Editor’s note: Second in a two-part series on Mycoplasma mastitis. Click here to read the first part: Mycoplasma mastitis: It's everyone's problem.

Accurate diagnostics are a critical element in detecting and diagnosing Mycoplasma mastitis. Before you can effectively help clients prevent and control the disease, you need to be confident that Mycoplasma is, indeed, the organism with which you are dealing.

Mark Wustenberg, DVM, Monsanto Dairy, Bay City, Ore., and Paul Blackmer, DVM, Veterinarian’s Outlet, Chino, Calif., emphasize the importance of sampling clinical cows and culturing them on both blood agar and Mycoplasma agar.  As Mycoplasma becomes a larger concern across the country, Wustenberg recommends that practitioners check out the diagnostic labs they are using, to make sure they can accurately perform cultures for Mycoplasma and get consistent results.

“Visit the lab personally, and make sure they do a high volume of milk samples on a regular basis,” says Wustenberg. “They should be able to show you their techniques, and particularly demonstrate their quality control measures. Finally, you should ask to see some examples of Mycoplasma-positive cultures and verify that the lab routinely cultures for Mycoplasma in milk samples, not just swine and poultry.”

Blackmer notes that there is a misconception in some regions that Mycoplasma is hard to grow in the lab. It isn’t.  He believes the misinformation stems from labs using an inappropriate media in the early days of Mycoplasma screening. 

Many labs read Mycoplasma results at three and seven days of growth, so this timeframe must be factored into the decision-making process in outbreak management. If Mycoplasma growth is detected in a new herd with no Mycoplasma mastitis history, Wustenberg also recommends the additional step of validating the sample to confirm that it is Mycoplasma and speciating the organism to determine which strain is involved.

Today, some labs offer enrichment – preincubating milk samples in a broth to enhance Mycoplasma growth, then plating samples from there – to provide extra insurance that Mycoplasma is detected. Although this procedure was developed to better detect and control a dangerous organism, Blackmer strongly believes that it actually clouds the diagnostic picture.

“The more we learn about Mycoplasma, the more we know that the organism is everywhere in the dairy cow’s environment,” he explains.  “The chances of pulling a few Mycoplasma bugs from the air into a milking system are great, even with impeccable sampling techniques. So enrichment will allow them to grow, creating false-positive results and diagnostic overreacting.”

Unlike Blackmer, Wustenberg sees value in enriching bulk-tank samples.  “We did extensive work on paired cultures to compare the results of enriched and non-enriched bulk-tank milk and found multiple instances where we detected low levels of positive cows after enrichment,” he notes. “The false-positives at the bulk-tank level we found were few, if any. The trick is to recognize that there is the potential for false-positives, and develop a strategy to confirm before panicking.”

But Udder Health Systems, Inc., Bellingham, Wash., owned by Allan Britten, DVM, MS, processes thousands of Mycoplasma diagnostic samples from across the country each year, with no enrichment ever used. “I have not seen any proof that enrichment yields a significant number of additional, true positive samples that would provide better control of the disease,” says Britten. “It adds time and expense to the diagnostic process and is not the most important variable in the management of Mycoplasma mastitis.”

Blackmer’s advice instead: “Do direct plating of all milk samples onto Mycoplasma agar.  If you have an intramammary infection, it will show up loud and clear.”

Prevention strategies
The highly contagious nature of Mycoplasma makes biosecurity an essential element in keeping the disease in check. 

Four groups of animals are at an exceptionally high risk of either contracting Mycoplasma mastitis or spreading it within the herd:

1. Incoming first-calf heifers

2. Other newly acquired animals

3. Fresh cows

4. Sick cows entering or leaving the hospital for any reason

There are a number of practical measures that the experts recommend to prevent Mycoplasma mastitis from entering and spreading in a herd:

