A 3rd lactation cow developed mastitis in the left hind quarter approximately 2 weeks after calving. The animal was housed. Although the milk gave a positive reaction in the California mastitis test, the animal did not show any systemic signs. A milk sample was submitted for culture.
Description of Specimen
My milk sample from case 18 was free of clots and was a normal colour.
Results
Blood Agar – There was medium white colonies exhibiting no haemolysis growing on the agar
MacConkey Agar – Pinpoint sized pink colonies exhibiting no haemolysis grew on the agar. Lactose fermenting colonies.
CAN – There was medium white colonies exhibiting no haemolysis growing on the agar
Sabourard Dextrose Agar – Growth on very small/pin point sized white colonies exhibiting no haemolysis.
Gram stain from original clinical sample – A lot of debris was present, some gram + cocci could be seen but again could be mistaken for debris.
Gram stain from colony growth (BA chosen) – purple cocci colonies forming in bunches.
Oxidase test – Negative
Catalase test – negative
Lancefield Grouping test – Group D Lancefield specific
PCR- Enterococcus Faecalis
Anti-microbial susceptibility test
1. Amoxicillin – sensitive
2. Ceftiofur – intermediate
3. Lincomycin – resistant
4. Tetracycline – resistant
Interpretation of Results
Taking into account both the clinical history and results of testing the milk sample it is evident we were dealing with a mastitis caused by a bacterial infection. As not all bacteria cause mastitis I was able to come up with a small list of mastitis causing bacteria both gram positive and negative, to narrow down my diagnosis. Due to no systemic signs being noted it looked to be a sub clinical infection. I carried out a gram stain from the original clinical sample to identify gram positive or gram negative bacteria and I could see a lot of debris as well as some gram positive cocci. This was pretty inconclusive and would require further testing to identify the bacteria. Next I tested the growth of the bacteria on different agars. The bacteria grew well on BA as well as CNA which is specific for gram positive organisms usually. However small lactose fermenting colonies grew on MAC as well which is usually specific for gram negative organisms. This result is usually characteristic of an enterococcus bacteria. From my cultures of the milk sample I was able to narrow down the possible causative bacterial agent. I carried out another gram stain using a colony that grew on the BA and I observed much clearer gram positive cocci that were in bunches. I then carried out an oxidase and catalase test that were both negative. Taking these results into account I could conclude the bacteria was a probable streptococci. More specifically an enterococcus. Next, I carried out a Lancefield Grouping test to identify which strep group the bacteria belonged to. Coagulation was observed in Lancefield group D and therefore I confirmed the bacteria was an enterococcus. PCR testing was then carried out to confirm. The PCR sequencing gave me the result I was expecting – enterococcus faecalis. Whilst I was already confident this was the true identity of the bacteria prior to PCR testing, the PCR test just confirmed this. I got this result from plugging the sequencing into BLAST. All my results pointed to an enterococcus infection and the PCR test only confirmed this. I then carried out an antibiotic susceptibility test which show that enterococcus faecalis was sensitive to amoxicillin and intermediate to ceftiofur. However the enterococcus faecalis was resistant to lincomycin and tetracycline making it a difficult infection to treat.
Assessement of Case
Enterococci are bacteria found in the environment and are spread to uninfected cows when they come in contact with contaminated bedding or dirty milking machinery. Many control mechanisms can be employed to reduce the risk of bacteria spreading to uninfected cows. The use of clean straw or sand as bedding will prevent bacterial spread. A proper sanitary milking routine including the use of pre milking teat disinfectant should be implemented. Cows in early lactation like the cow in this case are at a great risk of infection with enterococcus faecalis.1. This is due to increased stress and immunosuppression that accompanies the post partum period. Prevention is the best approach when dealing with these hard to treat enterococcus infections.