Mastitis

Erin Royster, DVM, MS ◆ royster@umn.edu

Introduction

Mastitis is one of the most prevalent infectious diseases of dairy cattle. Approximately 25% of cows on US dairies are affected by clinical mastitis each year (NAHMS Dairy Study 2014). As a welfare concern, mastitis causes pain, can lead to death in severe cases, and increases the risk of culling. As an economic concern, mastitis is costly to the producer. Losses due to mastitis, estimated to be between $100-400 per case (Bar et al, 2008; Rollin et al, 2015), include treatment costs, milk discarded due to treatment, decreased production due to subclinical mastitis, and increased culling and death. Although milk from cows with clinical mastitis may not be sold, milk from cows with subclinical mastitis is of poorer quality and can affect the quality of finished milk products. Lastly, the use of antibiotics for treatment or prevention of mastitis accounts for the majority of antibiotics used on dairy farms. (Pol & Ruegg, 2009) As such, mastitis is significant to concerns over antibiotic use in food animals and the potential development of antimicrobial resistance.

Mastitis Overview

Summary

1. Mastitis is inflammation of the mammary gland, caused primarily by bacterial pathogens.
2. Mastitis is significant due to its impact on animal welfare and dairy food quality, economic losses to the dairy producer, and associated antimicrobial use in a food animal species.
3. Clinical mastitis is defined by the presence of abnormal milk.
4. A cow with visibly normal milk, and a SCC greater than 200,000 cells/ml likely has subclinical mastitis.
5. Milk culture is used to diagnose mastitis and identify the causative organism.
6. The udder has defense mechanisms that protect against mastitis. The integrity of the teat end and teat canal help keep pathogens out, and circulating immune cells respond when pathogens gain entry. When these defense mechanisms fail or are overwhelmed, mastitis occurs.

Mastitis pathogens are typically characterized as Contagious/Environmental or Gram-postive/Gram-negative/Other, and these categories provide useful information about infection characteristics and outcomes.

Content

What is mastitis?

Mastitis is inflammation of the mammary gland, almost always due to infection. Most cases of mastitis are caused by bacterial pathogens, although some are caused by non-bacterial organisms like yeast or algae.

How do we know when a cow has mastitis?

Mastitis may be clinical or subclinical. Clinical mastitis is defined by the presence of visibly abnormal milk. Milkers detect clinical mastitis by stripping the foremilk from each teat before attaching the milking unit. Abnormal milk may be watery, thick, bloody, or may have clots or flakes. In clinical cases, the milk is abnormal because of damage to the secretory epithelium caused by the pathogen and the cows’ immune response. The milk has an increased concentration of serum components, inflammatory cells, enzymes and other inflammatory mediators.

A commonly used severity scoring system is used to further categorize clinical mastitis cases and to inform treatment decisions:         

Cows with subclinical mastitis have visibly normal milk, but have an increased Somatic Cell Count (SCC). Somatic cells are white blood cells, primarily neutrophils, that migrate out of the bloodstream into the udder when infection is present. A SCC cutpoint of 200,000 cells/ml is commonly used to indicate whether a cow or quarter is likely to be infected. SCC can be measured or estimated in a variety of ways. Many U.S. dairy farmers utilize monthly or quarterly DHIA (Dairy Herd Improvement Association) testing, where a composite milk sample is collected from every cow and tested for SCC, fat, and protein. There are also cow-side tests that can be used on the farm to estimate the SCC, such as the California Mastitis Test (CMT).

How is mastitis diagnosed?

Milk culture is considered the gold standard test for diagnosis of mastitis. Further microbiological, biochemical or other molecular testing methods may be used to identify the exact species of microorganism causing the infection. Growth of one or two pathogenic microorganisms from an aseptically-collected quarter milk sample is considered a positive test. Growth of three or more species is usually indicative of sample contamination. Between 10 and 30% of milk samples from quarters with clinical mastitis yield no growth on aerobic culture. The majority of these are presumed to be cases where the cow’s immune system cleared the infection before symptoms were detected and the sample collected (“spontaneous cure”). A small percentage of these may be false negatives due to low or intermittent shedding of microorganisms in the milk of an infected quarter, or they could be infections caused by Mycoplasma, which does not grow in aerobic culture conditions.  

Why do cows get mastitis?

Understanding what puts a cow at risk for mastitis is important for prevention. At a very basic level, a cow gets a case of mastitis for two reasons: overwhelming exposure to a pathogen and/or failure of the cow’s immune system. Exposure to mastitis pathogens occurs at the teat end. With the exception of Mycoplasma, which can enter the udder through the bloodstream, the only way the pathogen gains entry to the mammary gland is through the teat canal.

