Nguyen Pham Thao Nhi, Tran Duy Thanh, Pham Trang Thanh Nguyen, 

Nguyen Khanh Thuan, Nguyen Phuc Khanh, Nguyen Thanh Lam*

1. Introduction

Bovine mastitis is an inflammatory response of the udder tissue in the mammary gland caused due to physical trauma or microorganism infections. It is considered the most common disease leading to economic loss in dairy industries due to reduced yield and poor quality of milk (Gomes and Henriques, 2016). 

Mastitis is a complex disease, with a number of factors contributing to the level of mastitis in a herd, including environment, management, udder physiology and cow health. This is readily demonstrated by the low incidence of mastitis in suckled beef cows and the difference in mastitis pathogens observed in hand-milked cows. Although individual clinical cases may be encountered by the practitioner, it must be remembered that mastitis is a herd problem and control measures must be directed at the herd level (Scott et al., 2011).

Bovine mastitis can be classified into 3 classes based on the degree of inflammation, namely clinical, sub-clinical, and chronic mastitis. A clinical bovine mastitis is evident and easily detected by visible abnormalities, such as red and swollen udder, and fever in dairy cow. The milk of the cow appears watery with presence of flakes and clots (Khan and Khan, 2006). Clinical mastitis can be further sub-divided into per-acute, acute, and sub-acute depending on degree of the inflammation (Kibebew, 2017). Severe cases of clinical mastitis can also be fatal (Gruet et al., 2001). Contrary to clinical mastitis, sub-clinical mastitis shows no visible abnormality in the udder or milk, but milk production decreases with an increase in the somatic cell count (SCC). The loss contributed by sub-clinical mastitis is very hard to quantify, but experts agree that it accounts for more financial losses in the herd than do clinical cases (Romero et al., 2018). Contrarily, chronic mastitis is an inflammatory process that lasts for several months, with clinical flare-ups occurring at irregular intervals.

2. Aetiology

Although numerous species of bacteria, mycoplasma, fungi, algae and yeast have been isolated from clinical cases of mastitis, the major pathogens can conventionally be separated into two groups: contagious (or cow-associated) and environmental pathogens. However, the distinction between contagious and environmental pathogens has become blurred in recent years by research findings showing that ‘traditional’ environmental pathogens (e.g. Escherichia coli and Streptococcus uberis) can persist in a recurrent chronic host-adapted form, and that traditional ‘contagious’ bacteria such as Streptococcus dysgalactiae can persist in the environment (Scott et al., 2011).

Contagious pathogens usually live in the udder or teat skin and are transferred to the teat and spread during milking. They then grow up through the teat canal and into the udder. The three most important contagious pathogens are Staphylococcus aureus, Streptococcus agalactiae and Streptococcus dysgalactiae, Mycoplasma spp., Corynebacterium bovis and coagulase-negative staphylococci are less common, but may cause significant problems on individual farms. A high proportion of strains of Staphylococcus aureus produce β-lactamase. Streptococcus agalactiae is highly contagious and is readily transmitted between cows during the milking process. It is usually brought into the herd via purchase of milking cows (Scott et al., 2011).

Environmental pathogens survive in the cow’s environment and enter the udder by propulsion through the teat canal (e.g. during milking, by capillary action, insertion of antibiotic tubes, insertion of teat canulae) or by passive penetration of the teat canal immediately after milking. E. coli and Strep. Uberis are the important environmental pathogens, although Pseudomonas aeruginosa, other coliforms, Bacillus cereus, yeasts and moulds and Pasteurella spp. are less common. Various environmental factors, such as poor housing and hygiene, may result in the multiplication of E. coli and hence an increased incidence of coliform mastitis. Recent studies have demonstrated the importance of environmental infections during the dry period (especially coliforms) (Scott et al., 2011).

Table 1. Prevalence of pathogens isolated from milk of dairy cows in percentages (Cobirka et al., 2020).

3. Pathogenicity

3.1 Bacteria

Table 2. Classification of mastitis-causing pathogens(Cobirka et al., 2020).

Staphylococcus aureus

The primary reservoir of this mastitis pathogen is the udder, although it can persist on the teat skin. It has particularly strong adhesive properties such that a cow shedding infection in her milk can infect the next 6–8 cows to be milked by the same cluster. It is primarily spread from cow to cow during the milking process. The majority of strains of Staph. aureus produce β-lactamase. Three separate disease syndromes are recognized with Staph. aureus infection:

• Severe acute gangrenous mastitis. Cows initially have a high rectal temperature (41–42°C), with heat, pain, redness and severe swelling of the affected gland(s). The cow is systemically ill and inappetent, has toxic mucous membranes and will become recumbent with severe depression. Over 24 hours the affected gland becomes cold, developing a sharply demarcated blue-black discolouration from healthy tissue. The udder secretion is cold, reddish brown and watery, with gas sometimes produced. Cows that recover eventually slough the affected quarter some weeks later and are culled from the herd.

• Moderate and mild clinical mastitis. Similar to other forms of acute mastitis, with clots in the milk and inflammation of the udder.

• Chronic mastitis. Staph. aureus infections of the udder are notoriously difficult to treat, leading to the formation of chronic infections with extensive fibrosis and induration of the udder (Scott et al., 2011).

