OIE Collaborating Centre Reports
Activities in 2012
Title of Collaborating Centre:
Address of Collaborating Centre
Validation, Quality Assessment and
Quality Control of Diagnostic Assays and
Vaccine Testing for Vesicular Diseases in
Europe
Veterinary and Agrochemical Research
Centre (CODA-CERVA)
Groeselenberg 99, B-1180 Ukkel
BELGIUM
Tel.:
+32.2.379.04.00
Fax:
+32.2.379.06.66
e-mail address:
website:
Name of Director of Institute
(Responsible Official):
kris.de.clercq@coda-cerva.be
http://www.coda-cerva.be
Dr. Pierre Kerkhofs
Name (including Title and
Position) of Head of the
Collaborating Centre (formally
OIE Contact Point):
Dr. Kris De Clercq
Name (including Title and
Position) of writer of this report
(if different from above)
Dr. Kris De Clercq
Annual reports of OIE Reference Centres, 2012
Head of Unit
Head of Unit
1
Validation, Quality Assessment and Quality Control of Diagnostic Assays and Vaccine Testing for Vesicular Diseases in Europe
Summary of activities specifically related to the mandate of
OIE Collaborating Centres
1.
Activities as a centre of research, expertise, standardisation and dissemination of techniques
within the remit of the mandate given by the OIE
1.1. Diagnostic Assays for foot-and-mouth disease (FMD): Evaluation of NSP-ELISAs with samples from
animals infected/vaccinated with foot-and-mouth disease virus (FMDV) strains belonging to the 3 SAT
serotypes.
The study was done in full collaboration between this OIE CC, the Onderstepoort Veterinary Institute in SouthAfrica (OIE FMD Reference Laboratory) and the Istituto Zooprofilattico Sperimentale della Lombardia e
dell'Emilia Romagna (IZSLER), Brescia, Italy (OIE SVD Reference Laboratory). The genetic heterogeneity of the
FMDV 3ABC-coding region of the SAT type viruses indicates that the current tests may not be sensitive in the
southern African sub-region. Thus, developing a 3ABC ELISA using SAT-type antigens might exhibit a higher
level of sensitivity towards the SAT types. This study also aimed at verifying whether the current available FMD
NSP-ELISAs could be used in regions where vaccination is practised against FMDV strains belonging to the 3
SAT serotypes and/or these strains are circulating.
A truncated peptide of a SAT-type 3ABC (tr3ABC) was successfully replaced in the type O recombinant 3ABC
antigen of the IZSLER DIVA ELISA format (IZSLER-3ABC-ELISA). Validation of the assay included testing the
SAT-adapted ELISA (SAT-3ABC-ELISA) against a total of 1946 bovine sera: naive (n=601), FMDV
experimentally infected (n=215), and FMDV vaccinated and sampled during a SAT 1 outbreak (n=1130). Samples
were tested in parallel with the IZSLER-3ABC-ELISA and the commercial PrioCHECK® FMDV NS (formally
Ceditest FMDV-NS). Results were compared using Cochran’s Q and McNemar’s tests and a Bayesian latent class
analysis was performed to estimate sensitivity and specificity within vaccinated cattle group.
Specificity of the three ELISAs within the naïve cattle group was similar i.e 99.3% (Priocheck-ELISA), 98.0%
(IZSLER-3ABC-ELISA) and 96.5% (SAT-3ABC-ELISA). The sensitivity within the experimentally infected
group varied significantly with Priocheck-ELISA (93.3%) > IZSLER-3ABC-ELISA (85.6%) > SAT-3ABCELISA (76.7%). However, within the field vaccinated group exposed to-SAT1 infection, the sensitivity for the
SAT-3ABC-ELISA was the highest (89.3%) followed by IZSLER-3ABC-ELISA (72.2%) and Priocheck-ELISA
(69.3%). Additionally, the specificity was >96% for all assays within this group of cattle. It was concluded that the
accuracy of SAT-3ABC-ELISA was comparable to the commercial Priocheck kit and the IE assay. Thus, the SAT3ABC-ELISA ELISA is a viable alternative to the more expensive commercial kits. These results were presented
at the open EUFMD Research Group meeting in Jerez de la Frontera, Spain, 29-31October 2012 and will be
published in 2013.
1.2. Vaccine Testing: Refinement of a Foot-and-Mouth Disease Infection Model in Guinea Pigs (GPs) for the
evaluation of FMDV vaccines.
