OIE Reference Laboratory Reports
Activities in 2011
Name of disease (or topic) for
which you are a designated OIE
Reference Laboratory:
Bluetongue
Address of laborator:
100 Old Soutpan Road
Onderstepoort
0110
Tshwane
SOUTH AFRICA
Tel.:
Tel.: (+27 12) 529 9117
Fax:
Fax: (+2712) 529 9418
e-mail address:
LubisiA@arc.agric.za
website:
http://www.arc.agric.za
Name (including Title and
Position) of Head of Laboratory
(Responsible Official):
Dr Baratang Alison Lubisi
Name(including Title and
Position) of OIE Reference
Expert:
Dr Baratang Alison Lubisi
Name (including Title and
Position) of writer of this report
(if different from above):
Dr Baratang Alison Lubisi
Annual reports of OIE Reference Centres, 2011
1
Bluetongue
Part I: Summary of general activities related to the disease
Activities of the OIE Bluetongue (BT) reference laboratory at the ARC-OVI for the report period included the provision
of diagnostic services (Table 1) and routine disease certification of ruminants for breeders and exporters of susceptible
live animals and embryos. Animal species tested included bovine, caprine, ovine, giraffe, kudu, oryx, eland, gemsbok,
rhinoceros, sable, buffalo and springbok. Other activities included performance of research aimed at improving the
currently utilised Culicoides species trap and control methods, and generating a database of BT virus (BTV) full
genome sequences. These activities are aligned with the Agricultural Research Council’s (ARC) strategic objective on
enhancing the ability of the agricultural sector to manage and mitigate agricultural risks.
Table 1 Results of BT diagnostic services provided for South Africa
Province
No. Tested
Positives/Tests conducted
cELISA
CFT
nRT-PCR
Gauteng
682
222/493
22/189
8/16
Limpopo
56
19/23
5/33
0
North West
411
58/118
36/293
2/8
Northern Cape
35
22/33
1/2
2/22
KZN
9
6/8
0/11
0
Free State
133
82/132
0/1
8/38
Eastern Cape
303
123/179
12/124
3/68
Mpumalanga
150
4/6
65/144
0/1
Western Cape
1069
583/1055
10/14
29/60
Twenty one submissions consisting of varying numbers of blood in EDTA tubes and tissues were submitted for virus
isolation. These were mostly blood from vaccine reactors detected by PCR. Only 2 isolations were made.
A panel of 5 ovine blood samples were sent to the Western Cape Provincial Veterinary Laboratory in Stellenbosch, for
a virus isolation inter-laboratory test. Results are pending.
1.
Test(s) in use/or available for the specified disease/topic at your laboratory
Test
For
Specificity
Total
cELISA
Antibody
Group
2047
CFT
Antibody
Group
811
Serum Neutralisation (SN)
Antibody
Group
48
PCR
Genomic material
Group
203
ECE and BHK cell culture
Virus isolation
Group
21 submissions constituted
by varying numbers of blood
and tissues
Diagnostics project leaders: B.A Lubisi - BVMCh.MSc (classical virological methods) and Marco Romito –
BVSc.MSc (molecular based diagnostics).
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Annual reports of OIE Reference Centres, 2011
Bluetongue
2.
Production and distribution of diagnostic reagents
Type of reagent
Amount supplied nationally
(including for own use)
Amount supplied to other
countries
Control positive serum
None
N/A
Antigens for SNT
None
N/A
N/A - Not applicable for the report period
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
I. Participation in an International Proficiency Test Scheme
The ARC-OVI participated in the Veterinary Laboratories Agency’s (VLA) VetQAS proficiency test scheme for
Bluetongue serology in September 2011. The laboratory tested the provided panel of sera using a commercial
cELISA kit and obtained correct results (Table 1).
Table 1 Results of 8433/SE PT0138: Bluetongue virus (BTV) distributed on 20/09/2011
Samples
a)
(VLA) VetQAS
ARC-OVI
cELISA
Source not stated
cELISA
Commercial kit
Intended
% Inhibition
Result
% Inhibition
11/7693
Negative
Not stated
Negative
0.679
11/7694
Positive
Not stated
Positive
0.348
11/7695
Positive
Not stated
Positive
0.330
11/7696
Positive
Not stated
Positive
0.214
11/7697
Positive
Not stated
Positive
0.386
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
Not done.
