Diagnosis and Detection of Mastitis
■ Definitions Technically, mastitis can be defined as any inflammation of the mammary gland (including inflammation caused by injury), but almost all mastitis occurring in dairy cows is caused by pathogenic organisms that are mostly of bacterial origin but can also include yeasts, fungi, and algae.5 Like infections occurring in other tissues, mastitis begins with invasion by a pathogen, followed by a brief latent period and then progression to either subclinical or clinical states or resolution of the infection as a result of the cow's immune response.105 Detection of mastitis is almost never based on identification of the precise moment of infection, but is based on observation of the resulting inflammation.
Therefore recognition of mastitis is dependent on pathogen and cow characteristics that govern the magnitude of the immune response and the intensity and accuracy of the detection methods.Subclinical mastitis is characterized by inflammation that is usually detected by enumeration of inflammatory cells in the milk. By definition, milk obtained from mammary gland quarters of cows experiencing subclinical mastitis appears visually normal but contains an abnormal number of somatic cells, with or without detectable presence of pathogenic bacteria.5 In most instances, detection of subclinical mastitis is based on evaluation of the SCC in an appropriate milk sample. The SCC can be measured using milk collected from individual quarters or composite milk samples that are a mixture of milk from all functional glands of an individual cow. Composite milk samples are routinely used in DHIA programs for monitoring the SCC of individual cows. When composite SCC values are used, some subclinical infections will not be detected because the SCC of the composite milk will be reduced by dilution with milk from healthy quarters that contain few somatic cells.106 When cows are not routinely tested for individual SCCs, veterinarians cannot determine important epidemiologic characteristics of subclinical infections (such as prevalence or incidence) that are necessary for implementing mastitis control programs.107
The distribution of cell types found in bovine milk varies depending on infection status and milk fraction.108 The SCC of healthy quarters is quite low and usually remains well below 100,000 cells∕mL.5 However, based on detection of IMI (microbiologically positive milk samples), a threshold of less than 200,000 cells/mL is usually considered to be the optimal value to define a mammary quarter as healthy.109-112 The selection of an appropriate threshold for defining subclinical mastitis is not fixed but rather is dependent on the goal of the control program.
Use of a lower threshold will identify more animals with subclinical infections (increased sensitivity and fewer false negatives), whereas use of a higher threshold (increased specificity) will result in fewer false positives.112 Regardless of threshold, review of the SCC history of a cow is much more informative as compared to observation of a single monthly value.An SCC greater than 200,000 cells/mL does not always indicate that bacteria will be recovered from milk samples obtained from the affected gland.113,114 The immune response indicated by the influx of leukocytes is often effective in reducing the number of bacterial colonies in milk, and about 10% to 25% of quarters that are above a threshold of 200,000 cells/mL will be apparently bacteriologically nega- tive.109,110,112 As the prevalence of infected quarters has declined, the predictive value of SCC relative to detection of IMI has declined. As the prevalence of pathogens that maintain long periods of subclinical IMI (such as Streptococcus agalactiae and S. aureus) has declined, the proportion of apparent “false negatives” (inability to recover bacteria from quarters that exceed the SCC threshold) has increased.115 These culture-negative samples do not always indicate that the quarter is cured; they may indicate that the immune response has reduced the number of bacteria to below normal laboratory detection limits114 and are essentially indicative of the chronic state of failed inflammation (inability to achieve bacteriologic cure). It is useful to educate clients that about 30% to 50% of milk samples collected from mammary glands with high SCCs will be culture negative, and thus sufficient samples should be collected to ensure meaningful diagnostic test results.
The duration of the subclinical phase of IMIs varies depending on etiology, as the magnitude of the inflammatory response is partially a function of virulence characteristics that vary among bacteria.
In most herds, a greater proportion of subclini- cal mastitis is caused by gram-positive pathogens as compared to gram-negative pathogens (Table 36.1). Usually, gram-negative opportunistic pathogens such as Escherichia coli have a shorter subclinical phase as compared to gram-positive pathogens such as S. aureus or most IMIs caused by Streptococcus spp.116 In general, approximately 25% to 35% of cows experiencing chronic subclinical mastitis caused by gram-positive pathogens may eventually exhibit clinical symptoms.117 For opportunistic environmental pathogens, the likelihood of a clinical episode occurring in a cow with subclinical IMI varies among pathogens. For example, about 50% of IMIs caused by streptococci may progress to clinical signs, whereas more than 80% of coliform infections will exhibit clinical signs.116It is important to diagnose both prevalence and incidence of subclinical infections within a dairy herd. Cows with IMIs that remain subclinical are known to produce considerably less milk, and they maintain a reservoir of pathogens that can 13118 result in increased exposure of otherwise healthy cows.13,118 Subclinical infections that persist for long durations are
■ TABLE 36.1
Results of Selected Studies That Describe the Distribution of Bacteria Recovered from Milk Obtained from Cows With Clinical and Subclinical Mastitis in Modern Dairy Herds Located in Developed Countries
| Country | Herds | Milk Samplesa | Staphylococcus aureus | Other Staphylococci | Streptococcus agalactiae | Other Streptococci | Coliforms | Other | No Growth |
| Milk Samples Collected from Clinical Cases | |||||||||
| Holland275 | 274 | 2737 | 18% | 6% | 0% | 25% | 28% | NRb | 22% |
| UK142 | 90 | 480 | 3% | 13% | 0% | 25% | 21% | 11% | 27% |
| New Zealand250 | 28 | 1332 | 19% | 7% | 0% | 45% | NR | 4% | 27% |
