AM O’Connor, BVSc, MVSc, DVSc, MACVSc S Sorden, DVM, PhD ... · AM O’Connor, BVSc, MVSc, DVSc,...
Transcript of AM O’Connor, BVSc, MVSc, DVSc, MACVSc S Sorden, DVM, PhD ... · AM O’Connor, BVSc, MVSc, DVSc,...
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AM O’Connor, BVSc, MVSc, DVSc, MACVScS Sorden, DVM, PhD, ACVP (Dip)
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Dr. A. O’ConnorDr. S. SordenDr. T. DenagamageDr. K-J. Yoon
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•VIBSP–Recruiting 400 herds or 40000 calves for testing for BVDV in Spring 2006–From Iowa based beef cow-calf producers
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•What are the aims?–Evaluate pooled testing as a screening tool –Estimate prevalence of BVDV in Iowa–Estimate risk factors for BVDV in Iowa
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•What it’s not?–Not a certification program–Not state sponsored
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Motivation“..it is the resolve of the Academy of the Veterinary
Consultants that the beef and dairy industries adopt measures to control and target eventual eradication of BVDV from North America”
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Immediate impact–Provide beef breeders with cheap and effective methods for screening for BVDV–Sensitivity and specificity of the pooled RT-PCR screening program
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Screening tests–Cheap–Associated with false positive’s –Positive’s further securitized
PCR on Bulk milkSerology of sentinel heifers
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Diagnostic tests– Confirm or classify disease– Animals are abnormal and the challenge is to
identify the disease– Individual animal: IHC, AC-ELISA
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Long-range impact: – A cost-benefit assessment of BVDV
eradication program– Prevalence of BVDV-PI positive herds– Impact of eradication on performance
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•Enrollment phase: Oct 2005-Mar 2006–Recruiting veterinarians–Recruiting producers
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Ensure veterinarians are comfortable with BVDV control programs
– Available for veterinary meetings, extension, ICA, CVM
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•Recruiting veterinarians–Explain the goals, enrolling, sample collection, submission, receiving results–Explain BVDV to vets–NCBA BVD and AVC BVD materials
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•Vet contacts–3 mailings : brochures and publications–Phone call for each vet–Follow up letters for enrolled vets–Producer meetings for enrolled vets
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•Recruiting producers–IVMA vets–Iowa Cattleman's meetings and magazine–Iowa State University Extension officers
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After enrollment phase–Determine if there is sufficient interest to warrant continuation
–Ensure veterinarians are comfortable with BVDV control options
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•Study phase runs from Jan-July 2006 –Sample collection–Testing and interpretation–Analysis
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•Enrolled producers will receive –sample collection tubes–sample submission boxes –shipping labels –sample submission record sheet
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Calving
Window of opportunity for testing
Breeding
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•Sample collection either at–Birth when calves are ID–Spring processing pinkeye/blackleg vaccintion
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•Study phase –Producers notch calves–Freeze notches in tubes –Submit frozen notches to our lab within 1 week of collection
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•At the lab–Process and freeze samples–When collection complete then thaw and pool –Submit to pooled sample to VDL for PCR
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•Study phase–Pooled RT-PCR on PBS soaked ear notches–Follow-up of ACE on positive pools–Notify veterinarians for follow up sample
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Testing and interpretation–Available through veterinarian–Testing paid for by grant–Vet and client decide next step–Follow-up outcome
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AM O’Connor, BVSc, MVSc, DVSc, MACVScS Sorden, DVM, PhD, ACVP (Dip)
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Pooling ear notches
Retained ear notch and individual ID for further tests if needed.
