The importance of accurate mastitis diagnostics – unique profile for each farm

Mastitis is the most common and costly disease affecting dairy cows worldwide¹. Mastitis is defined as the inflammation of the mammary parenchyma, a nature’s own protection reaction against irritation from any source. Mastitis is most commonly caused by intramammary infections (IMI), resulting from bacterial pathogens entering the udder.Mastitis is most commonly caused by intramammary infections (IMI), resulting from bacterial pathogens entering the udder. Mastitis can significantly reduce milk production, lower milk quality, and increase the likelihood of culling infected animals leading to marked overall costs in addition to medicines and veterinary costs ^1–3. Estimations suggest that milk drop occurs 14 to 4 d before mastitis onset and can last until 15 to 25 d from the diagnosis ⁴. The presence of pathogens in the udder resulting poor milk quality, may lead to penalties for farmers and impact the overall profitability of dairy farms. The introduction of PCR technology has revolutionized the field of veterinary diagnostics and gained increasingly acceptance and ground in mastitis diagnostics. High sensitivity and capability of detecting DNA makes PCR useful in detecting infections caused by pathogens that may not grow well in standard culture conditions, but have raised questions over the years of how it compares to conventional culture, is it too sensitive and how should the results be interpreted in practice?

Udder health management and mastitis prevention depend on several cow, management and herd related factors, which are unique to each farm. Mastitis can be addressed at herd level and cow level. As mastitis is a production disease, it is not possible to get rid of if completely, but great results have been witnessed around the world, despite the size of the farm. Effective control of mastitis on farms needs planning, strategy, monitoring, and leadership, as any problem in any company. Most common   control measures in addition to management routines are typically ventilation, feeding, milking practices, transition period and and an increased focus on the immunity of cows.

Effective control of mastitis depends on early and accurate identification of the causative agents, which enables targeted treatment, reduces the spread of infection, and minimizes unnecessary antimicrobial use. Data of causative pathogens on farms should be saved electronically. With adequate data from a longer period of time, farms are capable of defining their bacterial profile (contagious, mixed or environmental) and address the possible problem accordingly with the help of a herd health veterinarian.  

Finding mastitis and the causative pathogen

Somatic cell count (SCC) is one of the most widely used and reliable indicators of mastitis⁵. SCC measures the number of macrophages, neutrophils, and lymphocytes (white blood cells), and mammary epithelial cells present in the milk, which increase in response to an infection in the udder. An elevated SCC typically indicates an underlying infection, but it does not provide specific information about the causative pathogen. To obtain a definitive diagnosis, bacterial culture has been the gold standard for identifying mastitis-causing pathogens from aseptically taken milk samples from the suspected mastitis quarters. However, traditional culture methods have limitations, including slow turnaround times, reduced sensitivity, subjectivity and difficulties in detecting certain organisms requiring special time or growth environment.

The introduction of PCR technology has revolutionized the field of veterinary diagnostics and gained increasingly acceptance and ground in mastitis diagnostics. PCR is a highly sensitive molecular biology technique that amplifies specific target segments of DNA to detect the presence of pathogens in a milk sample. Multiplex real-time PCR is a more advanced version of the traditional PCR technique. It allows for the simultaneous detection of multiple pathogens in a single test⁶. Some kits targeted for routine bovine mastitis can identify up to 15 of the most common mastitis pathogens, including Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, and Mycoplasma bovis in addition to the beta-lactamase gene, indicating resistance for beta-lactam antimicrobials. Unlike bacterial culture, which relies on growing live bacteria on a media, PCR amplifies DNA. Consequently, all pathogens must be broken down before the amplification process. High sensitivity and capability of detecting DNA makes PCR useful in detecting infections caused by pathogens that may not grow well in standard culture conditions, but have raised questions over the years of how it compares to conventional culture, is it too sensitive and how the results should be interpreted in practice?

The importance of proper sampling

Before sampling, we must define the reason for sampling. For surveillance or for diagnostics? For the herd or for the cow? Different sampling techniques, bulk tank milk sampling, cow composite sampling, and quarter-based aseptic sampling, serve distinct purposes in monitoring and managing udder health.

1 - Bulk Tank Milk (BTM) sampling

Bulk tank milk (BTM) sampling is a method where a sample is taken from the tank, including milk of an entire herd, presuming that milk of all lactating cows is included.

