Rabies
OIE Reference Laboratory Reports
Activities in 2011
Name of disease (or topic) for which you are a designated OIE Reference Laboratory: / RabiesAddress of laboratory / Onderstepoort Veterinary Institute
P Bag X05
Onderstepoort 0110
SOUTH AFRICA
Tel.: / +27 12 5299 439
Fax: / +27 12 5299 390
e-mail address: /
website: / www.arc.agric.za
Name (including Title and Position) of Head of Laboratory (Responsible Official): / Dr Wonderful Shumba (Veterinarian)
Name(including Title and Position) of OIE Reference Expert: / Dr Claude Taurai Sabeta
Senior Research Scientist and
Project Manager
Name (including Title and Position) of writer of this report
(if different from above):
Part I: Summary of general activities related to the disease
The Onderstepoort Veterinary Institute is part of the Livestock Business Division of the Agricultural Research Council (ARC). The activities of this institution are undertaken via 5 Research programmes namely Molecular Epidemiology and Diagnostics (MED), Feed Food and Veterinary Public Health (FFVPH), New Generation Vaccines (NGV), Parasites, Vectors and Vector-borne Diseases (PVVD) and Transboundary Animal Diseases (TADs). The activities in these programmes are underpinned by ISO Standard 17025 accreditation and support livestock and/or agro-industry in disease control: through the detection of pathogens using classical, serological, molecular tools. The work is pivotal in the surveillance & prediction to prevent future occurrence and prevention of disease outbreaks.
1. Test(s) in use/or available for the specified disease/topic at your laboratory
Test / For / Specificity / TotalFAT / Antigen / Group / 936
FAVNT / Antibody / Group / 2863
MAb typing / Antigen / Group / 117
Mouse inoculation test (MIT)
/ Virus isolation / 932. Production and distribution of diagnostic reagents
Type of reagent / Amount supplied nationally(including for own use) / Amount supplied to other countries
Immunofluorescent conjugate / 10 x 1 ml vials of biological conjugate / Nigeria and 14 Southern African Development Community (SADC) members.
Antigens for pathogenicity and challenge test / 80 mls of CVS and 25 mls each of a canid, mongoose and spillover rabies isolate for pathogenicity studies. / Allerton Provincial Laboratory, Pietermaritzburg, South Africa and own use
BHK cells / 4 x T25 flasks / Allerton Provincial Laboratory, Pietermaritzburg, South Africa
Neuroblastoma cells
/ 4 x T25 flasks / University of Pretoria and the National Institute of Communicable Diseases (South Africa)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
Participated in ring trials for the FAT and FAVNT, being profieincy tests organised by Anses (Nancy, France).
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
As a direct result of the recommendations from the bi-annual conference of the South East African Rabies Group (SEARG) which took place in Maputo (Mozambique, January 25-27, 2011), all participants (including public health officers, animal health officers, researchers, vaccine producing organisations and medical practitioners) resolved that laboratory capacity for animal rabies diagnosis in the SADC region should be enhanced. One of the ways advocated was to conduct a proficiency testing, with the Onderstepoort Veterinary Institute co-ordinating all activities. In performing this exercise, laboratories would use a standardised standard operating procedure (SOP) and biologicals including the anti-rabies conjugate. This exercise would also verify whether the rabies diagnostic training by the Onderstepoort Veterinary Institute from July 27-31, 2009 had been attained.
A panel of 11 freeze-dried mouse infected materials was sent to each of the SADC laboratories (except Angola, Madagascar, Mauritius and the Seychelles (who did not indicate their intent to participate). The panel of samples included two panels: panel 1 consisting of Lagos bat virus (LBV, genotype 2), Mokola virus (MOKV, genotype 3), Duvenhage Virus (DUVV, genotype 4), mongoose rabies virus (genotype 1) and a negative brain tissue derived from a bovine and panel 2 was composed of 5 positive samples, 2 strong positives and three samples diluted with uninfected brain tissues (1:5; 1:100 and 1:400) and a negative sample. Most of the laboratories produced satisfactory results. Collectively, the performance of the laboratories can be significantly improved by reducing the number of false negative (n=23) and false positive results (n=9) which impact on the sensitivity and specificity respectively. For this proficiency test, a specificity and sensitivity of 65.4% and 80% respectively were obtained. This means the laboratories that participated in this proficiency test exercise are at different levels of competence. In future and in the subsequent proficiency test exercises, all participating laboratories should therefore aim to improve on this. The specificity and sensitivity of the actual test (FAT) is approximately 100% and between 99.2% and 99.9%.
