Introduction

The dairy industry is the third largest rural industry in Australia that generates AU$ 4.7 billion in annual income. While the number of dairy herds in Australia is decreasing, the herd size and level of intensification is increasing. This trend ensures that mastitis remains the most economically significant disease in the dairy industry.

It leads to:

• loss in milk production
• increase in discarded or downgraded milk
• early culling of cows with the need to replace them sooner than expected
• treatment expenses
• reduced breeding performance.

In the non-dairy mammal, poor milk production and refusal to allow suckling leads to retarded growth and even deaths in suckling animals.

Please review notes or textbook chapters on the anatomy of the udder and the physiology of milk production (TV2102). I have included a picture of a section through half of an udder of a cow to assist you in that review. When asking questions on mastitis, I consider this as assumed knowledge and some of the questions I ask on mastitis and its predisposing factors may in part require a basic understanding of normal structure and function of the udder/mammary glands.

For general information available to dairy farmers refer to the Mastitis and Milk Quality webpage managed by Dairy Australia.

Mastitis and its importance to the dairy industry

Mastitis is defined as an inflammation of the mammary gland usually in response to a bacterial infection. Other infectious causes include fungal, algal or viral.

It can manifest as:

• Clinical: peracute, acute, subacute, chronic. (These pathological terms refer to the type of inflammation present – refer to your general pathology notes on inflammation to understand these terms and the clinical picture and type of lesion associated with them)
• Subclinical: No clinical signs. Only detectable by increased somatic cell count (& change in milk composition)- most common. There will also be a drop in milk production.

Impacts of mastitis

In recent years, with the increase in mastitis control programmes, the incidence of clinical mastitis has decreased and is more common in the first 60 days of lactation in older cows. However, the incidence of subclinical mastitis, where there is a raised leukocyte and bacterial count, some composition change in the milk and reduced milk production, is higher, affecting between 10-15% of cows.

Industry impacts:

• about 9 billion litres (AU$ 4.7 billion) of milk are produced per annum (1-2% annual growth rate) of which 45% are exported = 11% of the world market
• Queensland, the 2nd lowest producer at 485 million litres, has the smallest herds and the lowest milk production/cow
• about AU$ 40 million (about 8% of gross) income is lost due to mastitis.

In calves:

• Mastitis is also very painful and many mothers will not allow their young to suckle.
• Mastitis milk fed to bobby calves can result in diarrhoea caused by antibiotic residues or pathogenic bacteria. These bacteria may be antibiotic resistant if the cow was treated with antibiotics.

Public health:

The presence of unwanted bacteria in the milk affects public health, as some bacteria and their toxins can cause disease in humans and some changes to the milk’s appearance and composition affect milk processing and palatability. For example, about 50% of S. aureus in milk produce enterotoxins that cause diarrhoea and vomiting in people.

Below is a list of bacteria that can be found in milk that can cause disease in people if they ingest the milk:

• Staphylococcus aureus
• Escherichia coli 0157 (Shigatoxin) = enterohaemorrhagic pathology type
• Listeria monocytogenes
• Coxiella burnetii (heat-resistant: requires 71.7°C for 15 seconds)
• Yersinia enterocolitica
• Campylobacter jejuni, C. coli
• Salmonella – all serovars
• Cryptosporidium parvum
• Mycobacterium tuberculosis var. bovis, M. tuberculosis van tuberculosis (not Australia)
• Brucella (not Australia).

Should we drink raw milk? All milk must be pasteurised in Australia

Exceptions are goat milk where the sale of raw goat milk is permitted in NSW, WA, SA and Qld. Some farms sell raw cows’ milk for bathing which is legal. In the USA 60% of foodborne disease outbreaks are associated with the drinking or eating of raw milk or milk products i.e. soft cheeses. Bacteria responsible for these outbreaks include (highest to lowest prevalence): Campylobacter  (54%); Salmonella (22%), enterohaemorrhagic E. coli (13%), Brucella (4 %), Listeria (4 %), and Shigella (3%).