The udder has several defense mechanisms that help prevent entry through the teat canal, and also protect the gland in the event that pathogens do gain entry. First, the teat end is protected by a muscular sphincter that closes when the cow is not milking. The teat canal is lined with keratin, a waxy substance that inhibits migration of microorganisms through the canal. Second, somatic or immune cells are present in the mammary gland even in an uninfected state. These cells, primarily macrophages, circulate and act as surveillance and the first line of defense against invading pathogens. Once a pathogen is detected, these cells help recruit other immune cells to the gland, resulting in an increased SCC.

Anything that impairs or overwhelms these defense mechanisms will result in an increased risk for mastitis. There are three common issues that impact the udder’s defense mechanisms:

1. Damage to the teat end or teat skin, three main mechanisms:

• Hyperkeratosis predisposes the teat end to bacterial colonization
• Congestion or edema in the teat tissue caused by mechanical milking that prevents rapid closure of the teat sphincter after milking
• Infectious or non-infectious teat skin lesions that predispose the teat skin to bacterial colonization.

2. Generalized immunosuppression, which may be subtle to severe. Some common causes of immunosuppression include:

• Transition-related hormonal immunosuppression
• Nutritional deficiencies
• Stress
• Concurrent disease

3. Overwhelming exposure to pathogens, such as:

• Grossly contaminated dairy bedding, which has prolonged contact with teat ends.

• Teat end “impacts” during milking, where milk that may be contaminated with mastitis pathogens travels in a reverse flow and may be rapidly injected through the teat canal and up into the mammary gland. This can occur during milking, when a liner slip allows air into the milk unit, which is under vacuum, creating a pressure differential across the unit.
• Introduction of pathogens into the udder due to unclean infusion technique during treatment

        Source: https://www.youtube.com/watch?v=YwVaE4DBOmQ

Learning Activities

Review the preceding “Mastitis Overview” before class.

Additional Resources

• The National Mastitis Council: https://www.nmconline.org/
• MSU Top MIlk: https://topmilk.msu.edu/

Acknowledgments

References

NAHMS Dairy Study, 2014.

Bar, D., Tauer, L.W., Bennett, G., Gonzalez, R.N., Hertl, J.A., Schukken, Y.H., Schulte,H.F., Welcome, F.L., Grohn, Y.T., 2008. The cost of generic clinical mastitis in dairy cows as estimated by using dynamic programming. J. Dairy Sci. 91,2205–2214.

Rollin, E., Dhuyvetter, K.C., Overton, M.W. 2015. The cost of clinical mastitis in the first 30 days of lactation: An economic modeling tool. Prev. Vet Med. 122, 257-264.

Pol, M. and Ruegg, P.L., 2007. Treatment practices and quantification of antimicrobial drug usage in conventional and organic dairy farms in Wisconsin. J. Dairy Sci. 90(1), 249-261.

Mastitis Pathogens

Content

Mastitis pathogens can be categorized as either contagious or environmental. This is useful because the category tells us the likely source of the infection and thus, what control strategies are likely to be effective. The primary source of contagious pathogens is infected cows. The primary source of environmental pathogens is the environment. To control contagious mastitis, you must identify infected cows and reduce the risk of transmission to other cows (effective treatment, culling or segregation). Cow-to-cow hygiene and biosecurity practices are important. To control environmental mastitis, you must reduce teat end exposure to pathogens in the environment through bedding and barn management and pre-milking teat disinfectant. However, remember that the cow’s immune system is key to fighting off either type of pathogens. And finally, know that this categorization is not totally black and white. Some organisms which may originate in the environment can behave like a contagious pathogen and spread from cow-to-cow. This is a likely occurrence in herds with a significant number of cows chronically infected with “environmental” pathogens (particularly some species of Environmental Streps, Klebsiella, and Prototheca).  