| Canada143 | 106 | 2850 | 11% | 6% | 0% | 16% | 15% | 5% | 47% |
| USA6 | 51 | 706 | 3% | 6% | 0% | 14% | 35% | 13% | 29% |
| China572 | 161 | 3283 | 10% | 11% | 3% | 21% | 33% | 5% | 16% |
| Milk Samples Collected from Cows With Visibly Normal Milk | |||||||||
| Finland210 | 216 | 12,661c | 3% | 17% | hurdle, a number of indirect measures of SCC have been developed. The California Mastitis Test (CMT) is an example of an indirect test that has been used for more than 50 years to help guide mastitis control programs.124,125 The CMT was developed to test milk from individual quarters but is also useful for composite milk and samples obtained from bulk tanks. The CMT reagent is a mixture of a detergent and bromocresol purple, which is used as an indicator of pH. The degree of reaction between the detergent and the DNA of cell nuclei is a measure of the number of somatic cells in milk. The CMT is generally read using a five-point scale that begins with “negative” (SCC range of 0 to 200,000 cells/mL) and “trace” scores (SCC range of 150,000 to 500,000 cells/mL) and ends with a maximum score of 3 (SCC >5,000,000 cells/mL). The CMT was developed during the period when many cows had very high SCCs as a result of chronic subclinical infections caused by S. agalactiae and S. aureus. Through the adoption of standardized best management practices, the prevalence of these pathogens has decreased,115 and SCC values of IMI caused by minor pathogens are usually not as high.126 While the CMT continues to have considerable utility, it is important to note that even “trace” scores indicate the presence of subclinical mastitis and the test may be difficult to read when used on milk obtained from cows in the first few days of lactation.127 In addition to an increased SCC, a number of changes in milk composition occur as a result of inflammation that is part of the immune response to infection.45,128 Mild inflammatory changes can also be detected in apparently normal mammary gland quarters adjacent to quarters that are experiencing clinical mastitis.45 Compositional changes include reduced casein, lactose, and α-lactalbumin concentrations; an influx of sodium, chloride, and plasma proteins; increased proteolytic and lipolytic activity; an increase in lactoferrin concentration and enzyme activities; and a rise in pH.129 Many of these changes can be used as indicators of subclinical mastitis. Among the more common milk tests are those that detect albumin, sodium, chloride, lactose, or concentration of adenosine triphosphate; W-acetyl-β-D-glucosaminidase (NAGase) activity; antitrypsin activity; or pH. Plasma tests detect α-lactalbumin, casein, or lactose concentration.130-133 Milk concentrations of acute phase proteins amyloid A and haptoglobin are able to distinguish healthy quarters from those with subclinical mastitis.134 Although a variety of immunologic mediators are modified by IMI, measurement of SCC remains the most accurate method of identifying subclincally infected quarters.132 With increases in diagnostic technology and increased emphasis on production of high-quality milk, considerable research has focused on producing tools for continuous in-line monitoring of milk quality, and these tools will increasingly be used in future dairy herds.135 Increased use of automatic milking systems (robotic milking) has led to the search for automated methods that can consistently and accurately detect abnormal milk, but challenges remain to identify automated systems that are accurate throughout the lactation period.136 While current testing technology generally gives accurate results, the ultimate value of any test depends on the value of the intervention that is chosen based on the test result, thus veterinary practitioners should continuously review how such tests can be best used on dairy farms.Clinical mastitis is usually defined as production of visually abnormal milk, with or without secondary clinical signs. Detection of clinical mastitis is based on observation of foremilk prior to attaching the milking unit, but the working definition of clinical mastitis varies greatly among farm workers. While the majority of workers on larger farms routinely remove foremilk, about one third of farms containing fewer than 500 cows do not routinely remove or observe foremilk of all cows, and in these instances detection of clinical mastitis is limited to cases with more severe signs.21,137 To evaluate incidence of clinical mastitis, veterinarians should ask milking technicians to describe their definition and detection methods. Clinical signs of mastitis result from inflammation and may include abnormal appearance of milk (presence of clots or serum); swelling, redness, or edema of one or more quarters; and severe systemic signs such as anorexia, fever, or agalactia. As compared to subclinical mastitis, a much greater proportion of clinical cases are caused by gram-negative pathogens (Table 36.2). Like other bacterial diseases, many cases of mastitis occur as a syndrome with the infected quarters alternating between a clinical state and a subclinical state.138 The disappearance of clinical signs indicates that inflammation has decreased and does not usually coincide with bacteriologic cure.139 The ability to achieve a bacteriologic cure varies greatly among etiologic agents and is associated with variation in immune response among cows and the appropriateness of the therapeutic plan.140Recording standardized severity scores can help veterinarians better define the pattern of clinical mastitis on individual farms.139,140 One severity scoring system uses a three-point scale that combines the appearance of milk with the progression to additional clinical signs. A score of 1 (mild mastitis) = abnormal milk only; 2 (moderate mastitis) = abnormal milk and abnormal udder; 3 (severe mastitis) = systemic symptoms.140 This system is practical and simply recorded and can be an important method to assess detection intensity. In most herds, distribution of severity of clinical mastitis is approximately 40% to 50% mild cases, 40% to 50% moderate cases, and 5% to 15% severe cases.6,139 Veterinarians are rarely asked to treat mild and moderate cases of clinical mastitis and therefore may not understand the true incidence of clinical mastitis on many dairy farms.141 Veterinarians need to promote the use of clinical mastitis records that allow them to monitor the occurrence of mild and moderate cases and to evaluate outcomes of mastitis treatments.
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