Streptococcus agalactiae

Strep. agalactiae is a gram-positive pathogen causing contagious mastitis. It can be found in bovine gastrointestinal tract as well as in the environment of dairy cows. It can be transmitted via milking machine and through oro-fecal route, particularly through contaminated drinking water; therefore, a recent study showed that maintaining udder and milking sanitary are not enough to control Strep. agalactiae infection, but fecal and environment management should also be taken into account. Strep. agalactiae causes sub-clinical mastitis with high SCC and low milk production even though no abnormalities were shown in milk. It can survive indefinitely in mammary glands of cows, by forming a biofilm that allows them to adhere and persist in the mammary gland, concomitantly enhancing resistance to host factor and nutrient deprivation (Rosini and Margarit, 2015).

Streptococcus dysgalactiae

Although conventionally described as a contagious pathogen, Strept. dysgalactiae survives well in the environment and thus has some of the properties of an environmental pathogen. It is commonly found on the teat skin (as opposed to the udder), especially if the skin is damaged. It is present in the tonsils and can be transmitted by licking, especially in heifers. It commonly infects dry cows, prepartum heifers and even calves, and is involved in cases of summer mastitis (Scott et al., 2011).

Mycoplasma spp

Contagious mastitis caused by Mycoplasma spp. is less common than Staph. aureus and Strep. agalactiae infection. However, it is highly severe and damage secretory tissues, and induce gland and lymphatic nodule fibrosis and abscesses. Outbreak of Mycoplasmal mastitis is sporadic without any deliberate intervention. Although it is self-limiting, it produces biofilm and invades host cell, and does not respond to antibiotic treatment. The only control is by regular monitoring and rapid segregation or culling of infected cow (Nicholas et al., 2016).

Corynebacterium bovis

This bacterium was thought to be a teat end commensal and thus present as a contaminant in milk samples. However, it has been associated with subclinical mastitis and high SCCs, especially in relation to poor post-milking teat disinfection (Scott et al., 2011).

Escherichia coli

E. coli is the most frequently found gram-negative pathogen. It invades the udder through teat, proliferate and initiate inflammatory response in dairy cow. It can be found in the environment surrounding dairy cow, such as bedding of the herd, especially in a wet condition. Mastitis caused by E. coli is usually clinical and transient. Symptoms are varied, ranging from mild with only local signs (red and swollen udder) to severe with systemic signs (fever). Severe clinical mastitis caused by E. coli can cause irreversible tissue damage in the mammary gland, complete loss of milk production, sometimes even leading to the death of dairy cow.

E. coli rapidly induce an inflammatory response in the host. The virulence factor best known to trigger the inflammatory response is the endotoxin, which is found on the outer membrane of E. coli, known as lipopolysaccharide (LPS). The binding of LPS to toll-like receptor (TLR4) in association with other molecules, such as LPS-binding protein and cluster of differentiation 14 induce a series of signaling pathways.

Nevertheless, E. coli was classified as an opportunistic pathogen with different virulence factors, since its pathogenicity is not only mediated by single and specific virulence factor (Fernandes et al., 2011). In fact, combinations of several virulence factors such as toxins, adhesins, invasins, capsule production, ability to resist serum complement, and iron scavenging, are reported as being necessary to overcome the host’s selection pressure and to colonize, multiply, and survive in the udder and cause inflammatory responses (Kaper et al., 2004). Besides, E. coli can persist in the mammary gland, causing recurrent mastitis infections that are hard to treat, possibly due to the ability to produce biofilm at different levels (Gomes and Henriques, 2016).

Klebsiella pneumoniae

Klebsiella pneumoniae is associated with damp sawdust bedding causing peracute severe coliform mastitis (Scott et al., 2011).

Pseudomonas aeruginosa

Pseudomonas aeruginosa is associated with contaminated water and outbreaks of P. aeruginosa mastitis have been linked to contaminated udder wash, teat-dip and dry cow tubes. The clinical signs vary from peracute severe endotoxic mastitis to chronic recurrent cases (Scott et al., 2011).

Enterococcus spp

Enterococcus faecalis is the predominant Enterococcus spp., followed by Ent. faecium. They are environmental gram-negative pathogens present in the organic bedding material of the herd. Pathogenesis of Ent. faecalis was reported related to the biofilm formation. In addition, both Ent. faecalis and Ent. faecium were reported to be resistant to several antibiotics such as lincomycin, tetracycline, kanamycin, streptomycin, quinupristin/dalfopristin, erythromycin, chloramphenicol, and tylosin owing to the presence of biofilm. This leads to frequent occurrences of enterococci infections, both recurrent and persistent, which are difficult to treat (Elhadidy and Zahran, 2014).

Coagulase-negative Staphylococcus

Coagulase-negative Staphylococcus (CNS), for example Staph. simulans, Staph. chromogens, Staph. hyicus, and Staph. epidermis, represents an emerging mastitis pathogen that has been isolated in many countries. The infections caused by CNS are relatively mild, usually remain sub-clinical but can be persistent, and are associated with an elevated SCC and decreased milk quality (Taponen and Pyörälä, 2009). However, unlike Staph. aureus, reports show that their persistence in the udder has no relation with the ability of biofilm production (Simojoki et al., 2012). They can behave as both contagious and environmental pathogens. So, post-milking teat disinfection is an effective measure in reducing CNS infections; as well as antibiotic intervention. CNS responds better to antibiotic treatment than Staph. aureus (Taponen and Pyörälä, 2009).