If PCR is run from BTM samples, the reason is usually in mastitis eradication programs and identifying the presence of contagious pathogens in herds. This method may be particularly useful for herd-level surveillance and for detecting contagious pathogens that could spread aggressively, and affect multiple cows, such as Streptococcus agalactiae or Mycoplasma bovis ^7–9. By routinely testing bulk tank milk with PCR, farmers and veterinarians may monitor trends in pathogen prevalence, detect emerging contagious infections early, and take preventive measures before widespread infection occurs.

Bulk tank milk sampling does not provide information about the specific cows that are infected. In addition, the bigger the tank, the more diluted the sample gets, thus increasing the possibility of false negative results. To minimize false positives and the carry over effect from other farms, the sample should be taken aseptically from upper hatch, avoiding those parts where milk truck hoses are implemented when collecting the milk.

2 - Cow composite sampling or DHI sampling

The traditional purpose of composite samples is to determine content of certain milk solids and SCC at the cow-level for dairy herd improvement (DHI), but they might also be used to diagnose subclinical IMI with microbiological tests ¹⁰. Cow composite sampling involves taking milk from all four quarters of an individual cow and combining it into a single sample by hand or by technical device. Using non-aseptic samples instead of aseptic quarter-based samples increase the sensitivity of PCR, specificity staying on same levels ¹¹. Composite samples may be particularly useful when screening cows for specific contagious pathogens, such as Staphylococcus aureus, Mycoplasma bovis or Strep. agalactiae, according to the needs of the specific herd.

In DHI monitoring the sampling is automated and there is no disinfection of the quarters. Automatic systems may lead to a phenomenon called carry-over. Carry-over refers to the unintentional contamination of a milk sample by residual milk from a previous cow during milking or sampling. This can occur in automatic milking systems (AMS) or when using shared milking equipment, leading to the transfer of pathogens between samples. Carry-over can affect the accuracy of mastitis diagnostics, potentially causing false-positive results, as bacteria from one cow may be mistakenly from another cow’s sample. Ensuring proper cleaning of equipment and using aseptic techniques help minimize the risk of carry-over. In screening DHI samples with PCR, milking order and the CT-value of the preceding cow should be included in the diagnostic process ¹².

3 - Quarter-based aseptic sampling

The primary purpose of quarter-based aseptic sampling is to accurately diagnose clinical and subclinical mastitis at the quarter level. It is used in cases where there is a suspicion of infection in a particular quarter based on clinical signs (such as elevated SCC or visible abnormalities in the milk). Results gained from quarter-based sampling help to decide whether the animal needs an antimicrobial in addition to NSAIDs or not. The most valuable thing from aseptic quarter-based sampling, in addition being part of the diagnose of the single cow, is reliable data, forming a bacteriological profile for the herd guiding the herd health management plan into the right measures.

Comparing PCR and bacterial culture as diagnostic methods

Identification of pathogens causing IMI can be carried out using phenotypic or genotypic methods. Phenotypic identification by bacterial culture (BC) was assessed as the golden standard for years, but as stated by professors Adkins and Middleton ¹³ there is no gold standard for the diagnosis of mastitis or intramammary infection. PCR (Polymerase Chain Reaction) has been established as genotypic identification of target pathogens in the sample. The most comprehensive question differentiating these two methods is that when using BC, we ask, “What we are possibly having in the sample?” and with PCR we are asking “What are we finding?” as the possibilities are limited according to the target pathogens used in the kit. Both methods have their limitations and advantages.

Turn-around times

BC is time consuming: accurate identification of the causative pathogen is often possible only after 48 hours of incubation and further diagnostic tests. For some pathogens, like Mycoplasma bovis, correct identification requires special media and incubation times up to 6 – 10 days ¹⁴. Commercial multiplex real-time PCR kits currently available are able to provide results within 3 hours.

Detection capacities, sensitivity and specificity

Sensitivity and specificity have been debated: is culture not able to find pathogens it should or is PCR producing irrelevant findings by being too sensitive?

A significant part of samples from clinical and subclinical mastitis cases turns out to be growth negative in BC. Studies consistently show that real-time multiplex PCR is a more sensitive diagnostic method compared with culture (Table 1). For example, Taponen et al. (2009) demonstrated that PCR detected bacteria in 43% of clinical mastitis cases where culture results were negative, while bacterial culture failed to grow any pathogens ¹⁵. Similarly, Shah et al. (2018) found PCR to detect bacteria in 100 % of subclinical mastitis cases, while culture detected 90 % ¹⁶. Koskinen et al. (2010) demonstrated that PCR detected pathogens in 89% of clinical mastitis cases, compared to 77% with bacterial culture ¹⁷. In a German study by Spittel and Hoedemaker (2012), PCR identified mastitis-causing pathogens in 71% of subclinical cases, whereas culture detected pathogens in only 32% of samples ¹⁸. Samples from healthy quarters were positive for pathogens in 15 -28% with PCR and 15 – 17% with BC (Table 1)¹⁸.