Is it worth it, to drink raw milk? Heat treatment of milk targets the most heat-resistant pathogen = Coxiella burnetii.  Heating changes milk taste but not its nutritional value.

Pathogenesis of mastitis

Bacteria usually infect the udder via the teat canal. The teat, an invagination of the skin, is lined with squamous epithelium. During milking the teat lining is constantly being sloughed off and replaced. This allows bacteria trapped in the cells to be flushed out during milking. In the dry period, the sloughed off epithelium forms a keratin plug, which prevents access to the udder by bacteria. During the dry period the udder becomes very susceptible to infection with this keratin plug being the single most important protective factor. Zinc deficiencies are known to negatively impact on this barrier.

The narrow streak canal is surrounded by a thick muscle layer known as the teat sphincter and the musculoelastic layer, known as the Rosette of Furstenberg surrounding the teat cistern, serves to exclude bacteria from the teat canal.

During milking the teat can evaginate allowing bacteria in or bacteria can be propelled into the teat cistern by vacuum fluctuations of the milking machine at the teat end. Furthermore too prolonged a vacuum can cause congestion and oedema within the teat canal. Once bacteria have breached the teat defenses as little as 100 bacterial cells/mL can cause mastitis.

Lactoferrin produced in the milk binds iron making it less available to bacteria. It also binds to LPS (gram-negative bacterium) causing peroxide formation and damage to the bacterial cell membrane. Lactoferrin also protects cell membranes from viral attack and will oxidise RNA.  However, bacteria in high numbers can overcome the effects of lactoferrin.  IgG2 present on phagocytic cells in the teat cistern can bind to and opsonise S. aureus.  The milk within the gland cistern will allow bacteria to divide and produce toxins and enzymes. Some bacteria such as E. coli are able to adhere to and invade mammary epithelial cells. In this way they evade phagocytic cells. S. aureus is able to survive intracytoplasmically in neutrophils.

Mammary cells become damaged both by inflammation and by bacterial toxin- or enzyme-induced cell necrosis. This will lead to epithelial cell damage in the alveoli of the udder with loss in integrity of the blood-milk barrier. The udder will then appear inflamed (swollen, red and painful) and the milk abnormal (watery, grey or pink to red, clots present). When secretory cells are damaged, they are often replaced by non-secretory cells and consequently decreased milk production. The damaged epithelial cells and neutrophils will be present in the milk together with the bacteria and can occlude the milk ducts further reducing milk production. The body will wall off the inflamed areas with fibroblasts and fibrin leading to abscess formation.

What factors predispose to mastitis?

Any activity that leads to teat damage and the loss of its ability to act as a barrier will allow the entry, colonisation and invasion of bacteria and other pathogens in the udder.

1. Udder anatomy and physiology: The udder becomes distended as the milk accumulates placing internal pressure on the teat canal. Milk is a high quality nutritive source not only for mammals but also for microbes. Milk stagnant in the teat canal will allow bacteria present to overgrow.
2. Stage of lactation: Udder oedema occurring, especially in heifers, during early lactation predisposes to coliform infection. Milking of cows during the dry period when the udder defenses are poor will result in mastitis.
3. Milkers: Milkers can transfer pathogens on their hands between cows and can harbour pathogens in their throats. People with respiratory tract infections can cough and transfer pathogens to their hands. This is often how S. aureus and tuberculosis is transmitted to cows.
4. Milking equipment: These should be well maintained and fully functional with good “in-between” cow cleaning regimens, otherwise they act as a fomite for pathogenic bacteria. Cracked teat liners allow bacterial colonisation and incorrect pulsation pressures can cause excessive eversion of the teat canal during milking.
5. Environment: Environmental pathogens such as the coliforms and Streptococcus uberis can build up in muddy and faecally contaminated environments. They also splash on the udder increasing teat contact with bacteria. Wet wood shavings and straw can lead to fungal, Klebsiella (wood shavings) and S. uberis (straw) overgrowth.
6. Teat damage by injury or viruses (i.e. Exotic diseases – Lumpy skin disease and FMD), ectoparasites (ticks)  will allow the entry of pathogens.

 causes of mastitis and transmission

Infection of the mammary gland can be either via the haematogenous route or the more common ascending route via the teat canal. Mechanical trauma, thermal trauma and chemical insult predispose the udder to intramammary infection.