The final goal of this study was to develop a small animal model to assess the in vivo activity of vaccines and
antiviral products against FMDV. Guinea pigs (GP) are widely used to evaluate (experimental) FMDV vaccines,
but a standardized model suited to substitute the current PD 50 FMDV vaccine efficacy tests in cattle remains to
be validated. Firstly, we aimed to refine the GP model by investigating several infection/protection parameters.
Dunkin Hartley GPs were vaccinated IM with 125µl, 500µl or 2000µl of a purified DOE O 1 Manisa vaccine.
Three independent experiments were performed using a total of 8 animals per vaccine dose and 10 unvaccinated
control animals. Three weeks later, all animals were intraplantary challenged with GP-adapted O 1 Manisa and
euthanized at 3 or 4 dpi. Clinical signs were recorded daily and serum collected at 2 dpi was examined with realtime RT-PCR.
Within 2 dpi, 10/10 unvaccinated control animals developed vesicular lesions at the inoculation site and severe
reddening with possible vesicles at the other footpads, 8/10 animals developed mouth lesions. All control animals
showed depression and weight loss (9.0±6.8%), but no fever. Mean Cp values in serum were 20.5±2.6. Twentyone vaccinated animals developed moderate reddening with possible vesicles at the inoculation site, and 1/8
animal in the 125µl and 2/8 animals in the 500µl dose group showed reddening at the other footpads. All
vaccinated animals were protected against depression, weight loss and mouth lesions. In every dose group, 6/8
animals had viral RNA in their serum, but Cp values were significantly lower than in unvaccinated control animals
(39.2±2.8, 36.2±4.7 and 38.3±3.5 for the 125µl 500µl 2000µl dose groups, respectively). Serum collected for
serological tests, oral swabs and a series of organs collected for real-time RT-PCR are investigated at present.
It was concluded that the present standardized and reproducible GP infection model will be further validated in
order to allow substitution of target species in FMDV vaccine and preliminary antiviral efficacy tests.
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Annual reports of OIE Reference Centres, 2012
Validation, Quality Assessment and Quality Control of Diagnostic Assays and Vaccine Testing for Vesicular Diseases in Europe
These results were presented with a poster at the open EUFMD Research Group meeting in Jerez de la Frontera,
Spain, 29-31October 2012 and will be published in 2013.
1.3. Testing of FMD vaccine alternatives: Application of a refined foot-and-mouth disease infection model in
Guinea Pigs (GPs) for antiviral drug testing.
The CODA-CERVA (VAR) in collaboration the Rega Institute, Catholic University Leuven Belgium and Okapi
Sciences Belgium, optimised the in vitro anti-FMD activity of different classes of small molecules. Early antiviral
lead compounds will be evaluated in guinea pigs (GP). Preparatory, the activity of the pyrazinecarboxamide
derivate T-1105, a molecule with known anti-FMD activity (Furuta et al., 2008), was assessed in FMDV-infected
GP. Dunkin Hartley GPs were administered with T-1105 (200mg/kg po, twice daily) for 5 consecutive days. Two
independent experiments were performed using a total of 16 treated and 8 untreated animals. One hour after the
first administration, all animals were inoculated with GP-adapted O 1 Manisa. Eight treated and all untreated
animals were euthanized at 4dpi, the remaining at 10 dpi. Clinical signs were recorded daily. Serum, collected at 2
and 4 dpi was examined with real-time RT-PCR.
Serum concentrations peaked at ~140µM 2 hours after oral administration of 200mg/kg (in vitro EC 50 =25µM).
All untreated control animals showed severe generalized infection. Mean Cp-values in serum at 2 and 4 dpi were
24.39±8.78 and 30.55±4.15, respectively. Disease severity was markedly less pronounced in treated animals. At 2
dpi, 16/16 treated animals had moderate lesions at the inoculation site and 5/16 slight reddening at the other
footpads. At 4 dpi, 4/16 and 1/16 animals developed vesicular lesions at the inoculation site and mouth,
respectively. At 2dpi, 4/8 animals tested RNA positive in serum (mean Cp-value of positives 37.00±5.36). At 4dpi,
3/8 animals tested RNA positive in serum, of which 1 that was positive at 2 dpi (mean Cp-value of positives
34.56±7.47). Viral RNA load in serum, oral swabs and various organs is currently being quantified. It was
concluded that T-1105 offered substantial clinical and virological protection against O 1 Manisa infection in GPs.
