Molecular Diagnostics in Veterinary Medicine Helps to ... · Molecular Diagnostics in Veterinary...
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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
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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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
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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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
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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BVD virus:BVD virus: how to outwit the hosthow to outwit the host’’s immune systems immune system
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BVD virus:BVD virus: how to outwit the hosthow to outwit the host’’s immune systems immune system
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BVD virus:BVD virus: how to outwit the hosthow to outwit the host’’s immune systems immune system
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BVD virus:BVD virus: how to outwit the hosthow to outwit the host’’s immune systems immune system
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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)
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
BVD virus:BVD virus: how to outwit the hosthow to outwit the host’’s immune systems immune system
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Mucosal Disease:a virological accident – with a lethal outcome
Cells not destroyedCells not destroyed cytopathogeniccytopathogenic
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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U. Braun, ZürichA. Tontis, Bern
A. Tontis, Bern A. Tontis, Bern
©E. Peterhans, Institut für Veterinär-Virologie, Uni 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
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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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
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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 !
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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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©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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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©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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)
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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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 ??
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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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
Limit of detectionLimit of detection
Limit of detectionLimit of detection
© 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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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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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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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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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 !
33©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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 !
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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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 !
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern
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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
©E. Peterhans, Institut für Veterinär-Virologie, Uni Bern