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
Not done.
Annual reports of OIE Reference Centres, 2011
3
Bluetongue
4.
5.
Preparation and supply of international reference standards for diagnostic tests or vaccines
No.
Item
Description
Quantity
1.
Virus
BTV serotypes 1-24
Approximately 50 X 1ml vials of each serotype
2.
Sera
Anti-BTV serotypes 1-25
Approximately 50 X 1ml vials of each serotype
Research and development of new procedures for diagnosis and control
I. Full genome sequencing of BTV
The aim of the project was to obtain low passage, early reference strain viruses for amplification and full genome
sequencing. These would serve as standard reference sequences for all 24 BTV serotypes, for use by all interested
parties in various projects based on diagnostic tool development or vaccine production, and epidemiological
studies.
Early isolates of BTV, isolated between 1948 and 1984 were identified and selected. Some of these were early egg
harvests, while others were cell culture materials. The freeze dried isolates were inoculated onto BHK 21 cell
cultures and passaged once to obtain sufficient quantities of virus. In some of the cases the egg isolation material
had to be passaged twice in cell culture. Virus dsRNA was isolated and purified from all 24 serotypes of BTV.
The viral dsRNAs were obtained and purified to high degrees to ensure the efficiency of the ensuing steps. The
genomes were reverse transcribed to full-length cDNA copies using a sequence independent oligo-ligation
protocol. The cDNA was amplified using a high fidelity polymerase and a limited number of PCR cycles.
The amplified cDNAs were purified and assessed for quantity and quality and taken to the BecA hub at the
International Livestock Research Institute (ILRI) for sequencing on the 454 FLX sequencing platform. The results
were analysed at ILRI and ARC-OVI using appropriate software. Full genome data were obtained for all the
reference strains of BTV.
The genome data were analysed in two parallel streams. The first was with the de novo mapping software on the
454 FLX sequencing platform, while the second analysis employed the specialist 3rd party Genomics Workbench
software. In the latter case the software was used to sort the sequence reads according to the sequence ID tags and
then used to map the reads against a reference genome. For BTV, at least one full genome sequence was available
on GenBank. The mapping was successful for all the genome segments that do not vary between serotypes i.e. all
except segments 2, 6, and 10. These segments code for serotype specific proteins and the percentage nucleotide
identity between the reference and query sequences is lower than what is considered acceptable by the software for
reference mapping. In these cases the original 454 de novo assembly data was manually investigated for the
presence of assembled sequences of appropriate size for the particular segments. These reads were then used to
query the GenBank protein database with the blastx protocol. Once the open reading frame had been identified, the
consensus sequence was added to the genome data for the particular virus.
Involved in the project from OVI are: Otto Koekemoer (PhD) and Phelix Majiwa (PhD.
ILRI collaborators are : Drs Steve Kemp, Appolinaire Djikeng and George Michuki.
II. Development of improved molecular diagnostic tools for AHS and BTV
It was previously reported that a two hydrolysis probe based real-time PCR methods that can be utilised as a
duplex to detect all serotypes of AHSV and BTV dsRNAs in one reaction was developed, and found to be more
sensitive than the routinely employed BT nested real time RT-PCR for molecular diagnosis of the BTV. However,
when the two hydrolysis probes for AHSV and BTV were tested using all serotypes of each virus in the form of
reference and field isolates to determine group specificity, all AHS serotypes were detected but some BTV
serotypes could not be picked up.
The BTV full genome sequences mentioned under point 5.I above are now available and will be analysed for
purposes of designing new probes and re-testing all BTV serotypes.
Project leader: Otto Koekemoer (PhD).
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Annual reports of OIE Reference Centres, 2011
Bluetongue
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
I. The attraction range of the Onderstepoort 220 V light trap
Despite some limitations, suction light traps are the primary tools used for the collection of Culicoides species.