Streptococcus uberis

This pathogen is widespread in the environment, especially in straw yards, which may contain up to 10⁶ bacteria per gram of straw bedding. It is also widespread on the skin of the cow, but relatively rare in faeces (compared with E. coli). Outbreaks can occur in cows at pasture, especially in late summer, presumably by transmission from the skin of the cow, via the lying area, to the teat.

Like E. coli, new intramammary infections during the dry period have been shown to play an important role in the epidemiology of Strep. uberis infections. The clinical signs of Strep. uberis infection vary from subclinical infections to acute severe clinical mastitis with a hard, hot, swollen, painful quarter(s), pyrexia and systemic illness in the cow. Certain strains of Strep. uberis are highly resistant to phagocytosis by white blood cells in the udder and thus develop into chronic recurrent cases unless prompt treatment is undertaken (Scott et al., 2011).

4. Epidemiology

Susceptible hosts

Following are the factors that make animals susceptible to mastitis (Hamadani et al., 2013):
Age: The prevalence of infection increases with age, peaking at 7 years.

Stage of lactation: Infection rate is more in initial and last stage of lactation.

Milk yield: High yielders are more commonly affected than low yielders.

Breed: Incidence is more in exotic and crossbreds than the zebu cows. In Holstein Friesian cows, incidence has been seen to be more than in Jersey cows.

Milking rate and udder morphology: High milking rate and large teat canal diameter have been associated with increased incidence of intramammary infection. Differences in udder depth, teat length, teat shape and teat orifice morphology are also thought to be associated with differences in mastitis.

Herd size: The incidence is more in large sized herd.

Nutrition: Heavy protein feeding may be a predisposing factor. Vitamin E, A and selenium may be involved in resistance to certain types of mastitis.

Hygiene: Bad hygiene and sanitation help in bacterial multiplication.

4.3 Transmission

Mastitis is most often transmitted by repetitive contact with the milking machine, and through contaminated hands or materials.

In general, it is believed that mastitis pathogens gain entrance to the udder through teat opening into the teat canal and from the teat canal into the intramammary area during the reverse flow of milk due to vacuum pressure fluctuation of the milking machine. However, the detailed mechanism of mastitis pathogen colonization of the mammary gland may vary among species of bacteria and the virulence factors associated with particular strain in each species. An example of this is in some cases; it has been shown that E. coli can penetrate the teat canal without the reverse flow of milk. Some of the major mastitis pathogens, such as E. coli, Staphylococcus aureus, and Streptococcus uberis can adhere to and subsequently invade into the mammary epithelial cells. This adherence and subsequent invasion into mammary epithelial cells allow them to persist in the intracellular area as well as to escape the host immune defenses attack and action of antimicrobial drugs Compared E. coli strains known to cause chronic infections with strains known to cause acute infections and found that chronic strains were more invasive to the epithelial cells, leading to the difficulty in clearance and persistent infection compared to acute strains. S. aureus enters the mammary gland through the teat opening and subsequently multiply in the mammary gland where they may form biofilms, attach to, and internalize into the mammary epithelial cells causing inflammation of mammary glands characterized by swelling, degeneration of epithelial cells, and epithelial erosions and ulcers (Dego, 2020).

5. Pathogenesis

A comprehensive understanding of the pathogenesis of mastitis is the key for the development of appropriate detection techniques. The primary cause of mastitis is a wide spectrum of bacterial strains; however, incidences of viral, algal and fungal-related mastitis were also reported. Normally, the teat canal is tightly closed by sphincter muscles, preventing the entry of pathogens. The teat canal is lined with keratin, a waxy material, which prevents the migration of bacteria. However, the efficiency of keratin is restricted. Fluid accumulates within the mammary gland as parturition approaches, resulting in increased intra-mammary pressure, and mammary gland vulnerability is caused by the dilation of the teat canal and leakage of mammary secretions. Additionally, during milking, the keratin is flushed out, and there is distention of the teat canal. The sphincter requires approximately 2 hrs to return back to the contracted position. 

Once inside the teat, bacteria must also elude the cellular and humoral defense mechanisms of the udder. If they are not eliminated, they start multiplying in the mammary gland. They liberate toxins and induce leukocytes and epithelial cells to release chemo attractants, including cytokines such as tumor necrosis factor-a (TNFα), interleukin (IL)-8, IL-1, eicosanoids (like prostaglandin F2α (PGF2α)), oxygen radicals and acute phase proteins (APPs) (e.g. haptoglobin (Hp) serum amyloid A (SAA). This attracts circulating immune effector cells, mainly polymorphonuclear neutrophils (PMNs), to the site of infection. PMNs act by engulfing and destroying the invading bacteria via oxygen-dependent and oxygen independent systems. They contain intracellular granules that store bactericidal peptides, proteins, enzymes (such as myeloperoxidase) and neutral, and acidic proteases (such as elastase, cathepsin G, cathepsin B and cathepsin D). The released oxidants and proteases destroy the bacteria and some of the epithelial cells, resulting in decreased milk production, and release of enzymes, such as N-acetyl-b-D-glucosaminidase (NAGase), and lactate dehydrogenase (LDH). Destruction of most of the PMNs takes place by apoptosis once their task is fulfilled. Subsequently, macrophages engulf and ingest the remaining PMNs. The dead and sloughed off mammary epithelial cells, in addition to the dead leukocytes, are secreted into the milk, resulting in high milk SCCs. When the infection persists, there is internal swelling in the epithelium of mammary gland. The mammary gland alveoli become damaged and start losing anatomical integrity. The blood-milk barrier is breached, causing extracellular fluid components, such as chloride, sodium, hydrogen, potassium and hydroxide ions, to enter the gland and mix with the milk. When extensive damage to the blood-milk barrier has occurred, blood might be detected in the milk. This leads to visible changes on the udder, such as enhanced external swelling and reddening of the gland. Changes also occur in the milk, including increased conductivity, increased pH, raised water content, and the presence of visible clots and flakes (Pal et al., 2019).