The sample that was diluted 1:400 was the least detected and this possibly indicates the low sensitivity of some microscopes (may have a bearing on the service history and status of this very important piece of equipment) or could be the level of experience of some of the readers. This is given that all laboratories were provided with the same biological conjugate. The false positive results obtained by some laboratories for the negative control are a concern though. One possible reason for this is that some laboratories may not have included Evans Blue in the test procedure as indicated in the protocol.
4. Preparation and supply of international reference standards for diagnostic tests or vaccines
None
5. Research and development of new procedures for diagnosis and control
Currently there is one active project entitled “The Development and implementation of competitive ELISA for African lyssaviruses” being jointly undertaken by Onderstepoort Veterinary Institute, the Canadian Food Inspection Agency (Canada) and the University of Pretoria (South Africa). Ribonucleoprotein (RNP) for representative Lyssavirus isolates of African origin was initiated and in 2011 Mokola virus (252/97) RNP was successfully purified and verified by western blotting. The RNP purification for other African Lyssavirus species is underway and as soon as the required quantities are obtained for animal experiments will be done. Only Mokola virus (MOKV) RNP was successfully purified and verified by western blotting. One of the challenges encountered during the study includes the low yield of harvested RNP or that RNP could not be eluted into eluent buffer easily. The initial challenges were overcome.
6. Collection, analysis and dissemination of epizootiological data relevant to international disease control
We continued to report continuation of the rabies outbreak in Gauteng province of South Africa although fewer rabies positive cases were confirmed in 2011 than the previous year [17 positive in 2011, 38 in 2010]. It now appears that the control efforts implemented by the Veterinary Services in 2010 are probably bearing fruit.
7. Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the pathogen and the disease concerned
The two tests used in the OIE Rabies Reference Laboratory were accredited according to ISO Standard 17025 by the South African National Accreditation System (SANAS).
8. Provision of consultant expertise to OIE or to OIE Member Countries
None
9. Provision of scientific and technical training to personnel from other OIE Member Countries
This report covers aspects in the context of the twinning project between the ARC and the National Veterinary Research Institute (NVRI, Nigeria) which ended on 31 December 2011. One notable achievement is that the FAT has now replaced the Sellers method that was previously used at NVRI for rabies diagnosis as the first line method for rabies diagnosis. On this regard, 5 scientists from NVRI visited the Onderstepoort Veterinary Institute and were provided with relevant documentation and procedures for safety and other tests, and also put through various processes associated with for rabies diagnosis as prescribed by the OIE. These processes include sample receipt at a central diagnostic registration point, registration and documentation and in addition specimen preparation for FAT, reading stained slides and reporting of test results as routinely carried out at the OIE Rabies Reference Laboratory in South Africa. Inter-laboratory exchange of samples was undertaken between the two laboratories in order to assess the competency of the technical personnel. The results obtained by the technician were as expected (100% correlation) which indicates an acceptable level of competence to perform the FAT. It is against this background that NVRI participated in an external proficiency FAT in April 2011 and performed satisfactorily.
Contrary to the traditional practice of using only the Hippocampus (Ammon’s horn) for rabies screening at NVRI, a composite sample of the brain [that includes the brain stem, cerebellum, hippocampus, pons, and portions of the cerebrum are used for making smear]. Separate sterile scissors and forceps are used for individual specimens, painstaking disinfection and cleaning of utensils while making smears for FAT and all tissue processing for FAT is carefully carried out inside a biosafety cabinet as a standard bio-security practice in veterinary laboratories. NVRI is now attempting to store all their positive samples but the power disruptions seem to be a perpetual problem.