The utility of the GP model for the preliminary evaluation of anti-FMD drugs is confirmed.
These results were presented with a poster at the open EUFMD Research Group meeting in Jerez de la Frontera,
Spain, 29-31October 2012 and will be published in 2013.
1.4. Detection of virus in semen: Application of a validated duplex real-time RT-PCR for the detection of
bluetongue virus in semen of rams.
This study investigated if viral RNA was detectable in the semen of rams clinically infected with bluetongue virus
serotype 8 (BTV-8) by of a validated duplex real-time RT-PCR, and to what extent the amount detected may be
predictive of sperm quality. Semen samples were collected on six occasions from 93 BTV-8 infected rams
involved in two longitudinal (n = 12 and 27, respectively) and one cross-sectional (n = 54) field study. Semen
quality was assessed in terms of mass motility, concentration of spermatozoa, percentage of living and dead
spermatozoa as well as cytological features. An overall semen quality score (SQS) was established. Depending
upon the studied population, BTV RNA was detected in 75–100% of semen samples at initial testing 25–57 days
post-observation (DPO) of clinical signs, and was detectable up to 116 DPO in a proportion of rams undergoing
repeated sampling. Semen quality variables were significantly altered following natural BTV-8 infection and
correlated with the amount of BTV RNA present. The SQS did not return to normal when virus was no longer
detectable, suggesting that clearance of BTV precedes full recovery of sperm quality. In conclusion, viral RNA
may be transiently recovered from the semen of BTV-8 affected rams and may serve as an indicator in predicting
ram breeding potential following natural infection. This study was done in collaboration with the Faculty of
Sciences of Namur, Belgium and the Veterinary Faculty at the University of Liège and the results were
disseminated via publication in the scientific journal The Veterinary Journal (Leemans et al., 2012).
2.
Proposal or development of any procedure that will facilitate harmonisation of international
regulations applicable to the surveillance and control of animal diseases, food safety or animal
welfare
2.1. Development of any procedure that will facilitate harmonisation of international regulations applicable
to animal welfare: Alternative in vitro FMD vaccine potency test based on vaccine payload.
This study was done in full collaboration with the OIE CC CODA-CERVA, the OIE FMD Reference Laboratory
ARRIAH of the Russian Federation, the France National Reference Laboratory for FMD ANSES and the
laboratory of the vaccine producer Mérial, France.
This study addresses alternatives to the standard European and South-American in vivo vaccine potency test for
cattle. Alternative serology-based vaccine potency tests have been published. Now the effect of antigen payload on
vaccine potency was investigated. A single batch of the FMD O 1 Manisa reference strain was used to produce 5
batches of monovalent double-oil emulsion vaccine with different antigen payloads. Each vaccine batch was tested
in triplicate in a standard PD 50 test. Cattle serum samples were collected at 21 days post vaccination and antibody
Annual reports of OIE Reference Centres, 2012
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Validation, Quality Assessment and Quality Control of Diagnostic Assays and Vaccine Testing for Vesicular Diseases in Europe
titers were determined. The in vivo observations and serological data were analysed and logistic regression models
were constructed for assessment of alternative vaccine potency.
The challenge trials resulted in overall PD 50’s of 8.8, 4.3, 10.5, 20.1 and 17.7 for the respective antigen payloads
of 25%, 50% 100%, 200% and 400%. Different logistic regression models were compared for their ability to
differentiate between protected and unprotected animals. The model including all parameters (antigen payload,
vaccine dose and antibody titer) had a sensitivity and specificity of 76% and 90%, respectively. For the models
based solely on antibody titer or antigen payload the sensitivities were 79% and 47% and the specificities were
80% and 81%, respectively. For models created with the subset of data containing only animals that received the
full 2ml vaccine dose the model combining antigen payload and antibody titer performed best (sensitivity: 97%,
specificity: 100%), however differences with the model correlating only antibody titer to in vivo protection were
small (sensitivity: 93%, specificity: 100%) and the model based solely on antigen payload had a lower sensitivity
of 80%. It was concluded that vaccine potency can be assessed with alternative tests to the in vivo test. However
the confidence of the estimated potency will be influenced by the characteristics of the chosen alternative vaccine
potency test. These results were presented at the open EUFMD Research Group meeting in Jerez de la Frontera,
Spain, 29-31October 2012 and will be published in 2013.