6. Clinical signs and pathology 

6.1 Clinical signs

It is important to remember that not all cases of mastitis have obvious changes in the milk or udder. The presence of severe cases of mastitis (e.g. Staph. aureus or E. coli) tend to represent the ‘tip of the iceberg’, and the approximate incidence of mastitis cases is: fatal (1%); severe (29%); mild (70%).

Mastitis can be graded using a clinical scale to determine the severity of mastitis (Table 3). In subclinical mastitis, SCCs and milk bacteria levels may be raised and are detectable prior to clinical signs. Milk conductivity also increases prior to milk and udder changes, and has been researched as a means of automatic detection for preclinical mastitis (although it is prone to a high number of false-positive results). Similarly, measurement of acute phase proteins in milk (e.g. milk amyloid A) might also prove useful in the future for the detection of subclinical mastitis.

Changes in the milk are detectable in mild clinical (Grade I) mastitis, especially if foremilking is practised. Mastitis can lead to changes from caseous lumps, to clots in the milk, to watery secretions. Although some changes may be classical (e.g. yellow watery secretion in acute coliform mastitis), it is not consistently possible to determine the organism producing mastitis from clinical signs alone. This needs to be determined by bacteriology.

Changes in the udder during moderate (Grade II) clinical mastitis are detectable as hot, painful, swollen quarters. Severe (Grade III) clinical mastitis is observed when systemic illness in the cow develops, especially in mastitis caused by Staph. aureus, Strep. uberis and coliforms. Signs may include dramatic reductions in milk yield, inappetence, pyrexia, dehydration, elevations in heart and respiratory rate and other signs of advanced endotoxaemia (Scott et al., 2011).

Table 3 Clinical scale to determine the severity of mastitis (Scott et al., 2011).

Summer mastitis: Because of its unusual aetiology and epidemiology, summer mastitis is considered separately from the other causes of mastitis. It is a disease of late gestation in dry cows and heifers (but may even occur in the rudimentary udders of young heifers, bulls and steers), and occurs at grass during the summer months.

Clinical presentation: During the early stages the affected teat and associated mammary gland are swollen, but this stage quickly progresses to systemic disease. Severely affected animals are pyrexic, stiff and lame due to the painful quarter. Cattle are dull and inappetent and isolate themselves from others in the group. Oedema may extend around the udder and up the inside of the leg. In severe cases there are marked joint effusions affecting mostly the hock and fetlock joints. The affected quarter is swollen, hard, painful and hot, with a grossly enlarged teat. The udder secretion is thick and clotted with foul-smelling green/yellow pus. Affected animals may abort and even die if prompt treatment is not administered. Even after prompt treatment, the affected quarter is permanently damaged. Illness leads to the birth of weakly calves (acute intra-uterine growth retardation), which have a high mortality rate (Scott et al., 2011).

Acute mastitis: Peracute and acute mastitis are most commonly seen in the first few weeks after calving and are often the result of periparturient immune suppression. Disease may result from recrudescence of dormant dry period infections, or from new intramammary infections during lactation. Cases can occur throughout lactation at a lower frequency. In most cases peracute mastitis with toxemia results from coliform infections. Similarly, in acute mastitis, environmental organisms such as coliforms (e.g., Escherichia coli) or Streptococcus uberis are frequently involved. Immune suppression occasionally leads to acute disease from “contagious” mastitis organisms such as staphylococci, which are carried on the skin or in the udder of affected cows and transmitted to other cows during milking (Blowey and Weaver, 2011).

Chronic mastitis: Streptococcus agalactiae, S. dysgalactiae, S. uberis, staphylococci, Mycoplasma, Arcanobacterium pyogenes, and other bacteria can produce a chronic mastitis, manifested as “clots” in the milk, with or without palpable udder changes (Blowey and Weaver, 2011).

Infectious teat diseases papillomatosis (warts): Most teat warts are seen in pregnant heifers and usually resolve before these animals enter the milking herd. Fibropapillomas that present in milking cows can predispose to mastitis and cause mechanical interference with milking.

Clinical presentation: There are two main skin lesions: flat/rice-grain fibropapillomas, which are seldom of clinical significance; and the more florid-type of projecting fibropapilloma, which may cause problems should they interfere with milking (Scott et al., 2011).

Herpes mammillitis (bovine ulcerative mammillitis): Herpes mammillitis is normally seen during the autumn/winter months. In a naïve dairy herd, infection spreads rapidly, but more usually disease is seen when susceptible heifers enter the milking herd.