6.3 ml of PBS supernatantfrom 18 ear notches
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RT-PCR Pools/ AC-ELISA
+ RT-PCR Pool -RT-PCR Pool
One or more Positive AC-ELISA
39* 0
No Positive AC-ELISA
0 33
*Includes 36 samples spiked with a single known positive sample
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19 1801-1900 Positive1899 positive all others negative
20 1901-2000 not detected No Positives21 2001-2100 not detected No Positives22 2101-2200 not detected No Positives23 2201-2300 not detected No Positives24 2301-2400 not detected No Positives25 2401-2500 not detected No Positives26 2501-2600 not detected No Positives27 2601-2700 not detected No Positives28 2701-2800 not detected No Positives29 2801-2900 not detected No Positives30 2901-3000 not detected No Positives31 3001-3100 not detected No Positives*32 3101-3200 not detected No Positives*33 3201-3300 not detected No Positives*34 3301-3400 not detected No Positives*35 3401-3500 not detected No Positives*36 3501-3599 not detected No Positives*
POOL SamplesBVD RT-
PCR ResultsAC-ELISA & IHC
results by Sample #
*AC-ELISA correlated with IHC on first 3016samples afterward IHC was discontinued
POOL SamplesBVD RT-
PCR ResultsAC-ELISA & IHC
results by Sample #1 1-100 not detected No Positives2 101-200 not detected No Positives3 201-300 not detected No Positives4 301-400 not detected No Positives5 401-500 not detected No Positives6 501-600 not detected No Positives7 601-700 not detected No Positives8 701-800 not detected No Positives9 801-900 not detected No Positives10 901-1000 not detected No Positives11 1001-1100 not detected No Positives12 1101-1200 not detected No Positives13 1201-1300 not detected No Positives14 1301-1400 not detected No Positives
15 1401-1500 Positive1433 & 1442 positive all others negative
16 1501-1600 not detected No Positives17 1601-1700 not detected No Positives
18 1701-1800 Positive1718 positive all others negative
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RT-PCR pools/ AC-ELISA RT-PCR BVD +
RT-PCR BVD -
Pool contained positive AC-ELISA (1 or more)
49 0
Pool did not contain positive AC-ELISA
2 (one pool contained 50 samples* the other contained 38 samples**)
38
*Samples from herd where a PI was found: **testing not completed for herd
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•400 + herds•40 PI-BVDV positive herds
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AM O’Connor, BVSc, MVSc, DVSc, MACVScS Sorden, DVM, PhD, ACVP (Dip)
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•Identifying PI’s early allows them to be removed from the herd before the next breeding season; •Otherwise, PI suckling calves may infect cows carrying early-gestation fetuses and create more PIs in next year’s calf crop. •Only need to test dams of + calves (dams are rarely positive, but must test).
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Finding BVDV PI Calves
•Options for identifying BVDV PI neonates:–BVDV isolation (VI) from serum or buffy coat:
•False negatives due to interference by maternal Ab.
–BVDV antigen detection in serum with Ag-capture ELISA:
•False negatives due to interference by maternal Ab.
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•Options for identifying BVDV PI neonates:–RT-PCR on serum (individual or pools):
•Need blood samples, $$ unless pool.–Ear notch tests
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Three tests for BVDV can be performed on ear notches:
1. Immunohistochemistry (IHC):Fix notch in formalin, embed in paraffin, apply
antibody to section to detect antigen.
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•2. Ag-capture ELISA:–Soak notch in saline, (freeze), run ELISA on fluid to detect antigen.
•3. PCR:–Soak notch in saline, run RT-PCR on fluid to detect RNA. Can pool fluid of 100 notches.
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1. Immunohistochemistry (IHC):Sensitivity = ~100%, but a small % of acutely infected calves are positive.
2. Ag-capture ELISA:Sensitivity = ~100%, but a small % of acutely infected calves are positive
3. PCR:Sensitivity = ~100% on individuals and pools. May also detect acute infections
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• Producers notch calves at ear-tagging.• Place notches in numbered tubes with saline
and freeze.• After all calves have been notched, send
frozen notches to VDL.• At VDL, ear notch fluids for each herd
pooled into 1 or more pools of ≤100.• RT-PCR for BVDV RNA on each pool.
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VIBSP Testing Strategy
•If pool is PCR-negative, no PIs present.•If pool is PCR-positive
–Ag-capture ELISA will be done on fluid from each calf in pool.–Ag-capture ELISA positive calves should be confirmed PI by re-testing (buffy coat PCR) >30 days after notches obtained.
