What’s N New on t the e Ho Horizon: Central alStar ar PCR D Diag agnostics cs Kelly R.B. Sporer, PhD Research Scientist/Diagnostic Technical Specialist Kelly.Sporer@mycentralstar.com 517-908-0500
Our Team Research and Development (Lansing & Grand Ledge, MI) Director: Dr. Todd Byrem Associate Director: Dr. Casey Droscha Research Scientist/Diagnostic Technical Specialist: Dr. Kelly Sporer DVM/MS Student: Katy Kesler 2 Research Associates: Kelsey Brigham & Chaelynne Lohr R&D intern: Jessie Zenchak MI Lab (Grand Ledge): 4 diagnostic technicians; 1 summer intern WI Lab (Kaukauna): 3 diagnostic technicians
• Fecal samples • Whole Blood • Tissue (ear notch) • Milk Automated N Nucl cleic A c Aci cid Extract ction
Emerging Diagnostics • Leukosis (BLV) PCR: SS1 test • β -casein (A1/A2) genotyping
BLV PCR (SS1 as assay) y)
Bovine l leukosis B ovine L eukemia V irus 83-89% • Infects white blood cells of 87% cattle/incorporates its own DNA • Can cause abnormal cell growth/lymphoma • Most cows asymptomatic • Eradicated in 22 countries 45%
Why s should we c care a about BLV? Milk production Other infectious diseases Cow longevity Immune function/ COSTS Response to vaccinations Erskine et al., 2011; Bartlett et al., 2013; Norby et al., 2015; Frie et al., 2017
How is BLV spread?
How can we diagnose BLV? • ELISA = Enzyme-linked ImmunoSorbent Assay • Measures cow’s immune response to virus • Measured in blood (serum or plasma) or milk • Relatively cheap • PCR = Polymerase Chain Reaction DNA test: measures copies of virus (proviral • load or PVL) Measured in whole blood • More sample processing; expensive •
How c w can we diagnose l leukosis? • White Blood Cell Counts • Costs vary • Specificity?
What i is CentralStar doing about B BLV? • Helping herds determine/reduce their prevalence • Investigating when cows get infected/when they become most infectious • Developing better diagnostic tests to find the most contagious cows (“Super-Shedders”) • Identifying genetic markers for resistance/susceptibility
What i is CentralStar doing about B BLV? • Helping herds determine/reduce their prevalence • Investigating when cows get infected/when they become most infectious • Developing better diagnostic tests to find the most contagious cows (“Super-Shedders”) • Identifying genetic markers for resistance/susceptibility
Helping herds determine their prevalence
Helping herds determine their prevalence BLV Herd Profile Your Herd Michigan 2010 60% 50% 50% Percent BLV Positive 40% 45% 40% 35% 39% 30% 30% 33% 20% 29% 20% 19% 10% 0% 1st 2nd 3rd 4th and greater Average Lactation Group n = 10 freshest from lactations 1, 2, 3, and 4+ *40 cows total tested by BLV milk ELISA at no charge* Erskine et. al, 2012
Identifying and removing “Super-shedders” significantly decreases BLV herd prevalence 80 Farm J (160 cows) 70 Farm K (160 cows) 60 BLV Prevalence % Farm H (70 cows) 50 40 30 20 10 0 0.5 1 1.5 2 2.5 3 3.5 4 Years Enrolled
The SS1 Field Trial ELISA- DHI Test SCC and other components positive Leukosis ELISA value cows Blood Tube DNA Central Star SS1 PCR test
3 herds SS1 Field Trial Herd BLV Prevalence ~ 350 cows 80 Farm J (160 cows) 70 Farm K (160 cows) Farm H (70 cows) 60 Farm B (260 cows) % Prevalence 50 Farm W (900 cows) 40 Farm S (1300 cows) Farm L (550 cows) 30 20 10 7 herds 0 >3000 cows 0.5 1 1.5 2 2.5 3 3.5 4 Years Enrolled
Farm S (~1100 cows) Whole Herd BLV Distribution 84% 60 56 72% 50 31% Number of Cows 43 40 6% 12% 37 34 41% 30 2% 32% 24 20 21 16% 14 10 10 9% 6% 6 5 1 3 4 3 0 4 1 1 4 3 1 0 <25 BLV Copies/uL Blood • 25 cows responsible for over 1/3 of BLV shedding in herd
Her Herd D D: em empl ploying l lymphocyte c coun unt, E , ELISA, A, SS1 PCR t to er eradicate l e leuk eukosi sis s ~3300 cows Proviral Load (BLV copies/cell) Lymphocyte count (x1000)/mL blood
New infections: role of young stock Farm W Heifer BLV Prevalence 35% 33% Farm K Heifer BLV Prevalence 30% 600 lbs heifers n=10 26% 25% 25% Being bred n=16 % BLV Positive 23% Confirmed pregnant 30-60d n=10 20% >3 months pregnant n=13 Springers n=6 15% Total n=54 10% ALL BLV NEGATIVE 5% Farm Prevalence ~42% 0% Confirmed Far-off Close-up All Heifers Pregnant n=43 n=149 n=214 n=22 Pregnancy Stage Farm Prevalence ~32%
New infections: when are they happening? New Infections by DIM New Infections by Lactation 60% 80% Farm S (1300 cows) Farm S 70% 50% Farm W Farm W (900 cows) % of New Infections % of New Infections 60% Farm K 40% Farm L (550 cows) 50% Farm B Farm B (260 cows) 30% 40% 30% 20% 20% 10% 10% 0% 0% 1 2 3 4 5 6 <30 30-90 90-150 150-200 >200 Lactation DIM USDA NIFA AFRI grant proposal submitted 7/9/2019: “Establishing a comprehensive approach for eradication of BLV: Reducing BLV incidence in young stock while removing super-shedders from the milking herd”
β -cas asein (A1/ 1/A2) 2) genotyp otyping
A1/A2 Testing: In Demand Frequency of β -casein alleles Breed A1 A2 Guernsey 1-6% 88-97% Jersey 9-22% 49-54% Holstein 31-49% 49-62% Ayrshire 72% 28% *Frequency of B allele not shown; Taken from Kaminski et al., 2007
Reviewed by Pal et. al, 2015 Milk Intolerance Ischemic Heart Disease Diabetes mellitus Obesity Sudden Infant Death Syndrome
A1/A2 genotypes easily separate in blood and tissue (ear notch) using AntelBio PCR assay TISSUE A2/A2 A2/A2 A1/A2 A1/A2 A1/A1 A1/A1 Still need to compare to “known” results 98.7% accuracy* when compared to known results
A1/A2 A2 g genotypes D DO NOT e T easily s sepa parate i in milk A2/A2 A2/A2 A1/A2 A1/A2 A1/A1 A1/A1 DHI Milk Hand-stripped Milk
Emerging Diagnostics • Leukosis (BLV) PCR: SS1 test • β -casein (A1/A2) genotyping
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