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Molecular Diagnostics in Veterinary Medicine Helps toMolecular Diagnostics in Veterinary Medicine Helps toEradicate a Cattle Disease With a WorldEradicate a Cattle Disease With a World--Wide DistributionWide DistributionErnst Peterhans, Institute of Veterinary Virology, University of Bern

Biological background: the BVD virus and the diseasethe BVD virus and its host its host population

Epidemiological background: BVD is arguably the most widespread cattle disease

world-widethe situation in Switzerland and Europe

Is it possible to eradicate BVD ?diagnostics: the approachdiagnostics: the techniquesthe Swiss BVD eradication campaign

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The bovine viral diarrhea virus (BVDV)The bovine viral diarrhea virus (BVDV)

Genus Pestivirus, Family Flaviviridae• the closest relatives of BVDV are Border Disease

virus of sheep and and Classical Swine Fever virus•• a more distant relative is Hepatitis C virus of a more distant relative is Hepatitis C virus of

humanshumans•• two biotypes: two biotypes: cytopathogeniccytopathogenic / / nonnon--cytopathogeniccytopathogenic

100 nm

…you can park more than 100 million of theseviruses on a parking lot of 1mm2 area

©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern

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2 2 types of infection: types of infection: transienttransient (acute)(acute) // persistentpersistent

BVDV and its hostBVDV and its host

AB-neg → pos

Ncp or cpNcp or cp BVDVBVDV

AB-neg → pos

Fetal InfectionFetal Infection::ncpncp BVDV BVDV only!!only!!

Insemination

PI Ab+

gestationmonths 5-9

Early death Persistently infectedSpreads virus lifelong

immunotolerantimmunotolerant

malformationsAbortion

Month 1

Months 2- 4


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BVDV and host:BVDV and host: transient infectiontransient infection

Individual animal: moderate diarrhea, coughing

Beef production: intermittent diarrhea with coughingincreases use of antibiotics

Frequently observedFrequently observed

Rarely observedRarely observed

Bleeding in internal organs:USA/Canada 1989-92: 40‘000 animals perished of 120‘000 infected


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Acute hemorrhagic BVD


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BVDV and its hostBVDV and its host

AB-neg → pos


AB-neg → pos


Insemination

PI Ab+

gestationmonths 5-9




Month 1

Months 2- 4


What is so special about the persistent infection ? What is so special about the persistent infection ?

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BVD virus:BVD virus: how to outwit the hosthow to outwit the host’’s immune systems immune system


8©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern


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No antibodies against BVDNo antibodies against BVD--VirusVirus-- but virus carrier for life !but virus carrier for life !

candidate for lethalcandidate for lethalMucosalMucosal DiseaseDisease

AntibodyAntibody--positivepositive(protected)(protected)



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Mucosal Disease:a virological accident – with a lethal outcome

Cells not destroyedCells not destroyed cytopathogeniccytopathogenic


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U. Braun, ZürichA. Tontis, Bern

A. Tontis, Bern A. Tontis, Bern


Mucosal Disease:a virological accident – with a lethal outcome

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Persistent infection: the consequencesPersistent infection: the consequences

ca. 12,000 animals


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AB-neg → pos


AB-neg → pos


Insemination

PI Ab+

gestationmonths 5-9




Month 1

Months 2- 4


Why are there two types of Why are there two types of infection?infection?

Transient infection

Persistentinfection

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Why should the virus bother to have two Why should the virus bother to have two types of infection (transient and persistent) ?types of infection (transient and persistent) ?

Animals protected from infectionAnimals protected from infection BVD virus disappears from herdBVD virus disappears from herd©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern

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Persistent infection is a Persistent infection is a ““life insurancelife insurance””for the virus !for the virus !


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Persistently infected animals perpetuate and spread the Persistently infected animals perpetuate and spread the virus in the cattle populationvirus in the cattle population

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Epidemiology of BVDEpidemiology of BVD in in SwitzerlandSwitzerland

No ABs to BVDV (susceptible) 40%

ABs to BVDV (protected) 60%

Persistently infectedPersistently infected 0.70.7%%

(at least on PI animal on every eighth farm)(at least on PI animal on every eighth farm)


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BVD ERADICATION in Switzerland: criteria fulfilled ?BVD ERADICATION in Switzerland: criteria fulfilled ?

• Biology of infection known in sufficient detail √

• Epidemiology of BVD in Switzerland known √

• Unequivocal identification of animals √

• Concept of eradication clear √

• Diagnostic methods established ??


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BVD-Diagnostics: what is important ?