A second useful categorization is by Gram stain or organism type - Gram-positive, Gram-negative, or other (yeast, algae). This categorization gives us some general information about the infection characteristics and likely response to treatment. For example, in general, the common Gram-negative organisms (Coliforms) typically induce a robust host immune response, resulting in clearance of the infection regardless of treatment (spontaneous cure). This robust immune response is responsible for the increased severity of clinical signs associated with coliform mastitis. When the immune response results in a massive die off of Gram-negative cells, the LPS released from the cell wall causes moderate to severe local and systemic inflammation. On the other hand, Gram-positive bacteria, in general, elicit a less robust immune response, and are therefore more likely to result in chronic, subclinical infection and require the assistance of antimicrobial therapy to effect a cure. Of course, these generalizations are not universally true, as some Gram-positive pathogens also release toxins and may cause severe local and systemic inflammation, and some Gram-negative pathogens may become “host-adapted” and exist in a chronic, subclinical state. The “Other” category includes organisms like yeast and algae that are not typical mastitis pathogens, as well as Mycoplasma, a major mastitis pathogen. These organisms have no target for antibacterial therapy. There are many characteristics specific to each individual species, such as virulence factors, preferred site of infection within the udder, and antibiotic resistance, that influence infection characteristics and response to treatment.

For more information about the most common mastitis pathogens, see Mastitis Pathogen Factsheets at https://vdl.umn.edu/laboratories/laboratory-udder-health-luh/luh-factsheets-resources.

Learning Activities

Pre-work Assignment (individual):

1. Download/Copy the following charts and fill in the highlighted sections before class. Submit these to your instructor if indicated in the assignment. The remaining sections may be filled in by the student with the resources provided, or may be provided by the instructor.

Mastitis Bugs

                Mastitis Drugs

2. Make a list of questions that you have about mastitis treatment. Submit these to your instructor before class, if indicated in the assignment.

In Class:

Class time will be a discussion of issues in mastitis treatment, with the goal of students being able to develop a Mastitis Treatment Best Practices document afterwards. The issues to discuss include: pathogen characteristics, selection of antibiotic, duration of therapy, clinical mastitis severity score, systemic and supportive therapy, protocol inclusion criteria, monitoring, etc. Students should be prepared to discuss and ask questions about mastitis therapy.

Optionally or if time allows, students may complete mastitis treatment cases to practice applying treatment best practices.

Post-work Assignment (group):

Following class discussion, create a Mastitis Treatment Best Practices document. Use this Mastitis Treatment Best Practices template with suggested headings as a starting point, if desired. The goal is to have something to refer to in practice, or to use as client education material. The intended audience is dairy producers or managers.

Note, if mastitis-related protocols and SOPs have been assigned, these should be completed, or updated, after this class session to reflect what you learned in class.

Additional Resources for Students

• Websites:

• The National Mastitis Council: https://www.nmconline.org/
• MSU Top Milk: https://topmilk.msu.edu/ (see Resources > Treatment of Mastitis)
• LUH Mastitis Pathogen Factsheets: https://vdl.umn.edu/laboratories/laboratory-udder-health-luh/luh-factsheets-resources.

• Articles/Chapters:

• Treatment of Mastitis in Cattle, Royster & Wagner, Vet Clinics NA, 2015
• Antibiotic Treatment for Bovine Mastitis: Who, what, when, how and why? Pam Ruegg, AABP Proceedings, Sept. 2013
• Decision tree analysis of treatment strategies for mild and moderate cases of clinical mastitis occurring in early lactation, Pinzon & Ruegg, JDS 2011
• Can susceptibility testing help pinpoint treatment? Nydam, Royster, Godden, Gordon, Ruegg,  Hoards Dairyman, Dec. 2018

Instructor Resources

(These may be made available to students after class period or after assignments are submitted. Contact Erin Royster at royster@umn.edu to request access.)

• Completed charts: Mastitis Bugs, Mastitis Drugs
• Mastitis Therapy Discussion Points (guide for class discussion)
• Expert version: Mastitis Best Practices
• Mastitis Treatment Cases
• Examples: Mastitis Treatment Protocols