The more common bacterial, fungal and algal causes are discussed below and shown in the image below.

Agents that spread from the body to the udder are of interest as they enter the udder either via the bloodstream (haematogenous) or through the lymphatics. Bacteria such as Mycobacterium tuberculosis var. bovis and Brucella abortus are often trapped in the supramammary lymph nodes and if allowed to will spread to the udder from there. Note that M. tuberculosis var. bovis and Brucella abortus are notifiable diseases in Australia and both are considered to be absent. Leptospirosis is a common disease in Australia causing primarily abortions. However, the bacterium can also a reduction in milk production with a “flabby” udder and abnormal colostrum-like milk. Listeria and Coxiella enter the milk but rarely cause mastitis. They both can infect people and thus are of public health significance.

Bacteria entering via the teat canal that spread within the herd are described as contagious.  The most common bacterium in this group is Staphylococcus aureus. Bacteria originating from animal faeces and the environment such as Escherichia coli and Streptococcus uberis usually affect single animals. They are described as being environmental. Streptococcus dysgalactiae subsp. dysgalactiae shows both a contagious and environmental means of spread. Some bacteria such as Pseudomonas aeruginosa and fungi like the yeast Candida species only cause disease after a cow has been repeatedly treated with antibiotics for mastitis. Trueperella pyogenes and Corynebacterium pseudotuberculosis, common causes of abscesses in livestock can result in chronic mastitis. Antibiotics are not effective after abscesses have developed within the udder.

Below is a table showing the relative importance of the different causes of mastitis in SE Australia.^[1] The bacterial distribution may be different in Queensland, but it does indicate that the 4 most common bacteria are Streptococcus uberis, Staphylococcus aureus, Escherichia coli and Streptococcus dysgalactiae.

Some of the more common bacterial causes of mastitis are discussed below.

Staphylococcus aureus

This gram positive, catalase positive, beta-haemolytic coccus that clumps when it divides, can cause any clinical form of mastitis. It is also the most common cause of sub-clinical mastitis in Australia. It predominantly infects heifers and can cause persistent infections. Infected heifers are often responsible for in-herd spread of S. aureus. Being contagious, it is rapidly spread between animals via milkers’ hands and equipment. Feeding infected colostrum to heifer replacement calves is also a potential source of the bacterium (not proven). Good dairy hygiene, segregating and milking infected cows last, and dry cow therapy are important in controlling staphylococcal mastitis. Cows with persistent, chronic infections will not respond well to dry cow therapy and should be culled.

Streptococcus uberis

Streptococcus uberis is a non-groupable, non-haemolytic or alpha-haemolytic streptococcus that is commensal in the intestinal tract of cattle. It contaminates pastures and bedding such as straw for up to 3 weeks. Infections are more common in intensively-reared herds where there has been damage to the teat. The use of feed and calving pads increases the risk. Higher-yielding cows may be more at risk. Infection is more common in the drying off period and just before or after calving. Other than being a cause of sub-clinical mastitis, it causes sub-acute to chronic clinical mastitis that can become persistent. Intramammary treatment during lactation decreases clinical disease but does not cure it. The disease is prevented by the use of dry cow therapy and teat sealants at drying off.

Streptococcus dysgalactiae subsp. dysgalactiae

Bovine-associated strains of Streptococcus dysgalactiae subsp. dysgalactiae are non- or alpha-haemolytic on blood agar and have Lancefield Group C antigens in the outer membrane. They are carried on and in the teats of cows and are usually considered to be an environmental pathogen. However, in some outbreaks of mastitis they can spread rapidly between cows acting as a contagious pathogen. In Europe cattle flies (Hydrotaea irritans) may act as mechanical vectors of this bacterium. They cause both clinical and subclinical mastitis. Clinical mastitis is usually acute to sub-acute. S. dysgalactiae subsp. dysgalactiae can persist in the infected udder.

