How to use qPCR testing for bulk tank milk samples and contagious mastitis in individual cows

In the February 2019 edition of M2-Magazine, Dave Wilson of Utah State University, raised concerns about the sampling of milk aseptically for culture or PCR testing on robotic milking herds. I will try to explain how I recommend doing this, and how we worked with sampling in large automatic milking system (AMS) herds (200–900 cows) in Denmark when I was employed at the Danish farmer organizations as a mastitis advisor for all Danish dairy herds.

To avoid problems with contamination or carry-over, we always recommend using aseptically taken milk samples, also for PCR testing, for the diagnosis of mastitis and therapy decisions, with or without antibiotics.

That said, there is a great use of qPCR tests on bulk tank milk samples and individual cow Dairy Herd Improvement (DHI) samples in herds where aseptically taken samples are not easy to collect. For example, AMS herds or big herds where cows need to be locked up several hours for sampling, as mentioned by Wilson.


Bulk tank testing with qPCR

Bulk tank milk samples are generally used for testing of contagious mastitis or specific environmental bacteria, such as Klebsiella. qPCR can be used just as well as bacteriological culture for these samples, and in addition, Mycoplasma and non-hemolytic Streptococcus agalactiae are much faster and more easily diagnosed using qPCR.

Quantitative estimates of Streptococcus uberis and/or Staphylococcus aureus, for example, are very easy to evaluate in the qPCR test in comparison to cultures. The disadvantages of culture are overgrowth of background bacteria that can influence evaluation and the many different selective agars and dilutions needed, which increase the price.

We have used bulk tank milk samples in Denmark in our Streptococcus agalactiae program for screening. In 2012, I found that automatically taken milk samples in the milk truck can still be positive after loading 10,000 liters of milk from a negative tank from carryover from a former positive herd.  Now we always confirm a positive finding in a herd with negative status, by testing of a sample taken directly from the tank. Still, the general bulk tank testing is an excellent screening tool.


qPCR tests of DHI samples

Taking DHI samples with preservatives is an excellent way to easily check for contagious mastitis. If a bulk tank is confirmed positive with contagious mastitis bacteria, it is recommended as an excellent prevention tool to use segregation of infected cows, as recommended in the ‘5 point plan’ from 1969. The way I recommend finding cows with contagious mastitis bacteria in big farms and in AMS farms is to test DHI samples. If you expect the prevalence of your target bacteria to be lower than 5 per cent, you can choose to use pooled samples. The qPCR test takes 3–4 hours, which means that segregation can commence early on. When segregated, all segregated cows are tested for chronic infection and elevated SCC for three test days or more. Chronically infected cows are culled. The rest of the cows are clinically examined, palpated and controlled for normal milking in all four quarters. All teats are evaluated to for the presence of injuries or grade 3–4 teat end scores. Cows with any of these issues are also culled. Now the rest of the segregated cows are easily controlled, with aseptically taken milk samples, for evaluation of cull or therapy. Negative cows are left in the segregated area for the rest of their life. A negative status can be confirmed in weekly group samples, or pen samples, if possible. Bulk tank samples can be taken, when each group of cows are milked, and after the tank has been emptied and cleaned. Always start with the cows expected to have the highest chance of being noninfected. DHI testing can also be used to screen high SCC cows for problems with contagious mastitis bacteria, so arising problems in the herd can be identified.

Text: Jørgen Katholm (, veterinarian, certified in cattle herd health, market development manager at DNA Diagnostic A/S

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