• Grade 1, 2, 3
• Culture-guided or Blanket treatment

Mastitis Detection, Diagnosis and Screening

Summary

1. Mastitis tests may be classified as detection tests, diagnostic tests, screening tests, or some combination of those, depending on what question is being asked.
2. Detection tests answer, “Which cows are likely infected?” and detect the cow’s response to infection (inflammation), not the infection itself. Detection tests include: forestripping for abnormal milk, SCC, or the presence of other abnormalities in milk (elevated conductivity, inflammatory enzymes, etc.).
3. Somatic cells in milk from an individual cow may be measured as a total count (SCC) or differential count (Milk Leukocyte Differential, MLD), converted to a log score (LS), or estimated by cow-side tests (CMT). Individual cow SCC is used for individual cow management, or summarized in a variety of ways for use in herd-level monitoring (proportion new/clean/chronic/cured, cure risk, etc.).
4. SCC is an imperfect test, emphasizing sensitivity over specificity at a threshold of 200,000 cells/mL. Other thresholds may be used depending on the desired balance of sensitivity and specificity.
5. BTSCC is a herd-level measure of milk quality. While the US regulatory limit for BTSCC is 750,000 cells/mL, most US milk processors enforce the EU standard of 400,000 cells/mL (3-month geometric mean). A common goal for BTSCC is <200,000 cell/mL.
6. Diagnostic tests answer, “What pathogen is causing mastitis?”. The gold standard for mastitis diagnosis is milk culture. Milk culture is not a perfect test, and there are a variety of reasons why a culture result may be a false negative. Sample contamination is a major issue contributing to non-diagnostic results or false positives.
7. Screening tests answer, “Are contagious pathogens present in the herd?” and may be applied at the herd, pen/group, or individual cow level. At the cow level, milk culture can function as screening, detection and diagnostic test. At the herd level, bulk tank culture is the most common and cost-effective way to monitor for contagious pathogens.
8. Different screening strategies may be employed based on the infection status of the herd, what risk factors may be present, cost considerations, and the risk tolerance of the producer.

Content

Introduction

Detection tests identify which cows are likely to be infected, while diagnostic tests identify the pathogen causing infection. The term “diagnostic” is commonly applied to both types of tests, and some diagnostic tests can be used both to detect and diagnose mastitis. Detection tests can never be used to diagnose mastitis, despite some marketing claims to the contrary. A screening test identifies the presence of certain pathogens of concern, and may be applied at the cow, group or herd level.

Detection tests

The detection tests that will be discussed in this section all detect the cow’s response to infection (inflammation). The most common test used to identify cows with clinical mastitis is of course forestripping and examining the milk for signs of mastitis - clots, flakes, pus, watery, bloody, etc. The most common test to identify cows that may be subclinically affected is somatic cell count (SCC).

Somatic Cell Count (SCC)

SCC testing is usually performed as part of monthly or less frequent DHIA testing, where a composite sample from each cow in the herd is tested. A cutoff of 200,000 cells/mL is used to determine likely infection status. However, it is important to understand that SCC is not a perfect predictor of infection and this cutoff of 200,000 cells/mL is a trade-off of higher sensitivity (Se) and lower specificity (Sp).  

This diagram shows the proportion of true and false positives and negatives at two different SCC thresholds. At a threshold of 200,000 cells/mL, there are relatively few false negatives (infected cows who test negative, high Se), but many false positives (low Sp). At a higher threshold of 300,000 cell/mL, there are more false negatives (lower Se), but fewer false positives (higher Sp). From Erskine, R. 2001.

A cow with a composite SCC over 200,000 cells/mL is not always infected, and conversely, a cow with a composite SCC under 200,000 cells/mL is not always uninfected. First, there is the issue of timing. Very early in infection, the SCC in the infected quarter may not be elevated, or it may not be elevated enough to raise the composite SCC (infected quarter SCC is diluted by three healthy quarters). In fact, some infections may never elevate the composite SCC above 200,000 cells/mL, may take a much longer time to do so, or may revert to low SCC after an initially high SCC even when the quarter is still infected. In other cases, the SCC may stay elevated long after the infection is cleared. DIfferent pathogens provoke different levels of immune response, resulting in different SCC patterns. The cow’s ability to or efficiency in mounting an immune response also affects the SCC.

This figure shows how the composite SCC is affected by single quarter infection at different SCC levels (Ruegg, “Milk Quality & Mastitis Tests”). Note that the quarter level SCC must be at least 500,000 cells/mL before the composite SCC reaches the 200,000 cell/mL cutpoint.

This table shows the sensitivity and specificity of SCC as a predictor of infection, and demonstrates how the positive and negative predictive values are affected by herd prevalence of infection (Ruegg, “Milk Quality & Mastitis Tests”). At a threshold of 200,000 cells/mL a sensitivity of 73% means that the test will find 73 out of 100 infected cows, and a specificity of 86% means the test will correctly identify 86 out of 100 unifected cows. In scenario A, with a herd infection prevalence of 40%, a positive predictive value of 77% means that 77 of 100 positive tests will be correct (23 false positives), and a negative predictive value of 82% means that 82 of 100 negative tests will be correct (18 false negatives). In scenario B, with a much lower herd prevalence, the positive predictive value decreases and the negative predictive value increases.

DHIA Testing - milk samples are collected from meters installed for this purpose on test day.