Corynebacterium bovis

Corynebacterium bovis is a commensal of the teats and teat canals. It is a rare cause of clinical mastitis, but can cause mild to moderate elevation in somatic cells within the milk. Since it can be spread from cow to cow via the milking, high numbers of this bacterium are found on farms that don’t have good postmilking teat dipping practices and dry cow therapy. Often C. bovis is isolated after 2 days of incubation on blood agar as a milk contaminant rather than a cause of mastitis.

Bacterial causes of chronic mastitis

Unresolved acute and sub-acute mastitis can lead to chronic mastitis i.e. that cause by S. aureus. However, some bacterial and fungal agents, like Trueperella pyogenes and Actinomyces tend to result in the development of abscesses and granulomas. Lesions that they can cause is shown in the pictures below. Click on each picture to see the cause.

Peracute mastitis (blue bag) in ewes

Peracute mastitis or “blue bag” in sheep is caused by either S. aureus or Mannhemia haemolytica (gram-negative coccobacillus of the Pasteurellaceae family). This infection occurs shortly after partus (birth) or at weaning. Lambs and kids can spread the infection between mothers as they carry the bacteria in their oral cavities and will search for milk from another lactating female if their mother prevents feeding due to the severe pain mastitis causes.

Infectious mastitis in sows – part of the PPDS

Postpartum dysgalactia syndrome (PPDS) in sows is common (refer to notes on pig diseases) and has a multitude of both non-infectious and infectious causes. Sows, especially multiparous (many litters) sows if kept in faeces-rich environments may develop a mastitis or metritis in late gestation (pregnancy) (from day 111) due to coliform bacteria, mainly Escherichia coli or Klebsiella species. Endotoxin from these bacteria stimulate macrophages to release inflammatory cytokines which decrease the production of prolactin (also thyroid hormone) and hence colostrum and milk production. This means that even unaffected mammary glands will reduce milk production. (Read TV2102 notes on endotoxaemia to understand the pathogenesis of this disease better). Suckling pigs from these sows are smaller, are restless and cry a lot. Some may die. The sow will also try to lie down on her mammary glands to cool them and avoid suckling due to pain. She pants a lot and is restless.

Note that sows can also develop mastitis due to teat damage. In this case, a number of bacteria can cause infections including,  Staphylococcus aureus, Streptococcus species, Trueperella pyogenes and coliforms.

Diagnosis of mastitis

Examination of the affected animals

Clinical mastitis is evidenced by changes in the udder and the presence of abnormal milk. It can be divided into peracute, acute, subacute and chronic mastitis. Animals suffering from peracute mastitis often have a high fever, cannot get up and the udder takes on a purple discolouration. Milk is more serum-like or contains blood. Due to the presence of fibrin, the milk may clot in the presence of air and tends to form clumps. Peracute mastitis is often caused by coliform bacteria i.e. Escherichia coli and Klebsiella (gram-negative coccobacilli), and Staphylococcus aureus. Animals that have a coliform mastitis also have evidence of endotoxaemia. In ewes, Mannheimia haemolytica and S. aureus are more common causes of “blue bag”.

Acute mastitis is evidenced by fever, a reddened and swollen mammary gland that is warmer than the healthy mammary glands. Milk is abnormal, often yellow or red in colour. Several bacterial species can cause this form of clinical mastitis including S. aureus and S. dysgalactiae subsp. dysgalactiae. In subacute mastitis there are no systemic signs of disease.

Chronic mastitis is mastitis that is more than 100 days in duration and is evidenced by fibrotic changes within the mammary tissue. Abscesses may be present. Large abscesses in the mammary gland of cows are often caused by Trueperella pyogenes.

It is important to palpate the udder or mammary glands for heat, swellings and lumps.

Examination of the milk

1. Once one suspects mastitis either from the clinical picture or from a drop in milk production, milk samples should be collected into strip cups to determine whether it is abnormal. Examine for clots, abnormal colour, consistency and smell.

