Whats N New on t the e Ho Horizon: Central alStar ar PCR D - - PowerPoint PPT Presentation

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

Automated N Nucl cleic A c Aci cid Extract ction

• Fecal samples
• Whole Blood
• Tissue (ear notch)
• Milk

Emerging Diagnostics

• Leukosis (BLV) PCR: SS1 test
• β-casein (A1/A2) genotyping

BLV PCR (SS1 as assay) y)

Bovine l leukosis

Bovine Leukemia Virus

• Infects white blood cells of

cattle/incorporates its own DNA

• Can cause abnormal cell

growth/lymphoma

• Most cows asymptomatic
• Eradicated in 22 countries

87%

83-89%

45%

Why s should we c care a about BLV?

Milk production Cow longevity Immune function/ Response to vaccinations Other infectious diseases

COSTS

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

30% 20% 50% 40% 35% 19% 29% 39% 45% 33% 0% 10% 20% 30% 40% 50% 60% 1st 2nd 3rd 4th and greater Average Percent BLV Positive Lactation Group

BLV Herd Profile

Your Herd Michigan 2010

n = 10 freshest from lactations 1, 2, 3, and 4+ *40 cows total tested by BLV milk ELISA at no charge*

Helping herds determine their prevalence

Erskine et. al, 2012

10 20 30 40 50 60 70 80 0.5 1 1.5 2 2.5 3 3.5 4

BLV Prevalence % Years Enrolled

Farm J (160 cows) Farm K (160 cows) Farm H (70 cows)

Identifying and removing “Super-shedders” significantly decreases BLV herd prevalence

The SS1 Field Trial

DHI Test SCC and other components Leukosis ELISA value ELISA- positive cows Blood Tube

DNA

Central Star SS1 PCR test

3 herds ~ 350 cows

10 20 30 40 50 60 70 80 0.5 1 1.5 2 2.5 3 3.5 4

% Prevalence Years Enrolled

SS1 Field Trial Herd BLV Prevalence

Farm J (160 cows) Farm K (160 cows) Farm H (70 cows) Farm B (260 cows) Farm W (900 cows) Farm S (1300 cows) Farm L (550 cows)

7 herds >3000 cows

1 3 21 43 34 37 24 10 14 6 5 4 3 4 1 1 4 3 1 56

10 20 30 40 50 60

Number of Cows BLV Copies/uL Blood

6% 9% 32% 31% 12% 6% 2%

16% 41% 72% 84%

Farm S (~1100 cows) Whole Herd BLV Distribution

• 25 cows responsible for over 1/3 of BLV shedding in herd

<25

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

Lymphocyte count (x1000)/mL blood

Proviral Load (BLV copies/cell)

~3300 cows

23% 33% 25% 26%

0% 5% 10% 15% 20% 25% 30% 35%

Confirmed Pregnant Far-off Close-up All Heifers

% BLV Positive Pregnancy Stage

n=22 n=43 n=149 n=214

Farm Prevalence ~32%

Farm K Heifer BLV Prevalence 600 lbs heifers n=10 Being bred n=16 Confirmed pregnant 30-60d n=10 >3 months pregnant n=13 Springers n=6 Total n=54

ALL BLV NEGATIVE Farm Prevalence ~42%

Farm W Heifer BLV Prevalence

New infections: role of young stock

0% 10% 20% 30% 40% 50% 60% 1 2 3 4 5 6

% of New Infections

Lactation New Infections by Lactation

Farm S (1300 cows) Farm W (900 cows) Farm L (550 cows) Farm B (260 cows)

0% 10% 20% 30% 40% 50% 60% 70% 80% <30 30-90 90-150 150-200 >200

% of New Infections DIM

New Infections by DIM

Farm S Farm W Farm K Farm B

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”

New infections: when are they happening?

β-cas asein (A1/ 1/A2) 2) genotyp

• typing

A1/A2 Testing: In Demand

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

Frequency of β-casein alleles

Reviewed by Pal et. al, 2015

Milk Intolerance Ischemic Heart Disease Diabetes mellitus Obesity Sudden Infant Death Syndrome

A1/A1 A1/A2 A2/A2

TISSUE

A1/A1 A1/A2 A2/A2

A1/A2 genotypes easily separate in blood and tissue (ear notch) using AntelBio PCR assay

98.7% accuracy* when compared to known results Still need to compare to “known” results

A1/A2 A2 g genotypes D DO NOT e T easily s sepa parate i in milk

DHI Milk Hand-stripped Milk

A1/A1 A1/A2 A2/A2 A1/A1 A1/A2 A2/A2

Emerging Diagnostics

• Leukosis (BLV) PCR: SS1 test
• β-casein (A1/A2) genotyping