The range of attraction of the Onderstepoort light trap is unknown. An insight into the attraction range of a trap
will determine where the trap must be positioned relative to the hosts present, possible breeding sites and
environmental structures in the trapping vicinity. It will therefore contribute to a more meaningful interpretation
and comparison of results between trapping events. In the present study, the number of Culicoides midges
collected in a single trap was compared to that of midges obtained with additional traps at 1m, 4m and 8.5m away
from the first trap respectively. Treatments between sites were rotated in three replicates of 4 x 4 Latin square
design. While interactions were found between traps placed at 1m and 4m apart, no statistically significant
interactions were observed when they were 8.5m apart. The range of attraction, as indicated by the interaction
between two traps, will be between 2m and 4m. When interpreting light trap results, the limitations of the
collection method must be taken into consideration.
Project leader: Gert Venter (PhD); co-workers: D.M. Majatladi; K. Labuschagne; S.N.B. Boikanyo and L. Morey.
II. Insect repellents:
The use of insect repellents to reduce the attack rate of Culicoides midges on livestock may form an important part
of integrated control programmes against BT. The use of high frequency sound to repel Culicoides midges was
evaluated. The number of midges collected with two Onderstepoort white light traps fitted with electronic
mosquito repellents (EMRs) were compared with that of two untreated traps. Treatments were rotated in two
replicates of 4 x 4 randomized Latin square designs. Although less midges were collected in the two traps fitted
with EMRs, the average number collected was not significantly different. The EMRs were also found to have no
influence on any of the age groups of C. imicola or the species composition of the Culicoides population as
determined by the light traps. The results indicate that high frequency sound has no repellent effect on Culicoides
midges.
Project leader: Gert Venter (PhD); co-workers: K. Labuschagne; S.N.B. Boikanyo and L. Morey.
III. Comparison of light trap result with attack rate
As part of risk assessment, it is essential to monitor known vectors as well as potential vector species. In the
present study, two Culicoides insects sampling methods viz. overnight collections with the conventional
Onderstepoort light trap and mechanical aspiration (vacuuming) used at sunset on bait animals, were compared.
Culicoides imicola was confirmed as the predominant species using both trapping methods. Other species, mainly
C. bolitinos and C. gulbenkiani, were highly under represented in the light trap collections, but made a significant
contribution to the mechanical aspiration catches. The time for optimal collection differed between the trapping
methods, leading to the conclusion that mechanical aspiration is a useful addition to conventional light trap
collection and possibly the better choice when investigating insect vectors. An infection rate of 1.1% was
calculated for the midge population based on real-time quantitative reverse-transcription polymerase chain
reaction (real time qRT-PCR) assays of collected Culicoides midges, which exceeded previous estimates. This is
probably due to the increased sensitivity of the real time qRT-PCR assay used in this study as compared to the
virus isolation assays used in previous studies. Real time qRT-PCR positive midges were present in midge pools
obtained from both light trap and mechanical aspiration. Seven of the positive pools consisted of C. imicola only,
four contained mixed species and one pool contained no C. imicola, suggesting the presence of BTV in midges of
other species.
Involved in the project from ARC-OVI are: Gert Venter (PhD); co-worker: K. Labuschagne. Collaboraborators :
University of Pretoria, Faculty of Veterinary Science (UPFVS): Elli Scheffer (BVSc); P. C Page and A.J Guthrie.
University of California - Department of Entomology, Riverside, CA, U.S.A: B.A Mullens. University of
California, Davis - Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, CA,
U.S.A: N.J MacLachlan. Institut für Virologie, Fachbereich Veterinärmedizin, Freie Universität Berlin, Berlin,
Germany: Nikolaus Osterrieder,
7.
Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the
pathogen and the disease concerned
All diagnostic tests are performed by competent personnel; the laboratories participate in inter-laboratory tests,
and verified control reagents are employed. Tests are performed in BSL2 laboratories in biohazard cabinets, and
Annual reports of OIE Reference Centres, 2011
5
Bluetongue
personnel wear protective clothing in addition to exercising good laboratory practice. Biological waste is
autoclaved and incinerated.
8.