1. Persistently infected animals are crucial for persistence ofBVD virus in population

2. Prevalence of persistently BVD virus-infected animals is low

persistently infected animals must be detected withpersistently infected animals must be detected withthe highest possible sensitivity and specificitythe highest possible sensitivity and specificity

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Antigen-ELISA Real-time RT-PCRdetects protein detects nucleic acid

sensitivity ++ +++

specifity ++ +++

material: ear notch ear notch, blood, milkautomatized: yes yespools no yes

BVD-Diagnostics: two major methods

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Sensitivity of different methods of virus detectionA

mou

nt o

f vi

r us

in b

l ood Intake of Intake of colostrumcolostrum

ImmunohistologyImmunohistologyRealReal--timetime--RTRT--PCRPCR

AGAG--ELISA IELISA I

NS23NS23

Diagnostic gapDiagnostic gap

Limit of detectionLimit of detection

Diagnostic gapDiagnostic gap

AGAG--ELISA IIELISA II

EERNSRNS



© Institut für Veterinär-Virologie, Uni Bern

{Time

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© Institut für Veterinär-Virologie, Uni Bern

0.1

Antilope

BDV-3

BDV-2

BVDV-2

BVDV-1

BDV-1

Pyrenäen Gemse

Tunesische Schafe

Giraffe

BrasilianischerWasserbüffel

KSP

a

a

a

a

ac

i

jh

fg

b

b

b

b

b

kd

e

GifhornCH-BD2

Gi-1

Gi-6Gi-6

Gi-4

F

SH9

CH-R2000/95

CH-SM01/11CH-Maria

CH-SM05/27

CH-S03/897 CH-SM09/20

KS86

-1nc

pDe

er-G

B1

Deer-NZ1

519NADL C24V

C86

G

A

100

100 100

100

100100

100

100100

100

78

73

66

8177

90

7348

100

100

93

100

100

100

100

100

100100

100

100

100

100

100

100

100

83

LJ

W

23/15

RIT 4350

Osloss

26-V639

CH-Suwa

CH-Böhni

890

CH-BD1

17385/00 AZ 79 466 T1801/1L83/L84

X818

137/4

V2536/2

Alfort-T

Brescia

C-strain

BM01

SN1T

PG-2

Giraffe-1

D32/00 HoBi

Brz buf 9

CH-KaHo/cont

Schweinfurt

Development of a real-time RT PCR method for BVDV detection

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Real-time RT-PCR method for BVDV detectiontest originally developed by Swiss BVD reference laboratory, based on sequence information of >250 BVD field virus strains :Collaboration with Qiagen led to significant improvement of all test characteristics:

Exceptionally high sensitivity: detection limit close to Exceptionally high sensitivity: detection limit close to theoretical limit: 1.5 to 3 genome copies theoretical limit: 1.5 to 3 genome copies

No diagnostic gap: test can be used in animals of No diagnostic gap: test can be used in animals of all agesall ages

Test detects all pestiviruses that have been observed Test detects all pestiviruses that have been observed in cattle to date in cattle to date Suitable for automatized virus detection of virus in Suitable for automatized virus detection of virus in milk, blood, ear notches: test suitable for milk, blood, ear notches: test suitable for mass screeningmass screeningNo interference by antibodies: testing of No interference by antibodies: testing of pooled samplespooled samples::

negative poolsnegative pools animals uninfectedanimals uninfectedpositive pools positive pools identify !identify !

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Swiss BVD eradication programme

I Detect and eliminate persistently infected animals

II Detect and eliminate persistently infected animals born during years 1 and 2 after stage I

III monitor herds test selected animals for ANTIBODY

Diagnostic method adapted to stage of eradication: Diagnostic method adapted to stage of eradication:

test test allall animals for VIRUSanimals for VIRUS

testtest allall animals for VIRUSanimals for VIRUS

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The logistics of the Swiss BVD eradication campaign

In the first stage (3 months), appr. 1.5 Million animals will be tested for virus

Farms:All samples labelled with barcode (milk, ear notches, blood), linked with animal data base

Laboratory:recording of sample, pooling, RNA extraction, real-time RT-PCR, reporting of negative pools, identification of virus-positive animals in positive poolsreporting of virus-positive animals:

„robotics & electronics“

This is possible only when all steps of the process are streamliThis is possible only when all steps of the process are streamlined ! ned !


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The logistics of the Swiss BVD eradication campaign

In the first stage (3 months), appr. 1.5 Million animals will be tested for virus

Farms:All samples labelled with barcode (milk, ear notches, blood), linked with animal data base

Laboratory:recording of sample, pooling, RNA extraction, real-time RT-PCR, reporting of negative pools, identification of virus-positive animals in positive poolsreporting of virus-positive animals:

„robotics & electronics“

This is possible only when all steps of the process are streamliThis is possible only when all steps of the process are streamlined ! ned !


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Situation in the US:


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S, SF, N, DK close to BVD eradication

(D) national campaign in planning stage

(F) regional programmes

(A) national campaign started 2004

(NL) eradication by breeding assoc.

(I) regional programmes

??

Control of BVD: Situation in Europe


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STOPSTOPBVDBVD

National campaign to start autumn 2007

www.bvdwww.bvd--info.chinfo.ch

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