OIE Reference Laboratory Reports s10

OIE Reference Laboratory Reports Activities in 2011

Name of disease (or topic) for African horse sickness which you are a designated OIE Reference Laboratory:

Address of laboratory: 100 Old Soutpan Road Onderstepoort, 0110 Tshwane, SOUTH AFRICA

Tel.: Tel.: (+2712 ) 529 9117

Fax: Fax: (+2712) 529 9418

e-mail address: [email protected]

website: http://www.arc.agric.za

Name (including Title and Dr Baratang Alison Lubisi Position) of Head of Laboratory (Responsible Official):

Name(including Title and Dr Baratang Alison Lubisi Position) of OIE Reference Expert:

Name (including Title and Dr Baratang Alison Lubisi Position) of writer of this report (if different from above):

Annual reports of OIE Reference Centres, 2011 1 African horse sickness

Part I: Summary of general activities related to the disease

Activities of the OIE African horse sickness (AHS) reference laboratory at the ARC-OVI for the report period included the provision of diagnostic services (Table 1) and routine disease certification of equines for the import/export markets and relocations. Among the animals tested for the latter purpose were felines and canines. Other activities included performance of research aimed at improving the currently utilised Culicoides species trap and control methods, and generating a database of the full genome sequences of the related Orbivirus, Equine encephalosis virus (EEV). These activities are aligned with the Agricultural Research Council’s (ARC) strategic objective of enhancing the ability of the agricultural sector to manage and mitigate agricultural risks.

Table 1 Results of AHS diagnostic services provided for South Africa in 2011

Province No Positives/Tests Conducted

CFT iELISA PCR

Gauteng 1042 388/483 118/240 166/319 Limpopo 61 20/34 0 4/27 North West 119 41/52 6/6 39/61 Northern Cape 34 7/9 1/1 13/24 KwaZulu - Natal 228 84/156 1/1 26/71 Free State 138 61/112 0 17/26 Eastern Cape 385 276/342 0 30/43 Mpumalanga 148 54/74 0 40/74 Western Cape 234 10/19 32/39 76/176

Approximately 93 submissions consisting of various numbers of tissues and blood in EDTA tubes were submitted for virus isolation. Around 30 and 20 AHS and EE viruses (AHSV and EEV) were isolated respectively, with about 4 of the isolated viruses being West Nile (WNV).

Collaboration between ARC-OVI and CISA-INIA (Spain) was established in 2011 for the full characterisation of the 2011 outbreak viruses, using both molecular and classical virological methods. The work commenced in November 2011 and will continue until the project objectives have been reached.

ARC-OVI participated in 3 PCR based inter - laboratory tests organised by the Equine Research Centre of the University of Pretoria, Faculty of Veterinary Science (UPFVS). Each inter - laboratory testing exercise consisted of 10 samples, and the results obtained by both institutions correlated 100%.

1. Test(s) in use/or available for the specified disease/topic at your laboratory

Test For Specificity Total

indirect ELISA Antibody Group 287

CFT Antibody Group 1281

Serum Neutralisation (SN) Antibody Group 54

PCR Genomic material Group 821

Vero cell culture Virus isolation Group 93

2 Annual reports of OIE Reference Centres, 2011 African horse sickness

Diagnostics project leaders: BA Lubisi - BVMCh.MSc (classical virological methods) and Marco Romito – BVSc.MSc (molecular based diagnostics)

2. Production and distribution of diagnostic reagents

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

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

No Item Description Quntity Amount . dispatched 1 Virus AHSV serotypes 1-9 Approximately 50X1ml per serotype None

2 Antisera AntiAHSV serotypes 1-9 Approximately 50X1ml per serotype None

5. Research and development of new procedures for diagnosis and control

I. Full genome sequencing of EEV

Given the relatedness of EEV and AHSV and the closely related epidemiological patterns of the two diseases caused by each of the viruses respectively, EE is the primary differential diagnosis for AHS. Development and standardisation of EEV diagnostic tools is thus of paramount importance in AHS diagnosis since the two diseases are often tested for side by side.

The aim of the project was thus to obtain low passage, early reference strain viruses for amplification and full genome sequencing. These would serve as standard reference sequences for all 7 EEV serotypes, for use by all interested parties in various projects aimed at diagnostic tool development or vaccine production, and epidemiological studies.

Early isolates of EEV were identified and selected. Some of these were early mouse brain 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 isolates had to be passaged twice in cell culture. Virus dsRNA was isolated and purified from all 7 serotypes of EEV.

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 EEV.

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. At least one full genome sequence was available for EEV 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 (PhD); Appolinaire Djikeng (PhD); George Michuki (PhD).