SCC in Use

SCC is used on the dairy to identify individual cows who may need some management action taken to reduce their impact on the herd’s milk quality and production. Cows with a high SCC may have a milk sample collected for culture to identify if a pathogen of concern is causing infection, or they may be segregated to a high SCC pen, designated as a “Do Not Breed” (DNB) cow, or culled. SCC data is also used for herd level monitoring of milk quality and udder health status.

DHIA reports two measures of SCC - a raw count and a log or linear score (LS, or sometimes called Somatic Cell Score (SCS)). The log score is typically used as a summary measure, where the average LS is reported for a group or herd, as the logarithmic transformation reduces the impact of outliers (cows with very high SCC) on the average. The log score is also used to estimate the milk loss associated with the elevated SCC (Jones et al., 1984; Fetrow et al., 1988).

*Milk loss estimated based on lactation average SCC

SCC change from the previous month’s test to the current month’s test is used to categorize cows as new, cured or chronic infections, or uninfected/clean, primarily for monitoring purposes. See chart below for definitions and benchmarks. Similarly, SCC change over the dry period is used to monitor the efficacy of a herd’s dry cow program (dry off treatment, management and housing of dry cows, etc.).

This table shows the sensitivity and specificity of change in SCC from previous to current test to predict new infection (Ruegg, “Milk Quality & Mastitis Tests”). Note the sensitivity is quite low, thus this criteria should not be used, or be used with caution, for cow-level decisions such as treatment or culling. Rather, the proportion of cows in each category (new, clean, cured, chronic) is a useful herd-level monitor.

In addition to being an indicator of infection status at the cow or quarter level, SCC is used as a herd level indicator of milk quality when measured in bulk tank milk. Bulk tank SCC (BTSCC) has an inverse relationship to cheese yield and shelf life of pasteurized milk (Barbano et al, JDS 1991; Klei et al, JDS 1998; Ma et al, JDS 2000). The US regulatory limit for grade A milk is 750,000 cells/mL. However, some states, such as California, Washington and Idaho, have lower SCC limits (400,000 - 600,000 cell/mL). The European Union, New Zealand, Australia and Canada regulatory limit is 400,000 cells/mL based on a 3 month rolling geometric mean. Since 2012, any processors exporting to European markets must comply with this limit. In practice, this means that much of the milk produced in the US is subjected to a 400,000 cell/mL limit. However, the “goal” for BTSCC is much lower than the regulatory limit, with most producers targeting a BTSCC < 200,000 cells/mL. USDA APHIS periodically publishes a summary of BTSCC in the US (https://www.aphis.usda.gov/animal_health/nahms/dairy/downloads/dairy_monitoring/BTSCC_2018infosheet.pdf). In 2018, the US milk-weighted BTSCC was 172,000 cells/mL and 97.5% of milk, 91.4% of shipments and 73.3% of producers were less than 400,000 cells/mL.

SCC Tests

DHIA SCC testing is performed by fluorescent-based flow cytometry (FOSS Fossomatic™) in a laboratory setting (this is the only approved “rapid” method for regulatory testing). There are other available tests that measure SCC, though some have not been sufficiently validated. One that has been validated and is in use in smaller laboratory settings is the Delaval Cell Counter DCC, which uses fluorescent staining and a digital camera to count the cell nuclei present in the sample.

https://www.delaval.com/en-ie/our-solutions/milking/udder-health--hygiene/milk-testing/delaval-cell-counter-dcc/

California Mastitis Test and Other Tests that Estimate SCC

There are a few other tests that are similar to SCC in that they measure or estimate the levels of somatic cells in milk. The most common is the California Mastitis Test (CMT), a rapid, inexpensive, cow-side test that estimates the level of SCC in each quarter. With CMT, milk from each quarter is expressed into a paddle with four wells corresponding to each quarter, a reagent is added which lyses cells and the resulting reaction causes a thickening of the mixture. The degree of thickening corresponds to the number of cells in the milk samples. The scoring of the CMT reaction, shown in the figure below, indicates that any reaction should be considered a positive test (SCC > 200,000 cells/mL). Although CMT is relatively easy, it does take some training and experience to perform it accurately (https://www.youtube.com/watch?v=YRbH_E7JtTU).

