Monoclonal antibody typing

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OIE Reference Laboratory Reports
Activities in 2010
Name of disease (or topic) for
which you are a designated OIE
Reference Laboratory:
Address of laboratory:
Rabies
ARC-Onderstepoort Veterinary Institute
Private Bag X05
Onderstepoort 0110
REPUBLIC OF SOUTH AFRICA
Tel.:
(+27-12) 529.94.39
Fax:
(+27-12) 529.93.90
E-mail address:
[email protected]
Website:
Name of Head of Laboratory
(Responsible Official):
www.arc.agric.za
Dr Wonderful Shumba
Name of OIE Reference Expert:
Dr Claude Sabeta
Name of writer of this report
(if different from above):
Dr Claude Sabeta
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
1
Rabies
Part I: Summary of general activities related to the disease
1.
2.
Test(s) in use/or available for the specified disease/topic at your laboratory
Test
For
Specificity
Total
Fluorescent antibody virus
neutralisation test (FAVNT)
Antibody
Type
2522
Fluorescent antibody test (FAT)
Antigen
Group
1266
Immunohistochemistry
Antigen
Group
18
Mouse inoculation test (MIT)
Virus isolation
Group
110
Monoclonal antibody typing
Antigen
Type
157 (139 canid and
18 mongoose)
Production and distribution of diagnostic reagents
Prepared anti-lyssavirus conjugate and challenge virus standard (CVS) for rabies diagnostics.
Type of reagent
Amount supplied nationally
(including for own use)
Amount supplied to other
countries
Immunofluorescent conjugate
24 mls
16 mls
CVS
40 mls
2 ampoules each to the National
Veterinary Research Institute
(NVRI) and India.
LEP
None
2 ampoules to NVRI
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
Participated in international interlaboratory comparisons for the FAT and FAVNT tests both organised by Nanses
(France). The tests consisted of panels of 12 and 14 samples for the FAT and FAVNT tests respectively.
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
The project for the development and implementation of a competitive ELISA is a joint initiative of the
Onderstepoort Veterinary Institute and the University of Pretoria and the OIE Rabies Reference Laboratory in
Canada. Given the complexity of rabies in the African sub-region it is evident that the true prevalence and
epidemiology of rabies-related viruses is not well understood primarily due to the shortcomings of current
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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
Rabies
serological tools. The current recommended methods for determining the lyssaviruses specific neutralising
antibodies are relatively complex, expensive, use live virus and are tedious for large scale screening of field sera
for serological surveillance studies. The proposed study to develop and implement the competitive ELISA (cELISA) for rabies-related viruses of African origin will hopefully overcome the highlighted problems. A total of
six monoclonal antibodies (mAbs) were selected at the Centre of Expertise for rabies in Canada and labelled with
hydrogen peroxidase enzyme for use in the assay. The labelled Mabs are currently being evaluated in general and
genotype specific ELISA formats in which ribonucleoprotein from isolates from genotype 1 (canid and mongoose
rabies biotypes), genotypes 2, 3 and 4. It will also enhance the search of a natural and maintenance host species of
Mokola and Duvenhage viruses.
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
Epidemiological surveys:
The rabies laboratory continued with the primary diagnostic testing of brain tissues submitted from all the 9
provinces of the country. An unusually high number of dog rabies positive cases was diagnosed from Gauteng
province and in this regard a comprehensive sequence analysis of viruses recovered from this province was
undertaken. The positive rabies dog brain samples were subsequently characterised using a panel of 16 antinucleocapsid monoclonal antibodies obtained from the Canadian Food Inspection Agency, Ontario, Canada.
Molecular characterisation was performed by sequencing the G-L intergenic region of each of the rabies isolates
followed by phylogenetic analysis using standard algorithms. Previously characterised dog rabies virus isolates
were also included in the phylogenetic analysis. The outbreak virus isolates were shown to cluster with previously
characterised rabies virus isolates from dogs from KwaZulu Natal Province, a rabies-endemic region of the
country.
Project 1:
Dog rabies has been commonly associated with the eastern and southern border areas in Mpumalanga province.
However, the Nkomazi district in the east has been the mostly affected area. In other districts of Mpumalanga, dog
rabies has been under control for many years; but, in 2008, an increase in dog rabies cases was noted consequently
resulting in a widespread outbreak within the province. Selected rabies viruses (55) from this region and recovered
between 2000 and 2008 were genetically characterised in an attempt to establish the source of this outbreak. The
viruses were characterised through nucleotide sequencing of the cytoplasmic domain of the glycoprotein gene and
the G-L intergenic region. Genetic analysis of these viruses and previously characterised from Mpumalanga
province and neighbouring regions demonstrated that the recent emergence of rabies in Mpumalanga province
resulted from the spread of rabies from Nkomazi district. Furthermore, a comparative analysis demonstrated a
close genetic relationship among rabies viruses from dogs from Mpumalanga and KwaZulu-Natal provinces,
Swaziland and Mozambique. These data further cdemonstrate that rabies continues to pose a definite public health
threat in South Africa, a similar situation to other African countries and that concerted multinational efforts should
be focussed on control of the disease.
