Determining the type of pathogen that is causing subclinical mastitis in a single cow

Using the results that you have gained from the CMT to determine which cow has subclinical mastitis, in the laboratory you will culture that cow to determine which mastitis bacterial pathogen is causing her mastitis. As you learned in the lecture, there are two major categories of mastitis pathogens, environmental and contagious. It is important to determine the specific pathogen as each pathogen has different treatment, control, prevention, and herd management strategies.

Distribution of Mastitis Etiology

What pathogens are most commonly implicated in mastitis?

• 15-70% Gram-positive
• 8-44% Gram-negative
• 20-40% No growth

From these distributions, the 3 most common cultured pathogens are Streptococcus (24%), E. coli (18%), and Coagulase-negative Staphylococcus (13%).

Negative culture results

If a cow has subclinical or clinical mastitis, then why are 25-40% of the milk samples negative on routine culture (i.e. will not grow bacteria)?

• False negatives (rare):
  • Possibly not sensitive enough to detect at low shedding pathogens
  • Bacteria present do not grow or do not grow quickly enough, in culture media or the incubation environment (Mycoplasma, etc.)
• True negatives (most):
  • By the time the sample was taken the cow had already cleared the bacteria (possible/likely for Gram-negatives)
  • Not a real infection

Knowledge check

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.

Minnesota Easy® Culture system

In this laboratory, we will utilize the Minnesota Easy® Culture system – a rapid culture system designed to be used on-farm or in a veterinary clinic lab setting. The Minnesota Easy® Culture system has two plate options – a bi-plate and tri-pate – which utilize the following selective culture media:

• Factor® media selects for Gram-positive bacteria such as Staphylococci, Streptococci, Bacillus, and Corynebacteria.
• MacConkey media selects for Gram-negative organisms. The most common Gram-negative mastitis organisms belong to a group of organisms known as Coliforms. Examples are E. coli and Klebsiella.
• Focus® (formerly MTKT) media select for Streptococcus and Streptococcus-like bacteria, such as Enterococcus and Aerococcus species (Tri-plate only).

Individual milk culture procedure

Supplies

• • Milk sample from cow with suspected mastitis
  • Triplate
  • Cotton Swab
  • Sharpie
  • Disposable Gloves

Procedure

1. 1. Wear clean disposable gloves.
   2. Mix the sample vial well by gently inverting the sample approximately 15 times.
   3. Turn the plate upside down and label the bottom of the plate with the group ID, sample ID, and date. (normally this would be the cow and quarter ID)
   4. Use a new sterile cotton swab.
   5. Avoid touching the plate or cotton end of the sterile swab with your fingers or to any surface as this will result in contamination. (Open cotton swab packages such that the cotton end remains covered and only the stick is exposed.)
   6. Place a sterile cotton swab end in the milk sample, rolling the non-cotton end of the swab between index finger and thumb, for approximately 5 seconds, or until the swab becomes completely saturated with milk. It will help to bump the swab on the bottom of the tube to break up the cotton fibers.
   7. Try to avoid plating clumped milk. You can roll the swab on the side of the sample vial to knock milk clumps off the cotton tip. If milk clumps happen to get onto the plate use a marker on the bottom of the plate to note where they are so as not to confuse them with bacterial growth.
   8. Swab the sample onto each of the Minnesota Easy™ Culture System media. To do this, the media plate should be lid down on the counter, pick up the media side of the plate in the palm of your non-dominant hand. Take your cotton swab that you saturated with the milk sample and streak the milk over the entire surface of the plate section. Take care not to gouge the surface of the media with your swab. Swab each section in the pattern and order indicated below.
   9. Re-dip the swab in milk between each section of the culture plate.
   10. Once the plate has been swabbed, place the lid back on the plate and immediately re-seal the lid on the milk sample.
   11. Place the plate in the incubator upside down (media facing down or place the plate on its lid) so that any condensation on the lid will not drip onto the plated sample.
   12. The plates should remain in a 37° C incubator for up to 48 hours.

Knowledge check

Interpretation

The Minnesota Easy® Culture Bi-plate is used to differentiate between Gram-positive and Gram-negative bacteria. One side (bright red) has a Factor^® media (a type of Blood Agar Plate) to detect Gram-positive bacteria and on the other side MacConkey media to grow Gram-negative bacteria. Of the types of Gram-positive bacteria that result in mastitis, S. aureus is one of the only ones that will result in hemolysis on the Factor® media. However, we use the Tri-Plate and additional biochemical tests for confirmation.

The Minnesota Easy® Culture Tri-plate is used to further differentiate between Staphylococcus and Streptococcus sp. The plate is divided into 3 wells; Focus^® (formerly MTKT, maroon/deep red), MacConkey (pink), and Factor^® (bright red) media.

Limitations of selective culture medium

• Prototheca (algae) can grow on all media, but not always
• Bacillus (Gram-positive) grows on Factor, and will occasionally grow on MacConkey media
• Yeast grows on Factor™ media
• Mycoplasma will not grow on any media in Minnesota Easy®Culture System (requires special growth conditions)

Knowledge check