The other relatively common test used to estimate SCC on farms is the Portacheck SCC test strip.

https://www.portacheck.com/portascc

Milk Leukocyte Differential (MLD)

In an uninfected mammary gland, somatic cells, particularly macrophages, are present and act as surveillance and first responders to invading pathogens. When an infection occurs, more cells, particularly neutrophils, are recruited to the gland. The difference in proportion of cell types (macrophages, neutrophils, lymphocytes) between an uninfected and infected gland is the basis for this test. Though much effort has been made to identify a predictive algorithm for MLD that is more specific to mastitis compared to SCC, thus far the test performance of MLD has not been proven to be superior to SCC. With the emergence of an on-farm MLD test (QScout, Advanced Animal Diagnostics), there is renewed interest in this test and its potential uses in mastitis management.  

Milk Conductivity, Temperature, and Other In-line Detection Tests

The principle behind milk conductivity is that when a gland is infected, the inflammatory process results in greater capillary permeability and an increase in certain ions in the milk (primarily Na and Cl). The concentration of charged ions changes the electrical conductivity of milk. This concept has been around for a long time (Mas-D-Tec, handheld conductivity meter), but has gained more recent attention and implementation in automated milking systems where a variety of in-line sensors may be used. The conductivity of milk as an absolute measure has poor predictive values, in part because conductivity is affected by more than just mastitis (poor specificity). Differential conductivity, comparing between quarters of the same cow, is better.  More recent implementations utilize an algorithm that includes conductivity along with other factors, like milk temperature and production level, to flag cows as potentially infected (though this author is not aware of published data on the accuracy of this approach).  

Mas-D-Tect hand-held milk conductivity  meter. 

Diagnostic tests

Milk Culture

Milk culture is considered the gold standard test for diagnosis of mastitis and identification of the pathogen causing infection. Milk culture may be performed at several levels, and each level has value and limitations which must be acknowledged:

• Low-level: Typically rely on a combination of selective culture media to categorize culture results, such as Growth/No Growth, Gram-positive/Gram-negative; Rapid, inexpensive, and accuracy limited to broad categories; Performed on-farm, or in vet clinics.
• Mid-level: Utilize some additional microbiological techniques to identify some pathogens at the genus or species level, for example coagulase test for Staph aureus or microscopic wet mount for Prototheca; Performed in vet clinics or other service laboratories.
• High-level/Diagnostic lab: Technicians with advanced training and advanced diagnostic equipment allows accurate pathogen identification to the species level in most cases. Most expensive, may have delayed turnaround compared to low- and mid-level labs, but will be most accurate with a wider range of testing options.

Many diagnostic laboratories, such as the Lab for Udder Health in the UMN Veterinary Diagnostic Lab, utilize a MALDI-ToF (matrix-assisted laser desorption/ionization time-of-flight; a mass spectrometry instrument) for identification of bacteria growth from milk culture. It is important to understand that this is an identification method that follows standard milk culture, as opposed to PCR.

Although considered the gold standard, milk culture is an imperfect test which may result in both false negatives and positives for a variety of reasons. First, the test is only as good as the sample collected. To ensure that any microorganisms in the milk sample came from inside the gland, and not the streak canal, teat skin, collector’s hands, or other environmental sources, the sample must be collected using careful, aseptic technique. Second, many mastitis pathogens exhibit variable shedding in milk (for example, Staph aureus), so at any given time point, there may not be a detectable level of the microorganism in milk even though the gland is infected. Third, if a composite sample is used, where milk from all four quarters is commingled in the same tube, the milk from the infected gland(s) is diluted by milk from health quarters, decreasing the chance of finding the infectious agent. Fourth, if a milk sample is collected too soon following antimicrobial administration, microbial growth in the sample may be inhibited, and this undetectable, but not eliminated. Lastly, some microorganisms may not be identified using standard culture techniques - this is the case for Mycoplasma which requires special culture media and a CO2-rich incubation environment for 7-10 days. Other organisms grow slowly, and thus may be missed if the incubation period is too short.

A significant proportion(10-50%) of milk samples from clinical mastitis cases will yield a “No Growth” culture result. While some of these may be false negatives, for reasons outlined above, the majority of these should be considered true negatives in the absence of evidence to the contrary. In these cases, the cow was able to clear the infection by the time the sample was collected. Remember that clinical mastitis is detected when abnormal milk is present, and the milk abnormality is a result of the immune response to infection. This fact of a significant proportion of “No Growth” culture results is an important justification for the use of rapid culture for diagnosis of clinical mastitis, to identify cows that would not benefit from antibiotic treatment. However, in cases where there is some evidence or worry that a negative result is false, such as in a case that fails to cure/return to normal, or if Mycoplasma may be suspected, further investigation is warranted.  