  • Dedicating separate, sanitary facilities for maternity and sick cows – versus mixing them together or leaving them with the main herd – will help cut down on the amount of cross-contamination when a clinical case occurs. Ideally, the hospital area should have its own milking facility for fresh and sick cows. If this is not practical, fresh cows and the hospital string should be milked separately from the rest of the herd, with thorough sanitation of the milking clusters after they are milked.
  • Culturing every cow as she leaves the hospital will help identify new infections that may have occurred in the hospital, and keep a potential outbreak in check.
  • Quarantining incoming groups of heifers or individual animals and culturing composite, four-quarter samples before releasing them, will help prevent newcomers from seeding the rest of the herd with Mycoplasma
  • Regularly sampling the bulk tank and culturing for Mycoplasma. Britten suggests that bulk-tank samples be taken and cultured as frequently a once a week in herds larger than 1,000 cows and those with a history of Mycoplasma mastitis. Sampling monthly or every other week may be appropriate for smaller herds with no previous Mycoplasma episodes.
  • Scrupulous udder hygiene – including thorough pre- and post-dipping of teats, using individual cloth towels and maintaining excellent teat-end condition – also will prevent the spread of Mycoplasma
  • Good teat cannula management also cannot be overemphasized. “Poor treatment hygiene can turn an isolated clinical Mycoplasma case into a massive outbreak virtually overnight,” says Blackmer. “Even having more than one treatment cannula exposed in the hospital or parlor is risky, given the very high likelihood that it could be seeded with Mycoplasma from the air, flies, fingers, swishing tails, splattering urine or uterine discharge before it enters the teat orifice.”
  • Culturing every clinical quarter of every cow with clinical mastitis will help producers pinpoint Mycoplasma cases and deal with them knowledgably. Wustenberg also suggests monitoring individual-cow SCCs and culturing cows that convert from an SCC below 200,000 to above 200,000 in a one-month period.
  • Culturing composite milk samples from all fresh heifers (and also fresh cows if the herd has a history of Mycoplasma mastitis) also is advised.

The subject of backflush units also stirs some debate among the veterinarians. “Backflush units can easily create a false sense of security that causes a dairy to cut corners on the other fundamental practices that are important for effective Mycoplasma control,” states Blackmer. “They also tend to use an excessive amount of water and chemicals, and often the valves don’t stay clean. They can cause deteriorating milk quality, and their external surfaces remain fomites.” 

But Wustenberg and Britten aren’t so quick to cancel out the technology. Wustenberg likens backflush to a rider on an insurance policy. “You can buy different levels of insurance, and some dairymen are more risk-averse than others,” he says. “Backflush does not replace good, basic management practices, but, when used and maintained appropriately, it can be part of the solution.”

Britten adds that, like virtually any technology, backflush can be useful but only when it’s used appropriately. “It certainly doesn’t make any sense to put a dirty machine on a healthy cow,” he says, “but if backflush is going to be used for protection, it has to be functioning effectively and safely. It is an important role for the veterinarian to provide counsel to clients and communicate with equipment dealers and technicians. The practitioner should be regularly evaluating the system for alignment, leakage and germicide coverage and pointing things out that need improvement.”

Outbreak offense
When a routine bulk-tank culture shows positive for Mycoplasma, the best-case scenario is that it amounts to no more than a single, culprit cow being identified and culled. A positive tank alone does not automatically spell disaster, Wustenberg points out, because in this scenario the problem can be identified and eradicated very swiftly.

Detecting the perpetrator(s) may require various levels of investigation:

  • In some cases, a new heifer or clinical cow immediately sticks out and is found, confirmed and culled. Such animals will be even more obvious on dairies where routine cultures are performed on every cow undergoing intramammary therapy.
  • The next level of investigation is string sampling: culturing composite samples from milking strings and then performing individual-cow cultures only on the animals in the positive string(s).
  • In cases with heavy bulk-tank Mycoplasma loads (heavy growth) and/or a herd history of Mycoplasma mastitis, whole-herd culturing to detect the positive cow(s) may be warranted.

The other way that Mycoplasma typically surfaces in a herd is a high incidence of nonresponsive mastitis cows in the hospital. Wustenberg says that in those cases, the bulk tank may never even go positive, because clinical mastitis milk is withheld from the tank, and the Mycoplasma-positive cows are either culled or identified via culturing of clinical quarters. Or, the incidence of infected cows is low enough that it dilutes out in the bulk tank.

What to do with Mycoplasma-positive cows is another issue of some debate in the industry. If the problem stems from one or two cows, culling is deemed the most appropriate measure. Likewise, no one believes that chronic clinical cows should be kept around. 

But in the case of larger-scale outbreaks, “wholesale culling may be an extravagance that some dairies cannot afford,” says Blackmer. This is particularly true in expansion situations, when the dairy is under pressure to maintain cow numbers for collateral.