Sensitivity and specificity of PCR varies according to the CT (cycle threshold) cut-off value in PCR ^11,19 and there are some indications, that CT cut-off values could be feasible to adjust on a pathogen specific levels to improve the interpretation ¹⁹, but the best of my knowledge, there are no further research published to date. Hiitiö et al. (2018) demonstrated that PCR could detect Staphylococcus spp. several days after bacterial culture results were negative. This highlights the importance of interpreting PCR results in the context of the cow’s clinical signs and inflammation indicators.

Table 1. Detection capacity and/or Se and Sp of BC and PCR from different studies. Sample collection type (quarter-based, cow composite/DHI or bulk tank), mastitis type and targets are presented. Target “not specified” means that all targets of the used kit were detected. In some studies, focus was to find only certain pathogens, using kits that include other targets too.

*PathoProofTM Mastitis Complete-12 Kit (ThermoFisher). **PathoProofTM Mastitis Complete-16 Kit (ThermoFisher). ^ TBC qPCR (DNA Diagnostics). ^^Mastit-4B (DNA Diagnostics)

Interpretation of the results – contamination and sample quality

The accuracy and reliability of PCR, as well as BC, results depend heavily on the quality of the milk sample collected. Aseptic sampling technique is crucial to avoid contamination, which can lead to false-positive results. In practice, contamination often occurs when bacteria from the cow’s skin, milking equipment, or the environment enter the sample during collection. Contaminated samples may lead into false actions, like unnecessary treatments or culling animals. Good quality samples for PCR testing are not a myth. Vakkamäki et al. 2017 published a study where over 240 000 routinely samples, taken mainly by farmers, where almost 50% was including only one pathogen, 24 % two and only 13 % were considered contaminated (more than two pathogens)²⁰. Number of negative samples have varied from 12 % to 18% in PCR sampling data (Valio Oy, 90-120 000 samples per year) ²⁰. In Picture 1. the quality of samples from five different studies are presented. There is no consensus of PCR results regarding contamination in aseptically taken quarter-based milk samples. We have suggested to follow the BC definition with > 2 pathogens considered contaminated, but with more studies with CT-value cut-offs and the origin of the pathogens could change this definition in future. If sample contains more than three pathogens detected with PCR, the quality of the sample is questionable. 

Table 2 shows the quality of samples regarding how many pathogens there have been detected per sample in different studies. Table presented is from Academic Dissertation of Hiitiö ¹⁹.

Table 2. Percentage of samples where no (0) target pathogen DNA was detected and samples containing target DNA of 1-5 different species per sample of aseptically taken quarter milk samples, studied with real-time PCR (PathoProof™ Complete-12 or Complete-16 Kit).

Timing of the sampling

In addition to aseptic technique, timing is critical for accurate results. PCR is most reliable when samples are taken during the acute phase of inflammation, when bacterial load is highest. Interpretation is easiest when one pathogen dominates the sample (>99%) and the CT value is low, indicating a strong DNA signal¹⁹.

Negative results

One of the main challenges with culture-based methods is that it can result in a high proportion of culture-negative samples. Studies^29-32 have reported that up to 27% of clinical mastitis cases and over 40% of subclinical cases can yield negative culture results, despite clear signs of inflammation such as visible changes in the milk and elevated SCC, respectively.

1. Low bacterial load:

   • The number of bacteria in the sample may be too low to be detected by culture, particularly in subclinical infections or during the early stages of infection.

2. Short duration of infection

   • Infections that are transient or have been recently cleared by the cow’s immune system may not yield positive culture results.

3. Inhibitory compounds

   • Mastitic milk contains several compounds, such as leukocytes and antimicrobial proteins, that can inhibit bacterial growth in culture.
   • In samples taken to follow the cure, antimicrobials may be present in levels inhibiting growth

4. Sampling and storage

   • The time and handling between sample collection and culturing can also affect the viability of bacteria, leading to false-negative results.

