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OIE Reference Laboratory Reports
Activities in 2011
Name of disease (or topic) for
which you are a designated OIE
Reference Laboratory:
Address of laboratory:
Infectious bovine rhinotracheitis/
Infectious pustular vulvovagenitis
Institute of Diagnostic Virology
Friedrich-Loeffler-Institut
Südufer 10
D-17493 Greifswald-Insel Riems
GERMANY
Tel.:
+49-38351-7-1223
Fax:
+49-38351-7-1275)
e-mail address:
website:
[email protected]
www.fli.bund.de
Name (including Title and
Position) of Head of Laboratory
(Responsible Official):
PD Dr Martin Beer
Name(including Title and
Position) of OIE Reference
Expert:
PD Dr Martin Beer
Name (including Title and
Position) of writer of this report
(if different from above):
PD Dr Martin Beer
Annual reports of OIE Reference Centres, 2011
Dr Patricia König
1
Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis
Part I: Summary of general activities related to the disease
1.
2.
Tests in use/or available for the specified disease/topic at your laboratory
Test
For
Specificity
Total
Indirect ELISA
BoHV-1-antibody
(serum, plasma, milk)
BoHV-1
592
gB-blocking-ELISA
BoHV-1-gB-antibody
(serum, plasma)
BoHV-1
1229
gE-blocking-ELISA
BoHV-1-gE-antibody, marker test
(serum, plasma)
wild type BoHV-1
5518
Indirect ELISA
BoHV-2-antibody
(serum, plasma, milk)
BoHV-2
350
Neutralisation test (NT)
Plaque reduction test
BoHV-1-antibody (serum)
ruminant HV
163
Cell culture (MDBK,
KOP-R)
virus isolation (semen, swabs,
organ samples, vaccines)
replication competent virus
10
IFAT
BoHV-1 antigen (cell culture)
BoHV-1
5
PCR
genome of BoHV-1 or ruminant
herpesviruses (semen, swabs, organ
samples, vaccines)
BoHV-1
HV
49
REFLP
genome differentiation of bovine
herpesviruses (virus isolates)
HV
2
Production and distribution of diagnostic reagents
11 reference sera were collected from calves experimentally infected with different ruminant herpesviruses. In
total, more than 150 reference samples (serum and milk) are now available.
80 reference sera and 15 reference milk samples were supplied nationally to the regional laboratories, and
766 reference samples (serum: 716; milk: 50) were supplied to other OIE member countries.
Type of reagent
Amount supplied nationally
Amount supplied to other
countries
Reference sera, milk
95 ml approx. 500 ml for own use
766 ml
Part II: Activities specifically related to the mandate
of OIE Reference Laboratories
3.
International harmonisation and standardisation of methods for diagnostic testing or the
production and testing of vaccines
66 commercial BoHV-1-ELISAs were tested for batch release by the OIE reference laboratory. Two indirect
BoHV-1-ELISA tests were examined for licensing on the German market. A main focus in the field of validation
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Annual reports of OIE Reference Centres, 2011
Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis
of diagnostic tests was the evaluation of a modified commercial gE-blocking ELISA on behalf of the German
licensing body. Internationally collected data were analysed and compared to validation data generated by the OIE
reference laboratory and by German regional laboratories.
A new triplex real time PCR assay for the rapid detection and differentiation of field and vaccine strains of bovine
herpesvirus type 1 was validated and published. A broad series of swab samples, semen and tissues were examined
with the BoHV-1 specific PCR as well as with a pan-herpes PCR which detects conserved sequences of the
herpesviral DNA-dependent DNA-polymerase to elucidate potential introduction of ruminant alphaherpesviruses
into cattle herds.
a)
Establishment and maintenance of a network with other OIE Reference Laboratories
designated for the same pathogen or disease and organisation of regular inter-laboratory
proficiency testing to ensure comparability of results
There is a close contact to the other BoHV-1-OIE-reference laboratory in UK. Both methods, data and
samples were exchanged and tested.
In addition, an international cooperation with national reference laboratories in Belgium and in the
Netherlands was established to improve exchange of experiences and conferencing concerning application
and licensing of commercial diagnostic tests.
b)
Organisation of inter-laboratory proficiency testing with laboratories other than OIE
Reference Laboratories for the same pathogens and diseases to ensure equivalence of
results
The OIE reference laboratory participated in a BoHV-1 proficiency test organised by the Coordination Centre
for Veterinary Diagnostics (CCVD, Brussels, Belgium). 100% of the serum samples were correctly