Clinical presentation: Initially, widespread vesicles (0.5–5.0 cm) form on the teats and base of the udder. These quickly rupture to form painful ulcerative lesions that become covered in dried serum exudate, forming thick brown scabs. Healing takes place over 2–3 weeks. Another manifestation of this disease, pseudo-lumpy skin disease (Allerton virus), has been described with multifocal raised circular lesions (up to 2 cm diameter) developing over the body. The lesions are raised, hairless and form scabs that slough but rarely ulcerate. Healing takes place without treatment (Scott et al., 2011).

Pseudocowpox: Pseudocowpox is a common disease of dairy cows. Immunity is short-lived, resulting in endemic infection.

Clinical presentation: Initially, erythematous and oedematous painful lesions appear on the teats and these soon become raised orange papules then small dark red scabs. Vesicles are rare with this disease. The scabs are shed after 10–12 days leaving the classic raised ‘horseshoe’ or ring lesion. Complete healing of teat lesions may take 4–5 weeks. The virus can cause localized painful nodules on the hands and arms of in-contact humans (Scott et al., 2011).

Udder impetigo/necrotic dermatitis (udder rot): Udder impetigo is a superficial skin infection of the udder, which although unsightly, is usually of minor clinical significance.

Clinical presentation: Necrotic dermatitis presents in the udder skin where it apposes the medial thigh and, occasionally, in the ventral midline immediately cranial to the udder. Severe infection can give rise to multiple small pustular lesions, which may sometimes spread onto the teats. The condition is more common in heifers, especially those with considerable udder oedema (Scott et al., 2011).

Non-infectious lesions of teat skin udder oedema: This is a common problem affecting periparturient dairy cattle, especially heifers.

Clinical presentation: Animals are clinically normal except for hindlimb abduction when walking. In severe cases there is extensive pitting oedema of the udder and teats extending to involve the ventral midline subcutaneous area. Most cases resolve soon after calving when milking is initiated, but in severe cases treatment may be required (Scott et al., 2011).

Teat chaps: Teat chaps are very common in both lactating beef and dairy cows.

Clinical presentation: Teat chaps appear as horizontal skin breaks in the teat skin. They may cause discomfort when the cow is milked or Suckled (Scott et al., 2011).

Milking machine-induced teat lesions: Milking machine-induced teat lesions will occur at low levels in the vast majority of dairy herds, although a prevalence of >20% of teat ends with hyperkeratosis would indicate a problem with milking machine function, which will predispose cows to mastitis.

Clinical presentation: Hyperkeratosis caused by prolapse/eversion of the streak canal lining may become traumatized and infected. Secondary infection with Fusobacterium necrophorum leads to dark scabby lesions known as blackspot (Scott et al., 2011).

Teat lacerations: Traumatic teat injuries are not uncommon in dairy cows with pendulous udders and are usually caused by treading on the teats. Most teat injuries are treated conservatively, but surgical repair can be attempted (Scott et al., 2011).

Teat cistern obstructions: Teat obstructions are encountered relatively commonly in dairy herds. They interrupt the milk flow, resulting in teat end damage and an increased risk of mastitis (Scott et al., 2011).

6.2 Pathology

The inflammatory lesions (mastitis) observed in mammary quarter were morphologically cassified inti mixwd, lymphoplasmacytic, suppurative pyogranulomatous, abscedativbe, necrosuppurative and granulomatous. The agents  identified in the lesions and listed and were subdivided according to the

histological pattern of the asociated mastitis. These patterns were associated with the same set of pathogens: Streptococcus spp., coagulase-negative Staphylococcus, Staphylococcus aureus, Streptococcusagalactiae, Streptococcus uberis, and Corynebacterium bovis. The pyogranulomatous pattern with distinct distribution based on the agent involved, mostly S. aureus and Nocardia sp. Abscedative mastitis was characterized by multiple abscesses in the parenchyma and was mainly caused by Trueperella pyogenes. Necrosuppurative mastitis were characterized by severe parenchyma necrosis and were caused by bacteria such as CNS and Escherichia coli. The granulomatous pattern was occasionally associated with Mycobacterium sp (Bianchi et al., 2019).

7. Diagnosis

Mastitis is an inflammation of parenchyma of mammary gland characterized by physical, chemical, and usually bacteriological changes in milk and pathological changes in glandular tissue. Mastitis occurs in all species but assumes major economic importance in dairy cattle and buffaloes due to its effect on quality and quantity of milk in high yielders. To avoid these economic losses due to mastitis, it is distinctly important to identify the disease in early stage. Unlike the clinical form, in subclinical form there is neither visual detection of abnormalities in milk nor in mammary gland. Therefore knowledge of routine diagnostic screening tests for early detection of mastitis is desirable to treat the condition and to avoid the subsequent economic losses.

Clinical mastitis

Mild (Grade I) clinical mastitis can be detected by foremilking, which should be included as part of the milking routine. Alternatively, Ambic in-line milk filters may also be used to detect any clots in the milk. Checking the milk sock/filter at the end of milking is a retrospective measure. Palpation of the udder is useful to detect heat, pain and swelling in clinically affected quarters during moderate (Grade II) clinical mastitis. Changes in the behaviour of the cow (e.g. altered position when entering the parlour, kicking while machine attached) may also be early indicators of disease.

Subclinical mastitis

Subclinical mastitis may be detected by the use of SCCs, detection of bacteria in the milk, altered milk conductivity or the measurement of acute phase proteins (e.g. milk amyloid A).

