Analysis of potential biomarkers for bovine mastitis using real-time PCR
Open Access
- Author:
- Taylor, Laura Marie
- Area of Honors:
- Veterinary and Biomedical Sciences
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Bhushan M Jayarao, Thesis Supervisor
Dr. Lester C Griel Jr., Thesis Honors Advisor - Keywords:
- mastitis
cattle
biomarkers
real-time PCR
IL-8
IL-10
TNFa
28S - Abstract:
- Bovine mastitis is a significant disease that causes an estimated loss of $2 billion per year in the United States. The current diagnostic methods for mastitis are based on bacteriological analysis and somatic cell count (SCC) of milk samples. These techniques have low sensitivity and require a significant amount of time for a diagnosis, and are therefore insufficient. A test using real-time PCR and cytokine profiles of milk could provide a faster, easier, and more accurate diagnosis. This study looked at three possible mastitis biomarkers, TNF-α, IL-8, and IL-10, in conjunction with 28S as the control. The real-time PCR reaction was standardized for the four target proteins using a serial dilution of mRNA from milk. Quarter milk samples were collected from six Penn State Dairy Herd 2nd lactation cows 45-60 days in milk for a total of 23 samples (one cow had three quarters). The samples were subjected to bacteriological analysis using PSU-QUAD plates and SCC to categorize them as healthy or mastitic. The quarter samples were then analyzed using uniplex RT-PCR. The relative expression of each target protein in the healthy and mastitic quarters was compared using comparative Ct analyses and a statistical t-test. The expression of TNF-α among the previous studies is similar to that observed in our study, where TNF-α expression was similar in both mastitic and healthy quarters. Therefore, it can be inferred that TNF-α is not a suitable marker for mastitis. Although expression of IL-10 was elevated in mastitic quarters, the level of expression (Ct value) was not sufficient to allow its use as a mastitis biomarker. The relative expression of IL-8 was consistently higher in the mastitic quarters (high SCC) than the healthy quarters (low SCC). The relationship between high SCC and IL-8 expression indicates that IL-8 could be a promising biomarker for mastitis.