One diagnostic concern is the cow that cultures Mycoplasma-positive but exhibits no clinical signs. Given the high number of colony forming units (CFUs) in a true clinical case, sample contamination can be a common problem. Blackmer describes a “trailer effect” that poor sampling technique can produce:   “In three samples taken in a row, the first cow may be clinical and show obvious, confluent growth. The next one may show moderate growth, and the third one a trace of Mycoplasma. What probably happened is that the second and third cows were normal, but their samples were contaminated in the collection process.”

Blackmer recommends re-culturing positive animals that appear healthy, to confirm their Mycoplasma status before taking further action. Similarly, Britten suggests physical examination and a CMT test for such animals, with another culture if they are CMT-negative. Most important, both practitioners emphasize the importance of carefully training the individuals taking the samples to reduce the incidence of contamination errors.

The other animals that present a quandary are known Mycoplasma cows that resolve the infection and remain productive. Rather than immediately culling these cows, Blackmer sometimes suggests permanently identifying and moving them into a segregated, “to-be-culled/do-not-breed/junk” string, along with cows that are confirmed positive but show no clinical signs. The main herd is then clean and pathogen-free, and the Mycoplasma cows can be milked until the end of their productivity, which may be years later.

Mycoplasma bacteria in pasteurized milk pose no known human health risk. Blackmer says cows that have survived one episode of Mycoplasma mastitis
often endure and resolve subsequent flare-ups. No antibiotic or supportive therapy is needed, but if the clinical state becomes chronic or their productivity drops to a point of negative profitability, they should be culled.

This approach to temporarily salvaging Mycoplasma-positive animals may sound easy. It’s not. Blackmer cautions that the individuals working with these cows must be thoroughly educated about the highly contagious nature of the disease and trained to protect the rest of the herd from contamination without fail. Steps in that process include:

  • Double-identify Mycoplasma-positive cows with leg bands, ear tags, ear notches, etc.
  • Milk the junk string at a designated sequence in the dairy’s milking schedule, with thorough sanitation of the milking system – including internal and external surfaces of clusters – after they pass through the parlor.
  • Never allow cows from the junk string to enter the hospital, where they could contaminate cows from the clean herd.
  • Never feed milk from known Mycoplasma-positive cows to calves, even if pasteurization is available. The risk of a breakdown in the process and subsequent infection of the calves is too great.

The decision of whether or not to temporarily keep Mycoplasma-positive cows on a dairy will vary from case to case and should be based on the dairy’s economic condition; number of animals involved; level of milk production and reproductive status of infected animals; and the availability and price of replacements. Dairy managers also need to seriously consider whether they believe their staff and facilities can accommodate effective isolation and management of Mycoplasma-positive cows.

Hospital hot spot
In the throes of a severe Mycoplasma outbreak, other vigilant management efforts are needed. Clinical cows should be moved away from the rest of the herd to an isolation area. Blackmer prescribes a strict halt to anything going into any cow’s teat end in the hospital – no mastitis tubes, no dilators, nothing.

“I am not a believer in the ‘no intramammary therapy’ approach to clinical mastitis management, but in the case of a Mycoplasma outbreak, I make an exception,” says Blackmer. “The value of protecting Mycoplasma-negative cows in the hospital outweighs the benefit of treating them for other types of mastitis.”

The practitioner describes some dairies’ hospitals in the middle of an outbreak as “myco city.” The heavy pathogen load that is shed from infected animals can quickly overwhelm the usually small hospital barn, to the point where virtually every exposed surface is contaminated. Blackmer suggests daily foam disinfection of the hospital’s walls and other exposed surfaces to reduce the environmental presence of Mycoplasma.

If the hospital has its own milking facilities, he also recommends a thorough cluster-dunking process to disinfect milking units between every hospital cow.

Learning to live with it
Although more needs to be learned about the organism, one thing is certain:   Mycoplasma mastitis is here to stay. Its highly contagious nature, ability to survive in the environment and massive pathogen loads shed from infected animals make it uniquely challenging to control. As it surfaces in new areas of the country, the disease will only add to the managerial mix in today’s changing dairy industry. 

“The penalty for error with this disease is pretty high,” says Britten. “It behooves us all as veterinarians to get a good handle on how to manage it before we have to deal with a clinical outbreak.”