2.2. Development of any procedure that will facilitate harmonisation of international regulations applicable
to animal welfare: Alternative in vitro FMD vaccine potency test based on post-vaccination serology.
This study was done in full collaboration with the OIE CC CODA-CERVA, the OIE FMD Reference Laboratory
ARRIAH of the Russian Federation, the OIE CC Institute of Diagnostic Virology, Friedrich-Loeffler-Institut (FLI)
Germany, and the laboratory of the vaccine producer MSD Animal Health, Germany.
Foot-and-mouth disease (FMD) vaccine potency testing involves hundreds of animals each year. Despite
considerable efforts during the past decades, a challenge-free alternative vaccine potency test to replace the in vivo
potency tests has not been implemented yet. The aim of this study was to further characterize the properties of
serological vaccine potency models. Logistic regression models were built for 5 serological assays from 3 different
laboratories. The serum samples originated from 5 repeated PD50 vaccine potency trials with a highly potent
A/IRN/11/96 vaccine. Receiver Operating Characteristic analysis was used to determine a serological pass mark
for predicting in vivo protected animals. Subsequently, an estimated PD50 was calculated and the serotype
dependency of the logistic models was investigated. Although differences were observed between the laboratories
and the serological assays used, the logistic models accurately predicted the in vivo protection status of the
animals in 74–93% of the cases and the antibody pass levels corresponded to 84–97% of protection, depending on
the serological assay used. For logistic models that combine different serotypes, the model fit can be increased by
inclusion of a serotype factor in the logistic regression function. It was concluded that the in vitro estimated PD50
method may be at least as precise as the in vivo PD50 test and may accurately predict the PD50 content of a
vaccine. However, the laboratory-effect and the serotype dependency should be further investigated. These results
were disseminated via publication in the scientific journal Vaccine (Willems et al., 2012).
2.3. Development of a post-vaccination surveillance procedure to demonstrate the absence of virus
circulation.
A surveillance procedure was proposed to check the absence of a virus in a cattle population after an epidemic, in
casu the absence of bluetongue virus. This method is also applicable to other viruses. Bluetongue virus serotype 8
(BTV-8) emerged in Central Western Europe in 2006 causing a large scale epidemic in 2007 that involved several
European Union (EU) countries including Belgium. As in several other EU member states, vaccination against
BTV-8 with inactivated vaccines was initiated in Belgium in spring 2008 and appeared to be successful. Since
2009, no clinical cases of Bluetongue (BT) have been reported in Belgium and BTV-8 circulation seemed to have
completely disappeared by spring 2010. Therefore, a series of repeated cross-sectional surveys, the BT sentinel
surveillance program, based on virus detection in blood samples by means of a validated pan real-time RT-PCR
(RT-qPCR) were carried out in dairy cattle from the end of 2010 onwards with the aim to demonstrate the absence
of BTV circulation in Belgium. This study includes the first two sampling rounds of this BT sentinel surveillance
program carried out in October–November 2010 and January–February 2011. In addition, the level of BTVspecific maternal antibodies in young non-vaccinated animals was monitored and the level of herd immunity
against BTV-8 after 3 consecutive years of compulsory BTV-8 vaccination was measured by ELISA.
During the 1st sampling round of the BT sentinel surveillance program, 15 animals tested positive and 2 animals
tested doubtful for BTV RNA by RT-qPCR. During the 2nd round, 17 animals tested positive and 5 animals tested
doubtful. The positive/doubtful animals in both rounds were re-sampled 2–4 weeks after the original sampling and
then all tested negative by RT-qPCR. These results demonstrate the absence of BTV circulation in Belgium in
2010 at a minimum expected prevalence of 2% and 95% confidence level. The study of the maternal antibodies in
non-vaccinated animals showed that by the age of 7 months maternal antibodies against BTV had disappeared in
most animals. The BTV seroprevalence at herd level after 3 years of compulsory BTV-8 vaccination was very
high (97.4% [95% CI: 96.2–98.2]). The overall true within-herd BTV seroprevalence in 6–24 month old Belgian
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Annual reports of OIE Reference Centres, 2012
Validation, Quality Assessment and Quality Control of Diagnostic Assays and Vaccine Testing for Vesicular Diseases in Europe
cattle in early 2011 was estimated at 73.4% (95% CI: 71.3–75.4). The results of this study were disseminated
through a publication in in Preventive Veterinary Medicine, (Vangeel et al., 2012).