PCR

In recent years, PCR tests for mastitis pathogens in milk have become commercially available. It is important to understand the advantages and limitations of PCR. In contrast to milk culture which detects living, viable organisms, PCR detects DNA in the milk sample. Thus PCR will yield a much lower proportion of negative results compared to culture. While PCR may logically be assumed to yield a positive result in some of the culture false negative scenarios outlined above (low shedding, growth inhibition), it may also yield a positive result in scenarios that would be considered true negatives on culture, where the cow cleared the infection, but some bacterial DNA is still present in the gland. PCR is also vulnerable to issues with sample contamination (from collection through testing) because it detects (and amplifies) DNA rather than viable organisms. As with culture, only samples collected using careful, aseptic technique should be used for diagnostic testing, despite the fact that some DHIA testing centers offer PCR on meter-collected samples. Another contrast to culture is that PCR only detects DNA for which a primer is included in the test kit, whereas culture will detect any microorganism that grows under the culture conditions. For these reasons, care should be taken when interpreting milk-based PCR test results. In particular, results from PCR testing for environmental mastitis pathogens are difficult to interpret. PCR testing is also more expensive than traditional culture, and is offered in fewer laboratories. However, one benefit of PCR testing is turn-around time. Depending on how often the lab performs PCR testing, results may be available on the same or next day from receipt of samples. This may be a significant advantage over culture, particularly for Mycoplasma detection (7-10 day culture incubation period to declare negative results).

Sample type - quarter or composite

As a diagnostic test, milk culture must be performed at the quarter or cow level. The choice of sample type - quarter or composite - depends on how the results will be used. Quarter samples should be used to diagnose a clinical case of mastitis, and in cases where greater diagnostic sensitivity is desired. Composite samples may be used if the action taken depending on the result will be at the cow level, such as culling. However, 30-50% of composite samples will yield a “Contaminated” result (growth of 3 or more organisms), which is not useful for decision-making as it is impossible to determine which, if any of the organisms came from the udder. Composite samples are typically used for screening/detection, such as screening fresh cows for contagious pathogens, where cost is a significant factor.

Screening Tests

Bulk milk testing

Bulk milk testing, culture or PCR, is a screening method and is used to identify or monitor for the presence of contagious pathogens in bulk tank milk (herd level). Bulk milk culture methods are not necessarily standardized between labs, and different labs will report the presence (+/- quantity) or absence of different pathogens or pathogen groups. Users should understand that if a particular pathogen of interest is not reported, it may be that it was not tested for. Most labs will report and quantify the presence of the Staph aureus, Strep agalactiae, Environmental Streps, Staph species, and Coliforms - and interpretation guidelines exist for these categories (see below). Some labs may additionally report a number of other microorganisms for which no interpretation guidelines exist. Again, Mycoplasma, requiring special culture conditions, may not be included unless specifically requested depending on the lab.  

Bulk tank culture interpretation guidelines from UMN VDL Lab for Udder Health.

Note that the use of bulk milk culture for quantification of environmental bacteria has a completely different purpose than screening for contagious pathogens. The presence of contagious pathogens in bulk milk indicates the presence of infected cows in the herd (with the possible exception of low levels of Staph aureus, which can conceivably contaminate bulk milk from being present on teat skin). The presence of environmental bacteria in bulk milk indicates only that the bulk milk is contaminated with environmental bacteria, which can come from multiple sources including teat skin and dirty milking equipment. While it is true that some environmental bacteria in bulk milk could come from a cow infected with those bacteria, the majority of environmental bacteria in bulk milk is thought to come from environmental contamination, NOT infected cows. Thus, the level of environmental bacteria in bulk milk is used as an indicator of milking time hygiene. Note also that there is not a predictable correlation between the quantity of contagious pathogens in bulk milk and the number of infected cows in the herd - the number of cells per mL depends on how many cows are shedding and at what level, and how many uninfected cows are in the sample.  

String Sampling

In addition to bulk tank milk testing, another type of bulk milk testing is string sampling, where milk from a group or pen of cows is commingled and tested. String sampling may be employed as a way to increase the chances of detecting a contagious pathogen by decreasing the number of cows in a sample (compared to bulk tank milk) and thus decreasing dilution of infected quarters. It also may help narrow down which cows on a dairy may be infected. String sampling is accomplished through installation of a sampling device in the milk line, such as the QualiTru sampling system:

https://qualitru.com/industries/dairy-farms/string-sampling/

Screening Cows

In addition to the bulk tank or string testing, individual cow or quarter milk samples may be used to screen for the presence of contagious pathogens in a herd. In this case, the testing performs both the screening and detection function because when you get a positive result you know which cows are infected. Note some laboratories offer screening cultures for a reduced price, where only the pathogen of interest is identified (typically the contagious pathogens).