Somatic cell counts

SCCs are a measure of the number of cells present in the milk. In a healthy udder the SCC is made up predominantly of epithelial cells. In response to inflammation in the udder (typically mastitis), white blood cells enter the udder to combat the infection and the SCC rises.

Numerical SCCs are measured using the automatic Fossomatic method, which is used to determine SCCs in bulk milk and individual cow samples. Alternatively, the California Mastitis Test (CMT) is a simple cowside test, which crudely estimates the SCC via a gelling reaction (‘slime’) with a detergent reagent. It can be performed during milking and the results are available immediately. It can help to identify individual quarters with a high SCC in order to take samples for bacteriology and decide on treatment options. The major disadvantage is that it only detects relatively high SCCs (>400,000/ml).

SCCs can either be performed on bulk milk (BMSCC; presented as monthly, three-monthly and annual averages) or on individual cows (ICSCC). BMSCCs are important as in many countries farmers are paid for the milk partly on the basis of hygienic quality (SCC and bacteria level). Some regions also have government regulations on milk hygienic quality. For example, EC Regulation 853/2004 (detailing ‘Specific hygiene rules for food of animal origin’) states that all bovine milk for human consumption in the European Union must have a SCC <400,000 cells/ml based on a three months rolling geometric mean.

ICSCCs are an average using a composite sample taken from all four quarters, and are a useful tool in identifying high cell count cows. Regular monthly ICSCC recording is provided by commercial organizations. Single ICSCC results should not be taken in isolation, but regular (usually monthly) sampling and trends should be used for interpretation. ICSCC values can rise to 20,000,000/ml in clinical mastitis. A suitable threshold (such as 200,000/ml) is used to interpret ICSCCs, with low values under this threshold indicating healthy udder status, while high values above this indicate subclinical mastitis. Various computer programs (e.g. NMR Herd Companion in the UK) can then be used to assess the relative number of cows that have chronic intramammary infections (shown by a persistently high ICSCC), new infections (low ICSSC one month, rising to a high ICSCC the next month), recovery from infection (high ICSSC one month, dropping to a low ICSCC the next month) or uninfected cows (persistently low ICSCC).

Bacteria levels in milk

Conventionally, bacteria levels in milk are determined by the total bacteria count (TBC), which measures the number of bacterial colonies grown from milk after 72 hours incubation. In some countries (e.g. the UK) the TBC has been replaced by the Bactoscan test, which measures the total number of bacteria (viable and nonviable) present in a milk sample. Bulk milk tests that show high bacteria levels are penalized by milk purchasers, as they are more prone to spoilage.

Potential sources of bacteria in the milk include mastitis pathogens from the udder (especially if Strep. agalactiae and Strep. uberis are involved), environmental contamination and poor pre-milking teat preparation, poor cleaning and sanitization of the milking equipment after use and poor refrigeration of milk after collection.

Current trends in diagnosis of mastitis involves following routine diagnostic tests: 

1) Physical examination of udder 

Examination of mammary gland is important for successful detection of mastitis. It is emphasised to view the shape, size, consistency and contour of the udder properly. Detailed examination of the teat and teat orifices should be made to assess inflammation, hot painful swelling and loss of function. From his studies reported that the physical examination of the udder was quiet informative about the size, shape, and consistency when conducted immediately after milking. 

2) Strip cup test

In individual animals and in herds, strip cup or strip plate test is routinely used in milking parlor for detection of clinical mastitis. In herd health management practice operators of the milking machines visually examine the fore milk for gross abnormalities by squirting few stripes of milk on strip cup where the abnormalities are usually manifested in the form of blood, flakes, clots and wateriness suggestive of mastitis. The use of strip cup test bears some additional benefits apart from general identification of clinical mastitis as: i) stripping the first stream of milk stimulates milk letdown, resulting in faster milk letout; ii) fore milk is higher in bacteria than subsequent milk; removal of this milk may reduce bacterial contamination of the milking machine and udder. 

3) California mastitis test (CMT)

California mastitis test is simple, inexpensive and rapid screening test. It estimates the number of somatic cells present in milk. Somatic cells are composed of approximately 75 per cent of leucocytes (white blood cells) and 25 per cent of epithelial cells (secretary and lining cells). Leucocytes’, being the bodies of primary defence, there increase in mastitis is enviable. However, rise in epithelial cells is due to an injury to mammary epithelium. Procedure for conducting CMT test is by mixing the test reagent (CMT reagent) with an equal quantity of milk. The reagent reacts with DNA of the nuclei of the somatic cells in the milk to form a gel. The reaction is then visually scored as 0, T (Trace), 1, 2, or 3 depending upon the amount of gel formation. Formation of more gel indicates higher somatic cell count.

4) Wisconsin mastitis test (WMT)

Wisconsin mastitis test is primarily a laboratory test which is generally conducted on bulk tank milk samples. In both WMT and CMT same type of reagent is used. In CMT the test result reaction is qualitatively estimated while in WMT the test result reaction are measured (mm). The test is conducted by combining a measured quantity of milk with an equal amount of reagent. The milk and reagent are then mixed for 8 to10 seconds. The mixture is drained for a period of 18 seconds and returned to an upright position. After waiting for one minute, the amount of fluid remaining in the tube is measured. WMT scores are generally calculated in millimetres (mm) and used to predict the average number of somatic cells present in the milk.