Project 2:
In a study with the Eastern Cape Veterinary Department, where rabies is a definite public health hazard despite the
concerted efforts being made to control the disease. In this study, characteristics of the canine population in the
Emalahleni municipality in terms of body condition, age group, sex, breed, and vaccination status were
investigated. Rabies neutralising antibody titres were determined in a proportion of the dog population and only a
third of the animals sampled had neutralising antibodies of ≥0.5 IU/ml. Eighty dogs were also vaccinated and
resampled 30-60 days later. Of these, 82% had seroconverted to ≥0.5IU/ml and no significant correlation between
antibody response and body condition, age or previous vaccination status were found. Animal Health Technicians’
efficacy during the rabies vaccination campaigns were compared amongst each other and in terms of the whole
district and no differences between individual AHT efficacies but that the AHT’s did not reach the target of 70%
vaccination coverage in their respective areas. It appears that the main obstacle in rabies elimination in this
municipality studied does not seem to be animal response to vaccination but delivery of vaccine within the
communities and this could represent a common problem in this country.
7.
Provision of consultant expertise to OIE or to OIE Members
Responded to specific technical queries from the OIE and individual Member countries.
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
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Rabies
8.
Provision of scientific and technical training to personnel from other OIE Members
OIE twinning project between the ARC and NVRI
The twinning project between the ARC and NVRI (RSA-Ni(Rab)181109) was initiated in February 2010 with a
meeting and the following defined as the key activity areas to be pursued throughout the duration of the project.
1.
the fluorescent antibody test (FAT),
2.
the fluorescent antibody virus neutralisation test (FAVNT),
3.
Rabies tissue culture isolation test (RTCIT) and
4.
Mab typing of rabies positive samples. Within the FAVNT and serology objective, it was agreed that a mini
project for the evaluation of exposure to lyssaviruses (such as bats and other wildlife populations) or
surveillance should be undertaken.
Several exchange visits between the two laboratories were undertaken in 2010 starting with the one for FAT:
For instance, the two personnel from NVRI were provided with relevant documentation and trained on the
procedures for safety and diagnostic tests. In the training they were put through various processes for rabies
diagnosis and these included sample receipt, registration and documentation. In addition, specimen preparation for
FAT, reading stained slides and reporting of test results as routinely carried out at Onderstepoort was done. As part
of the training, the technician from NVRI was evaluated by two individual assessment tests to evaluate her
competence. The first test included a panel of 4 selected samples from the archive at Onderstepoort and the second
(a further 8 samples) were part of a proficiency panel that had been received from Nanses in France in 2008. The
results obtained by the technician were as expected (100% correlation) which indicates an acceptable level of
competence to perform the FAT. It is clear that the technician from NVRI is ready to take part in an external
proficiency for FAT in 2011.
Furthermore, a panel of 6 samples [4 from canine, and one each from a bovine and feline] was sent to
Onderstepoort for the inter-laboratory comparison and tested for the presence of rabies virus antigen by a
technician at Onderstepoort. The condition of the samples was very good demonstrating appropriate preservation
procedures for brain tissues being used at NVRI. There was no non-specificity observed on all test smears. The
results obtained at Onderstepoort were therefore comparable to those expected. During this first visit, the
activities/practices observed by the visitors at the rabies laboratory at Onderstepoort either confirmed some of their
practices (at NVRI) or enhanced their knowledge further. For instance, contrary to their (at NVRI) traditional
practice of using only the Hippocampus (Ammon’s horn) for rabies screening, a composite sample of the brain
including the brain stem, cerebellum, hippocampus, pons, and portions of the cerebrum are used for making a
smear, as prescribed by the OIE standards for this test. 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 biosafety practice in veterinary
laboratories. The reading of FAT slides for rabies diagnosis is routinely carried out by two readers to improve
reliability and where discrepancies arise, the test is repeated. All FAT positive rabies specimens, whether fully
characterised or not are stored frozen for future epidemiological, immunological and genetic studies. Suckling
rather than three-week old mice are used for mouse-inoculation for ease of handling and amplification of the
rabies virus. Strict disposal procedures are followed - all utensils used for rabies post-mortem and FAT procedures
are first incinerated before autoclaving. Every activity and performance of all pieces of equipment should be
recorded as a matter of routine.