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•Aim:–Estimate the prevalence of BVDV PI in Iowa beef breeding herds–Evaluate the efficacy of pooled testing to cheaply screen herds for BVDV
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Enrollment phase: Winter 2005
Study phase: Spring and Summer 2006
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•Enrollments to date–Calves -15000–Producers-102 average herd size ~150–Vets-51–Clinic-40
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•Issues to date–What to do with fall and spring calving herds–What to do with spread out calving seasons–Concern about ears appearance after notching
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•Do you produce seedstock? 36/71•Do you background or feedlot calves? 49/71•Do you purchase replacements, bulls or cows?70/71•Do you exhibit etc? 38/71*
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•Do you have nose to nose contact?53/71•Do other animals have contact with cattle feed etc ? 71/71•Conception > 90%? 60/71•Weaning rate> 90%?62/71•BVDV vaccination yearly? 64/71
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•81% of non BVDV herds vaccinate?•90% of herds diagnosed with BVDV vaccinate?
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•Project issues of the outcomes–Sensitivity of Pooled PCR–Risk factor analysis–Prevalence estimates
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•Sensitivity–The PCR test on the calf subset–The calf subset as a herd test
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•Causes of imperfect sensitivity in the calf subset
–Excessive dilution–Inhibition
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•100 of 400 samples•Prevalence of false-ve pools- 1% •ACE=95% sensitive & 100% specific
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•If all samples test negative, there is 95% confidence that the true prevalence is false negative is < 2.76%
–1- false negative prevalence=sensitivity–95% confident the sensitivity of pooled PCR is greater than or equal to 97.24%
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•100 of 400 samples; prevalence of false -vepools- 1%
–100% sensitivity & 100% specificity of ACE–If all samples test negative, there is 95% confidence that the true prevalence is < 2.1%
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BVDV -
BVDV+
4 PVP=39/43=91%Test result 39+
PVN=356/357=97%1 356_
40 (sen=97%) 360 (spec-99%)
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39
BVDV+ BVDV -
1
0
360
40 (sen=97%)360 (spec-100%)
PVN=360/361=99.7%
PVP=39/39=100%Test result +
_
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•99% sensitivity & 100% specificity of ACE–From an infinite population testing 300–If all samples test negative, there is 95% confidence that the true prevalence is < 0.01.
•Prevalence estimates from: http://www.ausvet.com.au/epitools/content.php?page=Freedom
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•Sensitivity of the calf subset as a herd level diagnosis
–Follow-up of herds that decide to herd test after finding a calf PI–Next funding phase
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•Project issues of the outcomes–Risk factor analysis–Prevalence estimates
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BVDV -
BVDV+
160044%
20050%
Exposure factor
RR- 1.1
95%CI - 1.01-1.47
+
200 2000
3600 (90%)
_
400 (10%)
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2050%
BVDV+ BVDV-
16044%
Exposure factor
RR-1.1
95%CI - 0.6 - 2.1
+
20 200
360 (90%)
_
40 (10%)
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2870%
BVDV+ BVDV-
18050%
Exposure factor
RR-1.4
95%CI – 1.1 – 1.8
+
12 180
360 (90%)
_
40 (10%)
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_+
12244%
6050%
Exposure factor
RR - 1.2
95%CI- 0.8-1.6
+
60 158
280 (70%)
_
120 (30%)
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_+
8040%
10050%
Exposure factor
RR-1.2
95%CI – 1.02-1.48
+
100 120_
200 (50%) 200 (50%)
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_+
14050%
8066%
Exposure factor
RR-1.6
95%CI – 1.1-2.2
+
40 140_
120 (30%)
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•Where next?–Funding for longitudinal animal health and enterprise analysis–Collaboration and sample supply
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AM O’Connor, BVSc, MVSc, DVSc, MACVScS Sorden, DVM, PhD, ACVP (Dip)
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•The American Heritage® Dictionary of the English Language, Fourth EditionCopyright © 2000 by Houghton Mifflin Company•http://www.census.gov/population/www/socdemo/school.html