5. Laboratory processes

   • Lab process is not carried out as SOP (NMC 2017)Poor handling, incubation times are not as long as required
   • Agar plates are flawed

6. False interpretation

   • Plate discarded before visible growth

7. Sample does not contain any viable pathogens able to grow on agar

Negative PCR results may be due to:

1. Causative agent is some other than those included into the used kit
2. PCR process failed

   • Technical problems
   • Fault reagents

3. DNA has dissolved from the sample for some reason

   • Excessive amount of disinfectants in sampling

4. Sample does not contain any pathogen DNA
5. Too low amount of target pathogen DNA in the sample

   • Successful PCR amplification usually needs only 5 target copies ³³.

Subjectivity issues

The process of culturing involves growing bacteria from milk samples on agar plates, and the results are interpreted based on the appearance of bacterial colonies. This introduces several points of potential subjectivity like evaluation of the milk sample before culture, colony identification by their size, shape, color, and growth and further laboratory tests. This is a highly subjective process, despite the guidelines and standards of practices, both for laboratories and culture methods ^34,35.

The degree of subjectivity during both the interpretation and handling phases has been demonstrated in proficiency studies ^36,37. Norway and Sweden have carried out routine proficiency testing of their own and results are aligned with published ones. In Sweden, 39 % of the 112 laboratories got poor results in BC interpretation (scores 1-5/10, where 10 was correct identification for all) (NJK Nordic mastitis seminar 2024). The interpretive variability inherent in bacterial culture means that results differ depending on the skill and experience of the microbiologist/veterinarian and requires expert judgment. There is also variation in detection limits for IMI in culture, similarly as in PCR with Ct-value cut-offs. The decision of what constitutes a significant bacterial count for IMI (colony-forming units per milliliter) can vary between laboratories and it has differed also in studies from 100 cfu/ml to 500 cfu/ml and in some varied according to the pathogen ^28,38,39

PCR diagnostics is regarded as an objective method in terms of the process itself. After sampling and manual handling of the sample before the PCR amplification, the PCR process becomes more objective under laboratory conditions with proper calibrations and the inclusion of negative controls in every run ⁶. Interpretation of the amplification curves is automated, and a software produces the results. This makes the process more objective, but the source of the pathogen (especially environmental) remains open, if the sample is not aseptically taken. The interpretation guidelines by NMC (2019) states that due to high contamination risk and difficulty to correctly interpret the presence of environmental bacteria, milk recording samples are not recommended to be used with PCR.

Benefits and limitations of PCR in mastitis diagnostics

Multiplex real-time PCR has several clear advantages over traditional culture-based methods in diagnosing bovine mastitis. According to National Mastitis Council (2019) PCR techniques can be a viable alternative to classical bacterial culture in the diagnosis of intramammary infections causing clinical and subclinical mastitis when using aseptically collected quarter or composite samples. PCR may be more sensitive, faster, and does not necessarily require refrigerated transport of samples.

Benefits of PCR diagnostics:

1. Speed

   • PCR can provide diagnostic results within hours, enabling faster decision-making for treatment.

     • Quick turnaround is especially critical for clinical mastitis cases where timely intervention is essential to prevent the spread of infection and minimize production losses.
     • Test numbers should be large enough and PCR run every day to gain the benefits of speed

2. High sensitivity

   • PCR is highly sensitive, capable of detecting even small amounts of bacterial DNA in milk samples. This is particularly advantageous for diagnosing subclinical mastitis, where bacterial loads may be too low for traditional culture methods to detect.
   • The ability to catch infections at an early stage allows for more proactive management of udder health.

3. Detection of dead bacteria may also be considered an advantag

   • Use of preservatives in milk sample is possible. This “freezes” the situation to the timepoint of sampling without a fear for example Escherichia coli growing over other pathogens during transportation, biasing the results.
   • When live bacteria are not detectable through culture for some reason

4. Broad application

   • A useful tool for herd-level surveillance and monitoring of udder health from BTM samples and cow composite or DHI samples. For example, regular testing of BTM samples can help detect contagious pathogens like Mycoplasma bovis early on, allowing for swift intervention to prevent herd-wide outbreaks.

Finding DNA instead of viable cells – how to interpret?

Despite PCR being sensitive and accurate, a debate on the relevance of the finding has occurred. One of the main challenges with PCR is that it cannot distinguish between live and dead pathogens. This can lead to false-positive results, particularly in cases where the infection has already been cleared, but bacterial DNA remains in the udder. At the same time, it is important to understand that the result from a milk sample is only a small, yet important part of mastitis diagnostics. CT values alone cannot provide a complete understanding of the infection’s severity or the cow’s clinical status. Veterinarians must consider the cow’s history, clinical signs, SCC, and other factors when making treatment decisions based on PCR results. They must also compare the diagnostic results to the severity of clinical symptoms of the cow. For example, if there are no signs of clinical mastitis and the result is “high amount of E. coli”, it means that there might be manure contamination in the sample.