The area of cell culture and the neutralisation test for rabies antibodies did not met with the same level of success as the FAT. Again, three scientists from NVRI visited Onderstepoort for training in cell culture. During the visits, the scientists were shown how to reconstitute cell culture media, maintain two important cell lines for rabies diagnostics: the BHK and MNA cell lines. These included the inability to resuscitate the BHK cell line and the lack of a dedicated functional fluorescent microscope. A panel of 200 serum samples collected from dogs in Nigeria was collected. This panel of samples is composed of samples collected from normal and apparently healthy, sick; pre-vaccination status (unknown) and follow up samples 4 weeks later. The fluorescent antibody neutralisation test continues to present problems to NVRI personnel. Some of these problems include malfunctioning equipment (particularly uniform distribution of carbon dioxide in the incubator) and this then affects virus infection as well as lengthening the time that cells take to become confluent.
The rabies tissue culture isolation test (RTCIT) is an in vitro method for isolating rabies virus in cell culture. Murine neuroblastoma cells (MNA cells) have been shown to be the best substrate for isolating southern African rabies variants and this cell line will be used for growing field strains of rabies. The RTCIT was successfully demonstrated at Onderstepoort and will be validated in 2012 at NVRI.
A demonstration on the use of a panel of monoclonal antibodies (Mabs) to differentiate lyssaviruses into their respective biotypes was undertaken in the two years of this twinning project. This is particularly important where there is a large diversity of lyssaviruses as in the case of the southern African sub-region and where more expensive diagnostic tools such as the real time PCR assays cannot be accommodated.
In 2011, dog viruses from Nigeria were subjected to antigenic characterisation using a 15-anti-nucleoprotein (anti-N) Mab panel [obtained from our collaborators in Canada] and several reactivity patterns were observed. None of these reactivity patterns appeared to resemble the patterns established for this southern African panel for Lagos Bat, Mokola or Duvenhage virus. Clearly, with the first 7 mAbs in the panel, the staining for all the samples in the panel seemed consistent with the canid and mongoose rabies biotypes of southern Africa. However, there was no variability observed in a single Mab, 32 GD 12 staining, which we usually noted for both the biotypes in the South African isolates. The patterns with the remaining 9 mAbs did not seem to correspond well to the patterns for either rabies biotype. This result was somewhat surprising given that dog virus isolates from Nigeria were previously shown to be antigenically similar. With the anti-P Mab panel used in this training, 5 of the 8 Mabs gave reaction patterns consistent with previously characterised isolates from West Africa.
During the training visit later in 2011 we utilised the anti-P Mab panel. Individually tailored training to five diagnostic personnel on strain differentiation of dog virus isolates passaged in mouse brain from Plateau State using a panel of 8 anti-P monoclonal antibodies was undertaken. The trainees were shown the following: how to titrate secondary antibody, prepare and fix smears, stain with primary, secondary antibody and Evans Blue counterstaining. In addition, the trainees were shown how to read the slides and score the antigen distribution and intensity of fluorescence. It appears that the samples from this west African country need to be screened with a bigger panel of Mabs. In preparation for this, a panel of 11 samples were inoculated into suckling mice and the brain materials harvested for shipment to the Canadian Food Inspection Agency, Canada. The fact that mice were taking long to succumb could indicate that the temperature at which the samples were being stored is not appropriate.
In all the diagnostic work described above, quality management was emphasised. Although SOPS for key methods were developed, there were some deficiencies: there is a need to replace key equipment such as the microscope, and there is a need to obtain a new breed of mice. A draft quality manual for the institute is now available and was used during the visit and audit, inter-analyst comparison have been consistently been carried out on the FAT test only and should now be extended to the FAVNT. Other observations included the following: a) Lack of records for the biohazard cabinet, b) Temperature for the incubator not monitored, c) There is need for a back-up power in case of power failures, d) no calibration or verification records for the micropipettes, e) no logbook for certain equipment e.g. the pH meter, balances, need calibrated weights. In order to address some of the problems identified above, it was recommended that each piece of equipment should have its own calibration, verification record, should be uniquely identified and monitored, manuals should be readily available, gas tank supplying carbon dioxide to the incubators should be securely chained and that a fire proof cabinet for the reagents be available. These were all addressed by the end of the project.