3.
Networking
a)
Maintenance of a network with other OIE Collaborating Centres designated for the same
specialty, and
The CODA-CERVA (VAR) as OIE CC is Member of the OIE/FAO FMD Reference Laboratory Network
and several scientists of our CC attended the yearly meeting of this network at Jerez de la Frontera, Spain, 12 November 2012.
As indicated above the OIE CC CODA-CERVA has collaborations with the OIE FMD Reference Laboratory
ARRIAH of the Russian Federation, the OIE FMD Reference Laboratory Pirbright Laboratory UK, the OIE
CC Institute of Diagnostic Virology, Friedrich-Loeffler-Institut (FLI) Germany, the OIE FMD Reference
Laboratory Onderstepoort Veterinary Institute in South-Africa, the OIE SVD Reference Laboratory Istituto
Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER), Brescia, Italy.
b)
Should the need arise, maintenance of a network with Collaborating Centres in other
disciplines
Collaborators of the CODA-CERVA (VAR) as OIE CC also attended the yearly meeting of the bluetongue
reference laboratory network, organised by the European CRL in Guildford, UK on 3-4 December 2012.
4.
Placement of expert consultants at the disposal of the OIE
• Dr. Kris De Clercq, as Vice-President of SCAD, participated in:
- the SCAD meetings on 13-17/02/2012 and 26-31/08/2012,
- the meetings of the ad-hoc group to evaluate the FMD Members Status and to review the OIE FMD Code
Chapter on 31/01-03/02/2012, 02-05/07/2012, 09-12/10/2012 and 09-14/12/2012.
• Dr. Kris De Clercq represented the SCAD at the Selection Committee for the OIE international call for tender
relating to the establishment of a Rabies Vaccine Bank in Asia, 06 January 2012.
• Dr. Kris De Clercq, as Vice-President of SCAD, took part in a Mission to the Andean region (Colombia,
Ecuador, Peru) to evaluate and discuss the FMD control programme and the regional approach in the Andean
region on 8-21 January 2012.
• Dr. Kris De Clercq organised and attended the FAO Workshop on ‘Approved vaccine producers’ in CODACERVA, Belgium, 12-13 March 2012.
• Dr. Tom Willems of the OIE CC CODA-CERVA co-organised and attended the GFRA Workshop on
Surveillance, Epidemiology, Vaccination and Control of Foot-and-Mouth Disease, Item on ‘FMD Vaccine
Efficacy’ Hosted by the OIE FMD Reference Laboratory ARC-OVI in Hazyview, South Africa, 17 to 19 April
2012.
• Dr. Kris De Clercq, as Vice-President of SCAD, took part in a Mission to Venezuela to evaluate and discuss the
FMD control programme and the regional approach on 29/04-05/05/2012.
• Dr. Kris De Clercq, as Vice-President of SCAD, attended the OIE General Session. Paris, France, 20-25 May
2012.
• Dr. Kris De Clercq, as Vice-President of SCAD, was speaker at the OIE-FAO Global on FMD in Bangkok,
Thailand, 24-30 June 2012.
• Dr. Kris De Clercq, as Vice-President of SCAD, took part in a Mission to Mongolia to evaluate and discuss the
application fro zonal freedom of FMD on 18-24/08/2012.
• Dr. Kris De Clercq represented the OlE at the Open EUFMD meeting in Jerez de la Frontera, Spain, 29-31
October 2012.
• Dr. Kris De Clercq represented the OlE at the OIE-FAO FMD Reference laboratories meeting in Jerez de la
Frontera, Spain, 01-02 November 2012.
5.
Provision of scientific and technical training, within the remit of the mandate given by the OIE, to
personnel from OIE Member Countries
Annual reports of OIE Reference Centres, 2012
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Validation, Quality Assessment and Quality Control of Diagnostic Assays and Vaccine Testing for Vesicular Diseases in Europe
- A special technical 2 weeks training was organised by the IAEA in Vienna, Austria, in full collaboration with the
OIE CC CODA-CERVA (VAR) on Part 1: Pre-Sequencing, Sanger Sequencing and Post-Sequencing Raw Data
Analysis and Part 2: Sequence Retrieval and Sharing; Sequence Analysis and Phylogeny, from 10-21 December
2012. This training was attended by participants from Botswana, Cameroon, Dem. Rep. Of the Congo, Ethiopia,
Kenya, Mali, Nigeria, Senegal, Tanzania, Uganda.