Some examples of abnormal milk detected in strip cups are shown in the pictures below. Click on each picture to see a description.

2. Some milking machines and robotic milkers have in-line conductivity tests that detect an increase in conductivity of milk due to increased sodium and chloride ions and decreased potassium ions and lactose. Rapid tests that detect the enzyme N-acetyl-β-D-glucosaminidase (NAGase) or lactate dehydrogenase (LDH). They are markers for mammary cell damage as they are released by the lysosomes of mammary cells when the cells are damaged. These tests can only be used as screening tests and should be followed up by more accurate tests.

The pictures below show a few of these tests. Click on each picture to see a description of each test.

3. Recently a number of tests using sensors have been researched, many are still in the development phase. Immunoassays to detect increased albumin that indicates increased damage to secretory cells within the mammary gland.

Tests based on somatic cell counts

Somatic cell counts are done on milk either at the individual cow level or on bulk tank milk. They detect sloughed epithelial or secretory udder cells as well as leucocytes in the milk and high numbers are an indicator of mastitis. Cell counts can be based on direct or indirect methods.

The rapid (Californian) mastitis test (RMT) screens for high DNA content in individual cow milk samples. The DNA originates from lysed sloughed epithelial, secretory and inflammatory cells present in mastitic milk. The test uses ethanol to precipitate the DNA which causes gelling of the milk. The more viscous the milk gel, the greater the number of cells in the milk. A YouTube video [2:44], Using a Rapid Mastitis Test, shows how the test is done.

Direct somatic cell counts on milk either using 1) microscopy (direct method) or using 2) an automated cell counter such as the Fossomatic (gold standard) or Deval (DNA is stained with ethidium bromide or propidium iodide which fluoresces). Handheld impedance cell counters such as “Scepter™ 2.0 Cell” are commercially available.

Bulk tank somatic cell counts

The automated methods are usually carried out on bulk tank samples. Bulk milk tank counts (BMCC) are indicative of the presence of subclinical mastitis within a herd. If the somatic cells count (SCC) in bulk milk is greater than 4 x 10⁵/ml the milk is discarded. An interpretative table is below (it is also in the Practical Manual). These limits are present in the Food Standards Manual of Australia and New Zealand. Thus they are enforced by State Food Safety officers and monitored by the milk processors.

To see how to collect bulk milk tank samples, look at this YouTube video [3:44], Do you know how to collect a bulk tank milk sample? You will not be examined on this.

Individual cow somatic cell counts

At the individual cow level, direct microscopy using a haemocytometer, strip count or automated cell impedance/flow cytometry methods can be used. Individual cow milk with a count of less than 150 000 cells/mL is generally normal and a count of 250 000 cells/mL or more during lactation is indicative of mastitis. Note that SCC should be avoided within the first 20 days of lactation and in late lactation due to the naturally high numbers of cells during these periods.

You will be carrying out a microscope strip count in the mastitis practical.

When to culture the milk for bacteria or fungi

Bacterial cultures and identification of milk collected aseptically from each individual quarter (cows)/half (sheep and goats) is used where the SCC is >2x 10⁵/mL milk. You will have the opportunity to see and interpret growth of different mastitis pathogens on media used by a milk laboratory (either in a dairy practice, a dairy or commercial laboratory).

Antibiotic susceptibility testing is carried out on positive cultures.  Treatment outcome is poor with methicillin-resistant strains of S. aureus. These strains often originate from humans.

The type of bacteria present and its antibiotic susceptibility assist you in the control of mastitis at an individual cow and herd level.

mastitis control

There are several strategies for the control of mastitis

The Countdown farm guidelines for mastitis control produced by Dairy Australia is a useful reference.

All cows with mastitis should be milked last and the milk of clinical and or treated cases be discarded.