This is extremely important when we are thinking of administering antimicrobials, as PCR may continue to detect dead bacterial DNA. In such cases, it is essential to interpret PCR results in conjunction with other diagnostic information, such as the cow’s clinical history, SCC, and the presence of visible signs of mastitis. The accuracy and reliability of PCR results depend heavily on the quality of the milk sample collected. Aseptic sampling techniques are crucial to avoid contamination, which can lead to false-positive results. In practice, contamination often occurs when bacteria from the cow’s skin, milking equipment, or the environment enter the sample during collection.

In addition to aseptic techniques, the timing of sample collection is critical for obtaining accurate PCR results. Research has shown that PCR is most reliable when samples are taken during the acute phase of inflammation when bacterial loads are at their highest. Hiitiö et al. (2018) demonstrated that PCR could detect Staphylococcus spp. several days after bacterial culture results were negative. This highlights the importance of interpreting PCR results in the context of the cow’s clinical signs and inflammation indicators.

Interpreting PCR results: the role of CT values

PCR results are often reported as cycle threshold (CT) values, which indicate the number of PCR cycles needed to detect the target DNA from the sample. The CT value is inversely proportional to the amount of bacterial DNA in the sample—lower CT values correspond to higher bacterial loads, while higher CT values indicate lower amounts of DNA. Most commercial PCR assays use a cutoff value of 37 cycles; any result detected after 37 cycles is considered negative or inconclusive.

CT values are essential for interpreting the severity of infection. For example, a low CT value (e.g., <25) suggests a high bacterial load and an active infection, whereas a high CT value (e.g., >37) may indicate contamination or the presence of dead bacteria. Some laboratories report PCR results as high (+++), moderate (++), or low (+) bacterial loads based on the CT value. The plus signs and their corresponding CT value ranges have changed over the years, with increasing information. Certain real-time PCR assays report Str. uberis with one plus at a CT value of 28.1, whereas the same value used to be indicated with ++. The same applies to yeast. This result is not related to the symptoms of the animal. 14 % of Finnish veterinarians stated that they consider CT-values very helpful in evaluating the reliability of PCR results, and 21 % stated that they would use them if provided ⁴⁰. Scale of “+” or “++” considering the amount of DNA in the sample is wide. Without CT values, we might have a situation where the result is “low amount”, but CT value is near “intermediate”, which may bias our interpretation. Therefore, I would recommend that all laboratories would report the actual CT values alongside the “plus” signs. However, it is important to remember that CT values alone do not provide a complete picture of the possible infection. Results must be interpreted alongside other clinical and diagnostic data, such as the cow’s SCC, clinical signs, and treatment history.

In cases where PCR results do not align with the clinical symptoms or SCC, it may be necessary to retest the sample or perform a bacterial culture to confirm the diagnosis. This is particularly important when PCR detects multiple pathogens in the same sample, as co-infections can complicate treatment decisions.

Live experience from Finland

Our leading laboratory for mastitis diagnostics is owned by Valio Oy, the largest dairy in Finland covering 80% of the farms. Valio replaced BC with PCR in use in June 2010 and Finnish diagnostics was changed overnight. Since 2010, PCR has been a routine tool. Over 130 000 samples are run with PCR every year, when number of culture samples is around 2 000. The change was rapid, and the most difficulties was caused by the lack of interpretation guidelines. The introduction of PCR to the market caused fears that the use of antibiotics would skyrocket. However, the trend has been completely opposite, as shown by Fimea’s report about Finnish veterinary antimicrobial resistance monitoring and consumption: FINRES-VET 2022 ⁴¹.

This success is thanks to colleagues and producers: the mindset is, that we want a reliable result from aseptic sample hoping for a result, where we are able to manage with supportive treatments and NSAIDs, without costly antimicrobials. Udder health is considered more holistically, and the focus is on prevention. The number of treatments given unnecessarily or as a precaution seems to have decreased. PCR, like other tests, always has its own challenges and areas for development.

Further research is still needed. In my opinion, the routine use of PCR is justified in terms of logistics, speed, and objectivity. The Finnish way of taking samples and compiling them into an easily usable form as a tool for udder health is globally unique. This provides an excellent foundation to continue working for the benefit of producers and high-quality milk production.

My dissertation “Multiplex Real-Time PCR in bovine mastitis diagnostics” (2018) is available in its enterity at:

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