6.
Organisation of scientific meetings on behalf of the OIE
None
7.
Coordination of scientific and technical studies in collaboration with other laboratories,
organisations or collaborating centres
7.1. FMD Vaccine Efficacy.
A Session on ‘FMD Vaccine Efficacy’ was organised by the OIE CC CODA-CERVA (VAR) and the FMD OIE
Ref. Centre in Buenos Aires, Argentina at the GFRA Workshop on Surveillance, Epidemiology, Vaccination and
Control of Foot-and-Mouth Disease, Hosted by the OIE FMD Reference Laboratory ARC-OVI in Hazyview,
South Africa, 17 to 19 April 2012 with the aim to determine the parameters influencing the efficacy of Foot-andMouth disease vaccines/vaccination.
Vaccine efficacy is considered as the effectiveness with which a vaccine can prevent disease, given that it is a
potent vaccine that is properly administered to animals that are capable of responding. The In-Production Quality
Assurance (QA) through a system of Good manufacturing practice (GMP) by the FMD vaccine manufacturers will
determine part of the vaccine potency. Vaccine strain selection is another parameter of great importance on the
vaccine potency. In this respect testing the vaccine potency (direct or indirect) and evaluating the effectiveness of
the vaccination campaign is essential. In an FMD endemic region ways of assessing the vaccination efficacy will
be sero-conversion and monitoring of virus circulation through various analytical methods including DIVA
testing. The control of the cold chain during the campaign is of utmost importance. In an FMD free with
vaccination setting the goal will be to maintain the immune barrier. Therefore the vaccination coverage should be
checked through measurement of herd. Countries free of FMD without vaccination rely on high potency vaccines
assuring a broad spectrum protection and a fast increase of the individual immunity. Therefore these aspects
should be checked to guarantee the vaccine efficacy in this particular situation. Vaccine efficacy needs to be
assessed in a harmonised and independent way.
7.2. Dr. Kris De Clercq is member of the Advisory Board for organising the FMDV antibody and virological
proficiency test by the FMD OIE Reference Laboratory at Pirbright, UK.
7.3. The OIE CC CODA-CERVA (VAR) coordinates a research project (Altandi-3) in which it collaborate with
the OIE Reference Laboratory ARRIAH of the Russian Federation, the France National Reference Laboratory for
FMD ANSES, the German National Reference Laboratory for FMD FLI and the laboratory of Mérial for vaccine
potency test evaluation and the development of indirect in vitro FMD potency tests.
7.4. The OIE-CC of the VAR is the coordinator of a FMD Research project with 14 partners. Next to 9 European
partners (Belgium, two from UK, the Netherlands, Germany, Italy, France, Denmark, Switzerland) there is also the
Chinese Veterinary Research Institute (LVRI) at Lanzou, the Argentinean Network ‘RIIDFA’, the FMD Reference
Laboratory ‘Kimron Institute’ from Israel and 2 FMDV vaccine producers: Indian Immunologicals (IIL) and
Mérial, France.
8.
Publication and dissemination of any information within the remit of the mandate given by the
OIE that may be useful to Member Countries of the OIE
Leemans J, Raes M, Vanbinst T, De Clercq K, Saegerman C, Kirschvink N. (2012). Viral RNA load in semen
from bluetongue serotype 8-infected rams: Relationship with sperm quality. Vet J. 192(3), 304-310.
Vangeel I, De Leeuw I, Méroc E, Vandenbussche F, Riocreux F, Hooyberghs J, Raemaekers M, Houdart P, Van
der Stede Y, De Clercq K (2012). Bluetongue sentinel surveillance program and cross-sectional serological
survey in cattle in Belgium in 2010-2011. Prev Vet Med., 106(3-4):235-243.
Willems T, Lefebvre DJ, Goris N, Diev VI, Kremenchugskaya SR, Paul G, Haas B, De Clercq K. (2012).
Characteristics of serology-based vaccine potency models for foot-and-mouth disease virus. Vaccine,
30(40):5849-5855.
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Annual reports of OIE Reference Centres, 2012