Acute clinical mastitis

One of the ways to treat acute clinical mastitis is to use pathogen-based treatment. This requires early mastitis detection and pathogen identification in a milk sample from the affected mammary gland. To limit mammary tissue damage, antibiotic treatments are administered ASAP, preferably within 24 hours of mastitis being identified in the cow. Making speedy detection of pathogens and their antibiotic susceptibility critical.  Note for legal and public health reasons drug withdrawal times must be adhered to (see the APVMA PubCRIS database). This results in a large amount of milk being discarded. Intramuscular and intramammary preparations are equally effective against streptococcal (S. uberis, S. agalactiae, S. dysgalactiae) infections, but should be administered at least 6 times. Generally acute infections with gram-negative pathogens will self-cure so antibiotics are not usually recommended for them. In severe or peracute mastitis use an antibiotic that is known to be effective against coliforms such as E. coli. Intramuscular or intravenous therapy is preferred as the bacteria often enter the bloodstream. Furthermore, therapy directed against endotoxin is also required. Intramuscular antibiotics that have proven to be effective are tetracyclines and cefquinome (4th generation cephalosporin). Despite the poor distribution of ceftiofur to the udder as a result of protein binding, it is considered effective for the treatment of coliform mastitis. In the case of staphylococcal mastitis affected cows should be segregated and treated by both mammary and systemic therapy. Note that these animals may not fully recover.

Subclinical mastitis

Cases of subclinical mastitis are usually treated at drying off with a dry cow therapy. Dry cow therapy. Use a longer acting intramammary antibiotic on all quarters of all cows at the end of the lactation to eliminate persistent infections. Dry cow therapy has the advantage that the antibiotic will be retained in the udder for longer and there is a longer risk of antibiotic contamination of the milk. This is where the highest volumes of antibiotics are used on a dairy farm. One of the ways to reduce this is to identify low risk cows that don’t require dry cow therapy. These would have one of less mastitis episode during lactation and have 3 consecutive low ISCC prior to drying off.

The antibiotics used to treat mastitis in cows is shown in the Table below.

Clinical chronic mastitis

The cure rate of chronically affected mammary glands is poor. Cows that have had 3 or more clinical episodes in a lactation or where the somatic cell count of their milk is above 250 000 cells/mℓ in two consecutive lactations are considered to have a persistent infection, therefore they should be culled.  For valuable or high-producing cows, agalactia in a single affected quarter can be induced by the infusion of 60 mℓ of 2.0% chlorhexidine-diacetate twice.

Prevention

Link to  Dairy Australia podcast on prevention of mastitis on dairy farms

1. Keep the cow’s environment and udder as clean and dry as possible. Calving should occur in dry, clean paddocks. Milk as soon as possible after calving to avoid over-distention of the udder.
2. Detect mastitis early (individual teat foremilk visually checked in strip cups during the first month of lactation) and treat bacterial positive quarters with antibiotics
3. Keep mastitis cases separated and milk them last. Even use a dedicated cluster for them. Milk from these cows should be discarded.
4. Dip all teats of all cows with an effective and registered disinfectant after every milking to reduce exposure while the teat recovers from the effects of milking. (Protects the teats that can remain everted for up to an hour after milking)
5. Check for damaged or cracked teats. 10% glycerine can be mixed in the teat dips to condition the teats.
6. Ensure, by at least annual testing, that the milking machines are operating properly. (reservoirs of contagious pathogens; damaged/worn teat cup liners become contaminated and overmilking increases the time of teat recovery after milking)
7. Dry cow therapy and teat sealants at drying off.
8. There are currently no effective mastitis vaccines registered in Australia. An effective vaccine against S. aureus has proven to be elusive, but research into these vaccines is still ongoing.  (Commercial J-5 E. coli vaccine in the USA). Other control strategies that are used include the use of probiotics such as Lactococcus lactis and Lactobacillus perolens. Bacteriocins such as nisin and lacticin NK34 are used in teat dips. Bacteriophage therapy has been unsuccessful as milk whey proteins that bind to S. aureus prevent the binding of bacteriophage.  Breeding programmes for mastitis resistance are also ongoing.

END OF CHAPTER