The use of bacterial culture for an on-farm method of determining mastitis causing pathogens

 

Bacterial culture is used to determine the species of bacteria and helps producers make easier decisions about treatment methods. Depending on the species, Gram-positive bacteria respond to antibiotics and can be treated such as Streptococcus agalactiae. Mild to moderate mastitis cases that are caused by coliform Gram-negative bacteria, such as E. coli, resulted in a high spontaneous cure rate, and further limiting the use of antibiotics on-farm1 . The use of onfarm bacterial culture can help to quickly determine if the mastitis is caused by a Gram-positive and Gram-negative bacteria and allow producers to treat appropriately.

Environmental Mastitis

Environmental mastitis is mostly caused by unsanitary bedding material that contain bacteria such as E. coli and Streptococcus uberis2 . Environmental mastitis can be prevented, at least in part, by applying pre-dip before milking6 and removing manure from bedding.

Contagious Mastitis

Contagious mastitis is exposed through milking protocols not being followed and unsanitary milking equipment from an infected cow that was milked previously5 . Common contagious bacteria include Staphylococcus aureus and Streptococcus agalactiae which are Gram-positive species. The best way to prevent contagious mastitis is to use a teat disinfectant after milking5 and using correctly cleaning milking equipment.

On-Farm Bacterial Culture

Being the gold standard for identification of mastitis pathogens, bacterial culture is an on-farm method that can provide results in 24 h.

Materials needed for culturing:

• Incubator set at 37℃ (98.6ºF)
• Culture plates (Tri-plate, Bi-plate, blood agar, MacConkey etc.)
• Disposable gloves
• Milk sample vials
• Sterile cotton swabs
• Cotton swabs soaked in 70% ethyl alcohol for teat disinfection
• Cooler with ice (if transporting milk samples to lab)
• Permeant marker for labeling

Teats should be disinfected with a cotton swab soaked in 70% ethyl alcohol and scrubbed for 10 to 15 seconds7 to ensure a non-contaminated milk sample. If collecting from all four quarters, make sure to completely disinfect all teats with a different cotton swab to prevent contamination. Make sure milk vials are labeled with cow ID and which quarter the sample is collected from. Then direct milk streams into the vial until 1/3 full6 , while preventing the teat from touching the vial. If applicable, immediately place the sample vial in the refrigerator or samples can be kept frozen up to 60 days7 .

Next, a sterile cotton swab should be submerged in the milk sample until the swab is saturated with milk. Then apply the swab to the culture plate by the pattern shown below (Figure 1), and re-dip the swab in the milk for each of the three sections. After the milk samples are applied to the plate, label plates with cow ID and date. Then place the plates lid down in the incubator at 37℃ for 24 h7 .

Interpreting the tri-plates results are different for each agar. Factor agar selectively grows streptococci spp, while Focus agar selectively grow staphylococcus spp1 . MacConkey agar is designed to grow Gram-negative bacteria. The simple diagnosis can tell the producers if treatment with antibiotics is necessary. Figure 2 represents further interpretation of Staphylococcus aureus with growth on the Factor agar. Figure 3 represents growth of E. coli which grows on MacConkey agar because this bacterium is Gram-negative. If plates contain three or more bacteria, then it is considered contaminated, and plating should be completed again6 . If the plate results in no growth, then that can be considered a true negative (no infection) or there was an error in the collection7 . If more than 40% of results are no growth, it is best to consult your veterinarian about diagnosis and treatment protocols7 . Bacteria such as Mycoplasma species cannot be cultured, so further methods are needed for identification such as quantitative polymerase chain reaction (qPCR) or a cell stain.

 

 

Conclusion

On-farm bacterial culture provides benefits to the producer to overall reduce mastitis cases. This is useful in providing the producer with information

regarding where the bacteria source is coming from. If a cow’s culture contained contagious mastitis, then the producer can separate the cow from the herd to reduce the risk of transmission. If the cow’s culture contains environmental bacteria, then the producer can determine if bedding is the source. Bacterial culture overall reduces the on-farm use of antimicrobials and reduce cost of treatment. If the producer is using antimicrobials when not necessary, this can lead to antimicrobial resistant making future treatment of mastitis difficult8 . Using bacteria culture for identification of mastitis causing pathogens can reduce the cost associated with treatment of mastitis cases.

References

1. Lago, A., S. M. Godden, R. Bey, P. L. Ruegg, and K. Leslie. 2011a. The selective treatment of clinical mastitis based on on-farm culture results: I. Effects on antibiotic use, milk withholding time, and short-term clinical and bacteriological outcomes. J. Dairy Sci. 94:4441–4456.
2. Derakhshani, H., Fehr, K.B., Sepheri, S., Francoz, D., De Buck, J., Barkema, H.W., Plaizier, J.C., Khafipour, E. 2018. Invited review: Microbiota of the bovine udder: Contributing factors and potential implications for udder health and mastitis susceptibility. Journal of Dairy Science. 10.3168/jds.2018-14860
3. Liang, D., Arnold, L., Stowe, C., Harmon, R., Bewley, J. 2017. Estimating US dairy clinical disease costs with a stochastic simulation model. Journal of Dairy Science. 10.3168/jds.2016- 11565
4. Barkema, H., Schukken, Y., Zadocks, R. 2006. Invited review: The role of cow, pathogen, and treatment regimen in the therapeutic success of bovine Staphylococcus aureus mastitis, Journal of dairy science. 10.3168/jds.S0022-0302(06)72256-1
5. Hogan, J.S., Martin, M. 2016. Current concepts of bovine mastitis. Fifth edition. National Mastitis Council.
6. Enger, B., Fox, L., Gay, J., Johnson, K. 2015. Reduction of teat skin mastitis pathogen loads: Differences between strains, dips, and contact. Journal of Dairy Science. 10.3168/jds.2014-8622
7. University of Minnesota Laboratory for Udder Health, 2016. Minnesota Easy Culture System User’s Guide.