Provision of consultant expertise to OIE or to OIE Member Countries
The third meeting of the OIE BT Net was hosted by of Istituto Zooprofilattico Sperimentale dell Abruzzo e del
Molise (Istituto G. Caporale) OIE Collaborating Centre for Veterinary Training, Epidemiology, Food Safety and
Animal Welfare and OIE Reference Laboratory for CBPP, BT, WN and Brucellosis. Due to the strong research
record of the ARC-OVI on Culicoides species and the viruses transmitted by these insects, Dr. G.J Venter from the
ARC-OVI was invited by the Director of the Istituto Zooprofilattico Speimentale dell Abruzzo e del Molise as
chairman and secretariat of the OIE Reference Laboratory for BT to attend and participate in the meeting.
One of the objectives of the meeting was the creation of a working group on Culicoides vectors within the OIE BT
Net. The main aim of this working group was to provide recommendations/guidelines “from field to lab” to
member countries on the methodology relevant for the production of comparable and meaningful data sets
regarding vector surveillance systems. The Culicoides workers were represented by Miguel Miranda (Spain) and
Gert Venter (South Africa) supported by the workers in Italy (Maria Goffredo and Annamaria Conti).
9.
Provision of scientific and technical training to personnel from other OIE Member Countries
Senior technologist, Mrs. Hope Tshabalala (BTech), trained other technologists on intravenous egg inoculations at
a course held at UPFVS in November 2011.
10. Provision of diagnostic testing facilities to other OIE Member Countries
Country
Namibia
No. of
specimens
1
Test
Tentative
Confirmatory
Yes
No
Positive
Results
Yes
Positive results
reported to OIE
N/A
N/A - not applicable because the tests conducted were not confirmatory, or if confirmatory, the results were negative
11. Organisation of international scientific meetings on behalf of OIE or other international bodies
Dr G.J. Venter (ARC-OVI ), together with Me E. Veronesi (IAH Pirbright-UK) were invited to organise and comoderate a symposium on Culicoides vectors and arbovirus transmission at the 59th Annual meeting of the
Entomological Society of America, 13 - 18 November 2011, Reno, Nevada, USA. The meeting was by invitation
only and attended by sixty delegates. The proceedings of the meeting were not published
(http://www.entsoc.org/entomology2011. A second symposium will be held at the 60th Annual meeting of the
Entomological Society of America to be held in Knoxville Tennessee.
12. Participation in international scientific collaborative studies
I. BTV transmission between ruminant host and Culicoides vectors: the importance of insect saliva
proteins
The project (programme no. 139, project no.1361 and application no. A550. ) is managed by the Institute for
Animal Health (IAH) -Pirbright, and its objectives are to: 1) examine the response of ovine cells (in primary
culture) to Culicoides saliva proteins and their effects on virus infection / replication; 2) examine the effect of
saliva proteins from different Culicoides species on BTV structural proteins and infectivity, and possible
involvement in vector competence; 3) measure and compare the amount of virus in the blood (systemic) and skin
(local) and asses their relative importance for transmission; 4) examine the influence of Culicoides saliva proteins
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Annual reports of OIE Reference Centres, 2011
Bluetongue
on local BTV infection of the skin and the consequences for mechanical or non-systemic transmission; 5) examine
the inflammatory response to Culicoides saliva proteins in the skin.
Due to the ARC-OVI’s experience with research on Culicoides and the abundance of C. imicola in South Africa,
the ARC-OVI was invited to be a partner. The ARC-OVI will mainly be involved with the second objective,
which aims to examine the effect of saliva proteins from different Culicoides species on BTV structural proteins
and infectivity, and possible involvement in vector competence. As part of the collaboration between the IAH –
Pirbright and ARC- OVI, a scientist from the IAH (Eva Veronesi) and a collaborator from Balearic Island
University (Ricardo del Rio) visited the ARC-OVI during February-April 2011 to investigate aspects of BTV
dissemination and thermal limits in C. imicola
Project leader from ARC-OVI: Gert Venter (PhD); co-workers: D.M Majatladi and S.N.B Boikanyo
Collaboraborators: P Mertens; K Darpel and E Veronesi from Institute for Animal Health, Pirbright, UK.
II. Full genome sequencing of BTV project is a collaborative project between ARC-OVI and ILRI.
III. The study on comparison of light trap result with attack rate elucidated above is a collaborative effort
involving researchers from 3 countries.
