Bluetongue
OIE Reference Laboratory Reports
Activities in 2011
Name of disease (or topic) for which you are a designated OIE Reference Laboratory: / BluetongueAddress 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: /
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
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 conductedcELISA / 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 / TotalcELISA / 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 tissuesDiagnostics project leaders: B.A Lubisi - BVMCh.MSc (classical virological methods) and Marco Romito – BVSc.MSc (molecular based diagnostics).
2. Production and distribution of diagnostic reagents
(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 / (VLA) VetQAS / ARC-OVIcELISA / 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
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
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.
4. Preparation and supply of international reference standards for diagnostic tests or vaccines
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
5. 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).
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 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).