Screening Strategies

There are several approaches to screening - bulk milk or individual cows, which cows, how often, which pathogens to screen for, etc. The best strategy for a particular herd will depend on the infection status of the herd, what risk factors may be present, cost considerations, and the risk tolerance of the producer. At minimum, monitoring bulk milk regularly (monthly or at least quarterly) is a low-cost insurance policy, so that when a new contagious mastitis outbreak occurs it is identified quickly. It is a common misconception that a “closed” herd is at no or low risk of a new contagious mastitis outbreak. Risk factors that should be considered when selecting a screening strategy include:

• Contagious pathogens already present in the herd
• Replacement animals purchased, or raised off-site and commingled with animals from other herds
• Introduction of new bulls for breeding
• Animals exposed to other herds at fairs or shows where flies or shared equipment may transmit infections
• Youngstock and adult animals housed together
• High new infection rate or BTSCC
• Herd level changes in teat skin integrity or other challenges to immunity

Herds at moderate or high risk for contagious mastitis, herds in the midst of a contagious mastitis outbreak, or herds that have endemic contagious mastitis must employ a more aggressive screening and detection strategy that includes individual cow cultures. Cows may be selected for screening based on convenience of sampling, likelihood of being infected, or rapidity of identifying infected cows in the face of an outbreak. For example, in an outbreak situation, a small to mid-size herd may choose to test all cows in the herd to quickly identify infected cows. Understand that due to false negatives, testing all the cows in the herd only once will not identify all the infected cows. Additional testing will be needed following a whole herd test. A more common approach, especially in larger herds, would be to select a population of cows to test based on convenience or likelihood of infection. The cows most likely to be infected with a contagious pathogen are cows with clinical mastitis, and it is also convenient to collect a sample at the time the case is detected. The next most convenient time to sample is at freshening, when the cow is being processed (vaccination, bolus administration, colostrum collection, etc.), and it is thought that organisms may be more likely to be shed in milk during the immune suppressed period around freshening. As a screening strategy, if you test cows at freshening, nearly every cow in the herd should be tested yearly. Other approaches may include screening cows with high SCC, or at dry off. Based on the test characteristics of SCC, discussed in the previous section, understand that testing cows based on SCC will likely result in a high proportion of negative tests, depending on the threshold used. Again, herds should employ a combination of the discussed strategies, based on current status and risk factors, and the strategy should change as status and risk factors change, to balance cost and efficiency of detection.

Learning Activities

Pre-work Assignment:

Read the preceded section, “Mastitis Detection, Diagnosis and Screening”.

In Class:

TBD

Post-work Assignment:

TBD

Additional Resources

• The National Mastitis Council: https://www.nmconline.org/
• MSU Top Milk: https://topmilk.msu.edu/

Acknowledgments

References

Barbano, D. M., R. R. Rasmussen, and J. M. Lynch. "Influence of milk somatic cell count and milk age on cheese yield." Journal of Dairy Science 74.2 (1991): 369-388.

Dohoo, I.R. Setting SCC cutpoints for cow and herd interpretation. Pp 10 - 18 in Proc. Natl. Mastitis Coun. 40th Annual Meeting., Feb 11-14, 2001 Reno, NV, 2001.

Erskine, R. J. "Mastitis control in dairy herds. Herd health: food animal production medicine." WB Saunders Co., Philadelphia, PA 397 (2001): 433.

Fetrow, J., K. Anderson, S. Sexton, K. Butcher. 1988. Herd Composite Somatic Cell Counts: Average Linear Score and Weighted Average Somatic Cell Count Score and Milk Production. J. Dairy Sci. 71:257-260.

Jones G. M., R. E. Pearson, G. A. Clabaugh, C. W. Heald. 1984. Relationships between somatic cell counts and milk production. J. Dairy Sci. 67:1823-1831

Klei, Linda, et al. "Effects of milk somatic cell count on cottage cheese yield and quality." Journal of Dairy Science 81.5 (1998): 1205-1213.

Ma, Yinqing, et al. "Effects of somatic cell count on quality and shelf-life of pasteurized fluid milk." Journal of dairy science 83.2 (2000): 264-274.

Ruegg, P. L., & Reinemann, D. J. (2002). Milk quality and mastitis tests. The Bovine Practitioner, 36(1), 41-54. https://doi.org/10.21423/bovine-vol36no1p41-54