Melanie McFarland,¹
Dale Holcombe, Ph.D¹
Dalene King, ¹
Jessica Allen ¹
Doug Redelman, Ph.D²

1University of Nevada, Reno, College of Agriculture, Veterinary Medicine
²Sierra Cytometry

Clinical mastitis or inflammation of the mammary gland occurs in both sheep and cattle. Strong emphasis has been placed on control and management of mastitis in cattle but little has been done to determine the extent to which mastitis infiltrates sheep flocks.

Sheep producers realize the effects of mastitis most often in low weaning weights, lamb death, and culling of infected ewes. Producers do not readily observe chronic subclinical mastitis cases, which do not exhibit the lumpiness, puss production, and teat lesions or teat damage observed with cases of clinical mastitis. Low-grade infections fluctuate in severity over time and may result in acute clinical stages of mastitis.

To decrease the detrimental effects of mastitis in sheep, a clear, simple, effective method of testing for mastitis needs to be established. It is proposed that by using flow cytometry to examine sheep milk it is possible to determine by cell populations whether or not an animal is subclinically infected (cell populations £ 1 million/ml of milk). In this study, one experiment was conducted to examine the cell profiles to determine the incidence and severity of subclinical mastitis in sheep milk.

In this experiment, seven ewes with subclinical mastitis on at least one side of the udder and seven ewes without mastitis were milked at 30, 60, and 90 (± 4) days postpartum. Samples were taken after each teat was cleaned using dilute Nolvasan solution and an initial 2-ml sample was collected and discarded. Five-ml samples were collected from each teat at 0, 2, 4 and 6 hours with additional collections at 12, 18, 24 hours at 90 days postpartum. Samples were examined for cell counts using fluorescent dyes, Propidium Iodide-Detergent stain (stains dead cells only) and Syber Green Propidium Iodide stain (stains live and dead cells), and a Coulter Epics Profile II flow cytometer to determine cell profiles over time.

Microbiology was done at the Nevada State Diagnostic Laboratory. Samples that were considered manitol-salt plate positive and coagulase positive were determined to be Staphylococci aureus. Those samples that were considered manitol-salt plate negative and coagulase negative were determined to be Staphylococci species.

In this study, low and high cell counts in sheep milk were detected using flow cytometry and fluorescent staining techniques. Cell counts were found as low as 3,000 cells/ml and as high as 100,000,000 cells/ml. Average cell counts were found to be 150,000 cells/ml for sheep determined to not be infected with subclinical mastitis, whereas the average for cell counts in sheep determined to have subclinical mastitis was 14,000,000 cells/ml. It is undetermined at this time as to what the threshold value for cell counts should be to determine minimal cell count levels adequate to describe subclinical mastitis by flow cytometry

Bacterial culture revealed that Staphylococci aureus and Staphylococci coagulase-negative species caused the mastitis observed in ewes used in this experiment. Correlation between bacterial presence and incidence of mastitis has yet to be analyzed. However, it is theorized that possible infection by bacteria may preclude an immune response detectable by flow cytometry by several hours to several days.

In conclusion, it is possible to detect cell counts in sheep milk by using flow cytometry and fluorescent staining techniques, and that sheep milk exhibits very high cell counts in excess of 100,000,000 cells/ml. Bacterial culture revealed that Staphylococcus aureus and coagulase-negative Staphylococci species are major causes of mastitis in sheep. However, further analysis of data is required to determine the minimal cell count level for diagnosis and detection of subclinical mastitis and to determine the correlation between bacterial presence and cell counts and subclinical mastitis.