5) Modified white side test

6) pH determination test

7) Chloride test

8) Electrical conductivity test

9) Somatic cell counts of milk (SCC)

The count used includes the direct microscopic somatic cell count (DMSCC), the bulk milk somatic cell count (BMSCC) and individual cow somatic cell count (ICSCC). In direct microscopic somatic cell counts, milk smear is made on a clean glass slide in the area of 1 cm2 stained with 1% methylene blue and 60 fields are examined for the count. The average numbers of cells per field are multiplied by the multiplication factor of the microscope and the value so obtained is considered equal to the number of cells/ml of milk sample. Now a day’s electronic somatic cell counters are practiced for DMSCC, which has following advantages: 

a) This technique can be automated thus allowing centralization of laboratory procedures. 

b) Preserved samples can be used for counting.

c) The procedure is more precise and more objective.

d) The primary disadvantage of electronic somatic cell count procedures is requirement of costly equipments with trained individuals. Reported somatic cell count more than 250,000/ml were considered to be indicative of inflammation, whereas counts less than 100,000/ml was indicative of normal udder and counts more than 500,000 cells/ml was indicative of infection. Presently, Bulk milk somatic cell count is the universally adopted screening test for mastitis. Benefit of this test is that it creates awareness in the mind of farmer/dairy owners/ herd health medicine observers about existence of mastitis problem in herd.

10) N-Acetyl-B-D-Glucosaminidase (Nagase) test

11) Methylene blue reduction test (MBRT)

12) Milk antitrypsin assay (Maum test)

8. Treatment

Treatment protocols for mastitis are based on the following parameters:

• Severity of mastitis: mild (Grade I), moderate (Grade II) or severe (Grade III). Potential mastitis pathogens involved.
• Stage of lactation.
• Previous history and experience on the farm.

Intramammary antibiotic therapy

Mild (Grade I) cases will usually be treated using antibiotic intramammary tubes alone without a veterinary visit. Treatment should be continued to obtain a complete bacteriological cure (up to 5–7 days), although in most cases farmers treat until resolution of clinical signs. Treatment success rates during lactation vary widely according to the pathogen involved, with coliform and Strep. agalactiae having success rates over 90%, whereas treatment of Staph. aureus can have success rates as low as 35%. Age and number of quarters affected can also affect treatment outcomes.

Treatment using both intramammary and parenteral antibiotic therapy is indicated in moderate (Grade II) mastitis cases, as well as in cases of mastitis caused by organisms that can be difficult to treat effectively, such as Staph. aureus and Strep. uberis. Some antibiotic products are licensed for combined intramammary and parenteral therapy, although a number of other parenteral antibiotics can be used ‘off label’ in combination with intramammary antibiotics, provided that statutory milk withhold periods are observed. Tylosin has good reported success rates against Staph. aureus and other gram-positive infections. Penethamate hydriodide injection is useful for Strep. Uberis mastitis. Potentiated sulphonamides, oxytetracycline and framycetin can be used in moderate and severe coliform mastitis cases.

Treatment of severe (Grade III) mastitis. Severe mastitis caused by coliform organisms.

Generalized endotoxaemia results in hypovolaemia, reduced cardiac output and inadequate tissue perfusion. The treatment of choice is initial intravenous infusion of 3 litres of hypertonic (7.2%) sodium chloride over 5–7 minutes, after which the cow will drink up to 40 litres of warm water. A pressure pump device or a 12 gauge intravenous catheter with the infusion bag suspended as high as possible should ensure infusion within 5–7 minutes. Large volumes (16 litres) of non-sterile isotonic (0.9%) intravenous fluids administered using pressure pumps are used by some practitioners, but this treatment has largely been replaced by sterile hypertonic saline (Scott et al., 2011).

Severe mastitis caused by Staph. aureus

Clavulanic acid-potentiated amoxicillin or tylosin are the antibiotics of choice, otherwise supportive therapy as described for coliform mastitis. Removal of the teat once the affected gland becomes cold with blue-black discolouration of the skin in order to facilitate drainage is sometimes performed, but is of little benefit.

Severe mastitis caused by Strep. uberis

Treatment is as for coliform mastitis except that penicillin or penethamate are the antibiotics of choice reduces the severity of clinical signs and hence the number of clinical cases. It has greatest benefit in reducing the fatalities associated with severe coliform mastitis (Scott et al., 2011).

THE LIST OF PRODUCT IS RECOMMENDED BY VEMEDIM TO TREAT MASTITIS IN COW.

   * Click on the name of each product to explore more detail information           

No	Product name	Indication	Image
1	Anti Mastitis	Suspension for intramammary administration.
2	Ceptifi for DC	For Intramammary Infusion in Dry Dairy Cattle
3	CEPTIFI FOR LC	Highly-Effective Treatment of Subclinical Mastitis in Lactating Dairy Cattle
4	Cequin For LC	For intramammary infusion in lacting dairy cattle.
5	Cequin For DC	Intramammary suspension for dry cows.

9. Control and prevention of mastitis

9.1 Control and prevention

Research in the 1960s formed the basis of important mastitis control measures for contagious pathogens, in particular the ‘5/6-point mastitis control plan’:
- Regular milking machine maintenance.
- Post-milking teat disinfection.
- Dry cow therapy.
- Prompt treatment and recording of all clinical cases.
- Culling of chronic mastitis cases.
- Milking parlour hygiene (Scott et al., 2011).