6. Collection, analysis and dissemination of epizootiological data relevant to international disease control

I. Culicoides surveys in Namibia

Light trap collections were made on a weekly basis at five sites in central Namibia. Despite Namibia’s classification as an arid country, it is considered an endemic area for AHS. Knowledge of the species composition and seasonal abundance of potential Culicoides vectors of AHSV in Namibia is lacking, especially those prevailing in the dry winter months. The main objective of the present study was to determine if Culicoides midges are present and can survive the colder drier months as adults in the Khomas region (S22°24’063’’, E17°01’791’’; S23°32’617’’, E15°53’870’’) of Namibia. The survey highlighted midge species richness in the study area. During the winter months from 6 July to 21 September 2009, a relatively high diversity of midges consisting of 25 species were identified from 9 091 Culicoides individuals caught from the five trap sites. The relative abundance of the AHS vector, Culicoides imicola, varied from 94% near high altitude Windhoek with relatively high annual rainfall, to 12% at the South Western most site with the lowest annual rainfall. Culicoides midge activity did not cease over the dry seasons in the south-western Khomas, and the region’s species richness was high. The implication is that the Culicoides species are going to be abundant during periods of above normal rainfall in the summer This relative high Culicoides midge abundance, coupled with the presence of a virus replication permissive host (zebra) in the area, imply that AHSV may overwinter in the area. The high species diversity found and the presence of a number of species about which little is known e.g biology and vector potential for arboviruses, adds to the complexity of the epidemiology of AHS in Namibia.

Involved in the project from ARC-OVI are: Gert Venter (PhD); co-workers: K. Labuschagne Collaborators: School of Environmental Science and Development, North West University, Potchefstroom Campus (South Africa): E. Becker; H. Van Hamburg and T. Greyling.

II. 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.

III. 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 AHS. 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.

IV. 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 horses, 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% Culicoides midge population was calculated based on real-time quantitative reverse-transcription polymerase chain reaction (real time qRT-PCR), a quantity 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 employed 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 AHSV in midges of other species.

Involved in the project from ARC-OVI are: Gert Venter (PhD); co-workers: K. Labuschagne. Collaboraborators : UPFVS: Elli Scheffer (BVSc)-MSc student; 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,

V. The use of real-time quantitative RT- PCR for the detection of African horse sickness virus replication in Culicoides imicola

The second objective of Dr. Elli Scheffer’s Masters Degree project was to determine the use of real-time quantitative RT-PCR for the detection of AHSV replication in C. imicola. Despite its important role as vector of AHSV, little has been published about the dissemination of the virus in C. imicola. The study employed a very sensitive real-time qRT-PCR, which was able to detect AHSV in dissected midges. Ninety-six individual midges were fed AHSV-infected blood and dissected into head/thorax and abdomen immediately after feeding, and after 10 days of incubation. The majority of the midges (96%) ingested the virus successfully and there were no significant differences between the virus concentration in the thoraxes and the abdomens immediately after feeding. However, virus could only be detected in 51% of the midges after incubation, and it was confined to the abdomen in the majority of these midges. The fact that virus was only detected in the thorax of four Culicoides midges after incubation suggested the presence of a mesenteronal escape barrier. Replication in the salivary glands could not be demonstrated. Nonetheless, an increase in the average virus concentration in the abdomen after 10 days incubation implied localised viral replication. The real-time qRT-PCR is recommended for further studies aimed at investigating the replication and dissemination of AHSV in Culicoides midges.

Involved in the project from ARC-OVI are: Gert Venter (PhD); co-workers: K. Labuschagne. Collaboraborators:

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 engage in inter-laboratory tests, and verified controls are employed. Tests are performed 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

Not done

9. Provision of scientific and technical training to personnel from other OIE Member Countries

Not done

10. Provision of diagnostic testing facilities to other OIE Member Countries

Country No. tests Test Positive Positive results reported results to OIE Tentative Confirmator y

Mauritius 596 Yes No Yes N/A

Nigeria 203 Yes No Yes N/A

Swaziland 39 Yes No Yes N/A

N/A - not applicable because the testes 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 co- moderate 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

1. Full genome sequencing of EEV project mentioned above is a collaborative project between ARC OVI and ILRI.

2. Studies on comparison of light trap result with attack rate and use of real-time quantitative RT- PCR for the detection of African horse sickness virus replication in Culicoides imicola elucidated above are collaborative efforts 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

 VENTER G.J., 2011. Overwintering of African horse sickness virus (AHSV) in Culicoides species (Diptera: Ceratopogonidae). Abstract book of the GDARD Annual Research Agenda Symposium, 1 June 2011, Midrand Gauteng.  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.  BECKER, E., VENTER, G.J. & VAN HAMBURG, H. 2011. Culicoides species (Diptera: Ceratopogonidae) abundance during the dry winter months in the south-west Khomas region, central Namibia, and preliminary investigations into AHSV prevalence in Hartmann's zebra populations. In: Abstract book of the XVII Congress of the Entomological Society of Southern Africa, 3-6 July 2011, University of the Free State, Bloemfontein.

 Scientific publications in peer-reviewed journals

 VENTER, 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.  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.  Dr. BA Lubisi reviewed the proposed revised AHS chapter of the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals.  Dr. G.J. Venter reviewed papers on the Culicoides vectors for Epidemiology and Infection, Medical and Veterinary Entomology, Parasites and Vectors, PLos ONE, Veterinary Parasitology and Veterinary World Journal.

 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). ______

Annual reports of OIE Reference Centres, 2011 7