widespread antibiotic use have prompted a global push to reduce their use in dairy cattle and minimize antibiotic residues in dairy products. In response to these challenges, probiotic agents have emerged as a novel approach to managing mastitis. Probiotics are live microorganisms that provide health benefits to the host when administered in adequate amounts.

Research has demonstrated that probiotics can protect against microbial pathogens and enhance immune functions. The application of probiotic bacteria has primarily focused on the gastrointestinal and mammary glands. The beneficial effects of probiotics on host health are attributed to their ability to produce antagonistic substances, adhere to host tissues, and colonize different sites on the host surfaces. LABs have demonstrated potential as gastrointestinal ecosystem modifiers, potentially improving animal immunity and productivity. LABs are well-known for their antibacterial properties and generation of bacteriocin-like inhibitory compounds. Systemic immune activity can be strengthened by LAB, which are particularly well-known as possible probiotics for therapeutic use against mastitis.

Probiotic effects

Indirect interactions of probiotics with the local microbiota

Probiotics, commonly administered orally as fresh fermentation products or lyophilized bacteria, are primarily composed of Lactobacillus and Bifidobacterium species. These beneficial bacteria interact with the gut microbiota to restore balance and correct dysbiosis. Recent studies suggest that the mammary gland (MG), once considered sterile, may host a microbiota, although this is still debated. In animals, certain Lactobacillus strains isolated from milk have been used to treat mastitis, showing some efficacy.  Alternative mechanisms, such as direct probiotic-pathogen interactions, are being explored for mastitis control. Despite these uncertainties, probiotics offer a promising alternative to antibiotics for managing mastitis, potentially reducing the reliance on antimicrobials and promoting a healthier mammary environment.

Direct interaction of probiotics with the pathogen

Probiotic bacteria produce various antimicrobial compounds, including lactic acid, short-chain fatty acids, hydrogen peroxide, nitric oxide, and bacteriocins, which can inhibit pathogenic bacteria. Bacteriocins, peptides produced by bacteria, are particularly noteworthy for their role in competing with other bacteria and facilitating colonization. They have distinct mechanisms of action, different from antibiotics, and can act as antimicrobial agents, colonizing facilitators, and signaling molecules. However, their effectiveness against Gram-negative pathogens is limited unless produced by Gram-negative bacteria like E. coli. In the context of the mammary gland (MG), bacteriocin-producing LAB have been isolated from dairy cows. They might be used as teat canal probiotics to competitively exclude pathogens.

Enhancement of the epithelial barrier function

The interplay of probiotics with the epithelium has been particularly studied in the intestine, and it has been shown that probiotics are important mediators of intestinal barrier function and integrity. Probiotics may exert beneficial effects on the intestinal epithelium in different ways: (1) By improving the mucus layer thickness through stimulation of the mucin-secreting goblet cells (2) By stimulating the production of antimicrobial peptides (defensins and cathelicidins) by epithelial cells and particularly Paneth cells (3) By enhancing tight junction integrity

Modulation of the immune response

Probiotics exert immunomodulatory effects through interactions with epithelial cells and modulation of endogenous microbiota. They can dampen inflammatory pathways, such as NF-κB, and reprogram T cells, including Th17 and Treg cells, to counteract pro-inflammatory signals5. Probiotics also modulate helper T cell balance by influencing Th1, Th2, and Th17 pathways. Certain probiotic strains produce molecules like LTA variants, which can induce anti-inflammatory cytokines, while others produce proteins that modulate inflammation. These mechanisms contribute to probiotics’ potential in enhancing immune function and reducing inflammation.

Appraisal of published probiotic trials about mastitis control

Oral administration of putative mastitis probiotics

Oral probiotics have been evaluated in dairy cows for treating mastitis, with studies suggesting that lactobacilli isolated from milk can reduce bacterial counts and pain scores, offering an alternative to antimicrobials. The rationale for oral administration is based on the entero-mammary pathway, where gut bacteria are thought to be translocated to the mammary gland, but this theory is less applicable to dairy ruminants. Direct intramammary administration of probiotics might be more effective, as the oral route may not efficiently deliver probiotics to the mammary gland. Despite these challenges, probiotics like Lactobacillus fermentum and L. salivarius have shown promise in reducing mastitis symptoms and recurrence compared to antibiotics.

Topical application of bacteria at the teat apex

The teat canal serves as the primary entry point for most mammary gland (MG) infections, making the microbiota colonizing the teat skin and canal crucial in preventing new intramammary infections. Certain bacteria, such as Corynebacterium bovis, can interfere with mastitis pathogens by competitive exclusion. Studies have shown that some bacterial strains isolated from the teat apex can inhibit the growth of mastitis pathogens, particularly Gram-positive bacteria like Staphylococcus aureus. These findings suggest potential applications for teat probiotics to prevent pathogen colonization. However, technical challenges, such as maintaining probiotic viability and ensuring consistent manufacturing quality, need to be addressed. The use of probiotics as teat dips has shown promise but requires further investigation to confirm efficacy and overcome methodological limitations.

Concluding remarks and prospects

The concept of using probiotics for mastitis control in the mammary gland (MG) faces several challenges. Intramammary probiotics are problematic due to potential inflammatory responses and the need for stringent safety and efficacy standards. The use of probiotics at drying-off might be less inflammatory but still lacks evidence. Topical application at the teat apex shows biological plausibility, as it could leverage competitive exclusion of pathogens. However, understanding the teat apex microbiota is crucial for developing effective teat probiotics. Despite these challenges, modifying the teat microbiota through teat dipping is a promising area of research, given the teat canal’s role as a primary entry point for mastitis pathogens. Further studies are needed to fully explore these approaches.

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