Blackmer relates a Mycoplasma-education example often used by a former California extension agent. “He would stand in front of a room of veterinarians or dairyman holding up a vial containing no more than 10 mL of milk and say, ‘In this vial I have enough Mycoplasma bacteria to infect every cow in California.’

“He was right, but the example may not have completely hit home for the listeners until they dealt personally with a Mycoplasma outbreak,” Blackmer says. “Client compliance is much higher if a dairy producer has endured a bout with the disease. Unfortunately, a growing number of producers and veterinarians across the country are receiving crash courses in Mycoplasma management. It is not an impossible disease to control, but it’s certainly one that should keep us all on our toes.”

What’s a practitioner to do?

Following are basic, recommended measures by Paul Blackmer, DVM, which every dairy veterinarian can follow to help all dairy clients protect their herds from Mycoplasma mastitis:

  • Culture every clinical quarter of every clinical cow on both blood agar and Mycoplasma media.
  • Routinely screen bulk tank samples for Mycoplasma.
  • Sample and culture every fresh heifer and purchased animal.
  • Educate milkers on hygienic milking and mastitis-tube infusion practices.
  • Prohibit all use of bottle-sourced intramammary infusions.

For a primer on aseptic milk sampling procedures, visit Blackmer’s Web site at www.vetoutletonline.com

One-day Mycoplasma diagnosis?

One of the challenges of managing Mycoplasma mastitis outbreaks to date has been waiting out the three-to-ten-day period that it took to achieve precise culture results. Those days may soon be over, with the advent of polymerase chain reaction (PCR) technology.

PCR is the quick method for generating unlimited copies of any fragment of DNA. Sometimes referred to as “molecular photocopying,” it can characterize, analyze and synthesize any specific piece of DNA or RNA. This exciting new technology is less than 15 years old.

Udder Health Systems, Inc., Bellingham, Wash., is one of the first mastitis diagnostic labs in the country equipped to perform PCR. Owner Allan Britten, DVM, MS, says PCR is a breakthrough method that could dramatically speed up the process of diagnosing Mycoplasma mastitis. “Using current methods, we don’t declare a sample Mycoplasma-negative until a full 10 days after culturing,” says Britten. “The hazard with that timeframe is that potentially infected animals are not dealt with until the results are back. Because Mycoplasma sheds in such high numbers from infected animals, every day is critical in making a decision about those cows.”

Britten and his staff are currently conducting an in-house research project to evaluate the sensitivity and specificity of PCR compared to standard culturing for Mycoplasma organisms. Although the outcome of such a comparison could vary from one diagnostic lab to the next – depending on the methodology used – a favorable outcome for PCR could mean much more efficient Mycoplasma diagnostics in the future.

“PCR results for Mycoplasma can be obtained in four to five hours, so a lab could deliver diagnostic results virtually overnight,” says Britten . Another appealing feature about the technology is that, when done with proper controls, PCR eliminates confusion with a confounding Mycoplasma look-alike. “When screening milk for M. bovis DNA with PCR, you don’t have to worry about getting false-positive Acholeplasma laidlawii results,” he notes.

The test is flexible, in that it can be manipulated to screen for Mycoplasma species in general or for individual, specific strains. Individual tests can be performed for speciation of each strain. PCR speciation can be done on isolated colonies off Mycoplasma agar or directly from milk before isolation.

In fact, Britten is so confident in the accuracy of PCR for Mycoplasma
speciation that it is the one service for which his lab currently offers PCR on a commercial basis.

Britten says his staff has embraced the method fairly quickly, noting that new microbiology graduates are now trained on PCR methods in school. “The more ‘seasoned’ technologists among us have had to go back to the laboratory classroom for PCR training, but it is not a difficult technique to learn,” he says.

Although the equipment and related accessories needed to perform PCR are becoming less expensive, Britten says cost is still a concern. Labs interested in adding PCR services should allocate a start-up budget of $30,000 to $40,000. He says the per-test cost still hovers over $15, but that he hopes the technology will continue to become more economical. 

Udder Health Systems routinely performs diagnostic work for veterinary practices across the country. 

For more information on PCR, contact Udder Health Systems, Inc., 6401 Old Guide Road, Bellingham, WA  98226, 360-398-1360, udder@az.com.