characterised for the presence of BoHV-1 gE- and gB-specific antibodies.
4.
Preparation and supply of international reference standards for diagnostic tests or vaccines
The German BoHV-1 reference sera R1 (positive), R2 (weak positive), R3 (very weak positive), R31 (negative)
and R32 (negative) were supplied to laboratories in Germany, Spain, Switzerland, Great Britain, Canada, and
Italy.
5.
Research and development of new procedures for diagnosis and control
After experimental inoculation of BoHV-1 antibody negative calves with BoHV-2, transient cross reactivity in
BoHV-1 ELISAs could be induced. Serological cross reactivity between BoHV-2 and BoHV-1 might explain
epidemiologically non plausible reactions in some regions in Southern Germany where BoHV-2 is highly
prevalent. Epidemiologically non plausible singleton reactors were found to be BoHV-2 antibody positive and to
harbour BoHV-2 DNA in their trigeminal ganglia. To further elucidate a potential cross reactivity of BoHV-2
antibodies under field conditions and to collect some more data of BoHV-2 prevalences in different regions, an
indirect BoHV-2 ELISA test was developed and compared to the plaque reduction test as gold standard. The major
advantage of the newly developed ELISA is the feasibility of mass screening in affected herds and regions.
Further research efforts were started to analyse cross reactivity pattern in bovine sera, including the testing of
ganglia for herpesviral genomes by PCR.
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
Data concerning the progress of the German BoHV-1 eradication program were collected and analysed using a
detailed questionnaire. In summary, more than 89% of the cattle holdings in Germany were BoHV-1-antibody or
BoHV-1-gE-antibody free at the end of 2010. This data were compared to available data from other countries.
31 BoHV-1 outbreaks were reported by the regional veterinary authorities to the OIE in the year 2011. The cases
of reintroduction of field virus showed stable or very slightly declining prevalences in comparison to the recent
years.
Annual reports of OIE Reference Centres, 2011
3
Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis
7.
Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the
pathogen and the disease concerned
Fully ISO-certified QM system (ISO IEC 17025) is in place since more than 5 years. Certifying body changed
from AKS to the new German agency DAkkS. Regular audits (internal and external) ensure tight update schedules
of QM measures. General as well as disease specific biosafety and biosecurity rules are in place following the
biosafety/biosecurity handbook of the Friedrich-Loeffler-Institut in agreement with the national/international
legislation.
8.
Provision of consultant expertise to OIE or to OIE Member Countries
Protocols, e.g. for BoHV-1-neutralization assays and the triplex real time BoHV-1 PCR, were transferred to
several international laboratories. Expert advices concerning BoHV-1 diagnostics and BoHV-1 eradication have
been required by colleagues from Spain, Poland, and Italy.
9.
Provision of scientific and technical training to personnel from other OIE Member Countries
Technicians from German regional veterinary laboratories were invited for a diagnostic training with focus on
virus isolation, serum neutralisation test and PCR techniques.
10. Provision of diagnostic testing facilities to other OIE Member Countries
BoHV-1 isolates from Italy were analysed and characterised by the OIE reference laboratory HV-1H. Numerous
serum and milk samples from Great Britain, Switzerland and Italy were subjected to confirmatory testing.
11. Organisation of international scientific meetings on behalf of OIE or other international bodies
Not applicable
12. Participation in international scientific collaborative studies
Not applicable
13. Publication and dissemination of information relevant to the work of OIE (including list of
scientific publications, internet publishing activities, presentations at international conferences)

Presentations at international conferences and meetings
None

Scientific publications in peer-reviewed journals
Wernike K, Hoffmann B, Kalthoff D, König P, Beer M. Development and validation of a triplex real-time PCR
assay for the rapid detection and differentiation of wild-type and glycoprotein E-deleted vaccine strains of Bovine
herpesvirus type 1. J Virol Methods. 2011 Jun; 174(1-2):77-84.

Other communications
Beer, M, Koenig, P, and Höreth-Böntgen, D. 2010. BHV-1. Tiergesundheitsjahrbuch 2010, Friedrich-LoefflerInstitut, Germany.
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Annual reports of OIE Reference Centres, 2011