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


LABUSCHAGNE, K. & SCHOLTZ, C.H. 2011. Distribution of Culicoides imicola and C. bolitinos (Diptera:
Ceratopogonidae) in South Africa. Proceedings of the XVII Congress of the Entomological Society of
Southern Africa (ESSA). 3-6 July 2011 University of the Free State Bloemfontein. P 92 – Oral presentation
MIRANDA, M.A., DEL RIO, R., VENTER, G.J., CARPENTER, S., LUCIENTES, J., VERONESI, E. &
ESTRADA, PENA, R. 2011. Review of the oral susceptibility results for determining vector status of
Culicoides spp. as transmitters of BTV in Europe and South Africa. In: Abstract book of the 5th Annual
Meeting Epizone, "Science on alert", 11-14 April 2011, Arnhem, The Netherlands – Oral presentation

Scientific publications in peer-reviewed journals
oVENTER, G.J., LABUSCHAGNE, K., BOIKANYO, S.N., MAJATLADI, D.M. & MOREY, L. 2011. The effect
of 1-octen-3-ol and 4-methylphenol on Culicoides midge numbers collected with suction light traps in South
Africa. Veterinary Parasitology, 10, 175 (1-2), 182-186.
o VENTER, GJ, LABUSCHAGNE K, BOIKANYO SN, & MOREY L. & SNYMAN, M.G. 2011. The repellent
effect of organic fatty acids on Culicoides midges as determined with suction light traps in South Africa.
Veterinary Parasitology, 181, 365-369.
o
VENTER, G.J., WRIGHT, I.M., DEL RIO, R., LUCIENTES, J. & Miranda, M.A. 2011. The Susceptibility of
Culicoides imicola and other South African livestock-associated Culicoides species to infection with
bluetongue virus serotype 8. Medical and Veterinary Entomology, 25(3):320-326.
o Drs. B.A. Lubisi and Gert Venter reviewed research papers on BTV and BTV and the Culicoides vectors
respectively, for Veterinary World Journal and Medical and Veterinary Entomology, Parasites and Vectors,
PLos ONE, Veterinary Parasitology, accordingly.

Other communications
Higher degree obtained :
DEBEILA, T.J. 2011. Characterisation of selected Culicoides (Diptera: Ceratopogonidae) populations in South
Africa using genetic markers. MSc thesis. T he University of Pretoria.
Mr Debeila is an ARC-OVI employee. The project was supervised by Otto Koekemoer (PhD).

Meetings attended
Dr BA Lubisi attended the 3rd meeting of the Office International Des Epizooties’ Bluetongue Laboratory
Network (OIE BTLabNet), which was held in Teramo, Italy, on January 21 2011. The following people
participated in the meeting: Yong Joo Kim (OIE, France); James Maclachlan (UCDAVIS, USA); Arnon
Annual reports of OIE Reference Centres, 2011
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Bluetongue
Shimshony (Hebrew University, Canada); Peter Daniels (CSIRO, Australia); Baratang Alison Lubisi and Gert
Venter (ARC-OVI, South Africa); Eileen N. Ostlund (Aphis-USDA, USA); Chris Oura (IAH- Pirbright, UK) and
Miguel Ángel Miranda Chueca (UIB, Spain). Attending the meeting from the host institute (IZS, Italy) were:
Giovanni Savini, Vincenzo Caporale, Rossella Lelli, Paolo Calistri, Andrea Carvelli, Annamaria Conte, Armando
Giovannini, Maria Goffredo, Federica Monaco, Daniela Morelli, Attilio Pini, Lucilla Ricci and Manuela Tittarelli.
Among the matters discussed were:
i)
Improvement of the BT chapter in the OIE Manual
ii)
Acceptability or non-acceptability of non-validated tests in the BT chapter of the manual
iii)
The importance of some of the tests in the manual and whether it was necessary for them to be included
iv)
The methods which were important and deserved inclusion in the manual
v)
Most suitable pathway to follow to validate the non-validated tests
vi)
The efficiency of the manual update process
vii)
BTV reference material for all 24 serotypes
Deliberations were made and agreements reached. A report was compiled and sent to the OIE.
_______________
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Annual reports of OIE Reference Centres, 2011