9.2 Vaccination

Vaccinating cows can be deemed as a preventive mastitis treatment in herds. Most vaccines are designed to target Staph. aureus, Strep. agalactiae, and E. coli. Vaccines targeting Staph. aureus and Strep. agalactiae are made up of either the whole organism (inactivated, high encapsulated or unencapsulated cells, and attenuated vaccines) or subunits (toxins, bacterial surface extract, and crude extract of polysaccharides); while for E. coli, the mutant core antigen J5 was used widely. However, vaccines are yet to provide reliable protection. These varying degrees of vaccine efficacy might be associated with varying management practices of different herds.

As mentioned before, mastitis is caused by a number of different bacterial pathogens; therefore, the lack of efficacy of vaccines might also be due to the multi-etiological nature of bovine mastitis. Not only the site of infection in the mammary gland varies among different bacterial strains, but their virulence characteristics and immunogenic capabilities can also be different. Hence, regardless of the type of vaccine, vaccination alone is not effective in preventing mastitis, especially in dairy herds that have high mastitis rates. Vaccination has to be coupled with other control procedures, such as hygienic milking, antibiotic treatment, infected cow culling, and so on, to reduce the incidence and duration of mastitis cases. Indeed, it is necessary to find a vaccine that is able to protect against a wide range of strains since multiple strains can be present within a herd and within an individual cow. It should also be easily implementable in the daily routine and be economically affordable (Cheng and Han, 2020).

10. References

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Bianchi, R.M., Schwertz, C.I., de Cecco, B.S., Panziera, W., De Lorenzo, C., Heck, L.C., Snel, G.G., Lopes, B.C., da Silva, F.S., Pavarini, S.P., 2019. Pathological and microbiological characterization of mastitis in dairy cows. Tropical animal health and production 51, 2057-2066.

Blowey, R., Weaver, A.D., 2011. Color Atlas of diseases and disorders of cattle e-book. Elsevier Health Sciences.

Cheng, W.N., Han, S.G., 2020. Bovine mastitis: risk factors, therapeutic strategies, and alternative treatments—A review. Asian-Australasian Journal of Animal Sciences 33, 1699.

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Derakhshani, H., Fehr, K.B., Sepehri, S., Francoz, D., De Buck, J., Barkema, H.W., Plaizier, J.C., Khafipour, E., 2018. Microbiota of the bovine udder: Contributing factors and potential implications for udder health and mastitis susceptibility. Journal of Dairy Science 101, 10605-10625.

Elhadidy, M., Zahran, E., 2014. Biofilm mediates E nterococcus faecalis adhesion, invasion and survival into bovine mammary epithelial cells. Letters in applied microbiology 58, 248-254.

Fernandes, J.B.C., Zanardo, L.G., Galvão, N.N., Carvalho, I.A., Nero, L.A., Moreira, M.A.S., 2011. Escherichia coli from clinical mastitis: serotypes and virulence factors. Journal of Veterinary Diagnostic Investigation 23, 1146-1152.

Ganguly, S., Shoukat, S., Wani, H., Ali, U., Ali, M., 2018. Chapter-6 Mastitis and Its Diagnosis: A Review.

Gomes, F., Henriques, M., 2016. Control of bovine mastitis: old and recent therapeutic approaches. Current microbiology 72, 377-382.

Hamadani, H., Khan, A., Banday, M., Ashraf, I., Handoo, N., Bashir, A., Hamadani, A., 2013. Bovine mastitis-A disease of serious concern for dairy farmers. Int. J. Livest. Res 3, 42-55.

Hoque, M., Sultana, M., Hossain, A., 2021. Dynamic Changes in Microbiome Composition and Genomic Functional Potentials in Bovine Mastitis. J Data Mining Genomics Proteomics 12, 232.

Kaper, J.B., Nataro, J.P., Mobley, H.L., 2004. Pathogenic escherichia coli. Nature reviews microbiology 2, 123-140.

Khan, M., Khan, A., 2006. Basic facts of mastitis in dairy animals: a review. Pakistan veterinary journal 26, 204.

Kibebew, K., 2017. Bovine mastitis: A review of causes and epidemiological point of view. J Biol Agric Healthc 7, 1-14.

Nicholas, R.A., Fox, L.K., Lysnyansky, I., 2016. Mycoplasma mastitis in cattle: To cull or not to cull. The veterinary journal 216, 142-147.

Pal, M., Regasa, A., Gizaw, F., 2019. Etiology, Pathogenesis, Risk Factors, Diagnosis and Management of Bovine Mastitis: A Comprehensive Review. International Journal 6, 40-55.

Romero, J., Benavides, E., Meza, C., 2018. Assessing financial impacts of subclinical mastitis on Colombian dairy farms. Frontiers in veterinary science 5, 273.

Scott, P., Penny, C.D., Macrae, A., 2011. Cattle medicine. CRC Press.

Simojoki, H., Hyvönen, P., Ferrer, C.P., Taponen, S., Pyörälä, S., 2012. Is the biofilm formation and slime producing ability of coagulase-negative staphylococci associated with the persistence and severity of intramammary infection? Veterinary Microbiology 158, 344-352.

Taponen, S., Pyörälä, S., 2009. Coagulase-negative staphylococci as cause of bovine mastitis—Not so different from Staphylococcus aureus? Veterinary microbiology 134, 29-36.