The FAVNT and cell culture:
Three scientists from NVRI visited Onderstepoort for two weeks in July 2010. During this period, the scientists
were shown how to reconstitute cell culture media, maintain both BHK and MNA cell lines, and perform the
serology test (FAVNT) and rabies isolation in tissue culture. All the tests worked as expected at Onderstepoort,
but back at NVRI the serology group met with some problems. These included the inability to resuscitate the BHK
cell line and the lack of a dedicated functional fluorescent microscope. However, it is refreshing to note that the
main pieces of equipment required for the FAVNT test have all been procured. A panel of 44 serum samples was
also collected from stray dogs close to NVRI and will be tested for rabies virus neutralising antibodies in 2011.
This panel is composed of samples collected from normal and apparently healthy, sick; pre-vaccination status
(unknown) and follow up samples 4 weeks later. Several things are outstanding and should be done this year: e.g.
production and titration of CVS, establish a system for tracking reagents, writing expiry dates of reagents to be
checked, calibration records of key equipment e.g. biohazard cabinet, monitoring of fridges. Cell culture media is
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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
Rabies
reconstituted at a central facility and for the benefit of this group we suggest that all members undertaking
serology tests be knowledgeable about maintenance of cells. All cell lines should be accompanied by
comprehensive information and temperature charts should be available for the incubator used for culturing cells. In
addition, passage history of some cell lines such as the BHK was not available [although that of other cell lines
such as the VERO cells was available].
A review visit in November 2010 demonstrated that there is a need for NVRI to procure key pieces of equipment
for the FAT test and surveillance in general. These include: a biological class II safety cabinet, a -70 freezer, a
working inverted fluorescent microscope and from a safety point of view the immediate use of sharps containers.
In 2011, more emphasis will be put on quality related matters. In general, the documentation in the FAT is
available and acceptable. However, there are some technical improvements needed for the test to conform to
international requirements. The use of Ms Access was presented to the whole group using an example of a
database from Onderstepoort.
There was limited progress on the Mab typing activity. A panel of 11 samples was selected from the archive at
NVRI and inoculated into individual families of suckling mice. At the time of the review, the families of mice
were still being monitored. The fact that mice were taking longer than usual to succumb could indicate a potential
problem with storage of the samples. The rabies isolates will be used for the selection of a panel of Mabs for
differentiating rabies positive samples in Nigeria. A material transfer agreement (MTA) between NVRI and the
Canadian Food Inspection Agency (OIE Rabies Reference Laboratory, Ottawa, Canada) has already been signed
to facilitate the provision of Mabs to NVRI.
9.
Provision of diagnostic testing facilities to other OIE Members
None.
10. Organisation of international scientific meetings on behalf of OIE or other international bodies
None
11. Participation in international scientific collaborative studies
This study is part of a larger program of anthrax research in Etosha National Park, Namibia, where seasonal
endemic anthrax affects Etosha’s scavenger community by generating large amounts of ungulate carrion during
the anthrax season is being studied in conjunction with the University of California. The focus is on how blackbacked jackal movement, diet, and population density are affected by these outbreaks. In this regard, 47 blackbacked jackal (Canis mesomelas) were captured, and 16 were fitted with GPS-UHF data-logging collars which
will record jackal location every hour for two years. During this period most of the captures serum samples from
immobilized animals is collected.
As rabies incidence in the park lags several months behind anthrax outbreaks, it is interesting to explore the
possibility that aggregations of jackals at carcass sites allows for higher degrees of rabies transmission than if
jackals foraged for smaller, more homogenously distributed prey items such as birds and small mammals. Jackals
are captured across a spatial gradient of anthrax incidence, and assessed whether serological indicators of exposure
to rabies virus vary accordingly. Dr Sabeta’s Team is also in the midst of capturing more jackals to assess whether
rabies virus exposure changes notably after rabies outbreaks. The Okaukuejo tourist camp has experienced a rather
high incidence of rabies this year with five jackals having been destroyed due to rabies thus far. A similar number
of jackal will be captured in 2011 and sera will be drawn from the captured animals.
12. 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
Presented progress at the mid-year European Virus Archive (EVA) meeting at Chandos House (London) 12-15
July, 2010.
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010
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Rabies
Presented the harmonised protocol for the FAT to the SADC laboratory sub-committee in Port louis, Mauritius, 29
October 2010.

Scientific publications in peer-reviewed journals
SJ van Sittert, J Raath; GW Akol; JM Miyen; B Mlahlwa & C Sabeta. Rabies in the Eastern Cape – where are we
going wrong? Accepted for publication in the South African Veterinary Journal December 2010.
Zulu, G., Ngoepe, E., Du Plessis, B., Reninghaus, B. and Sabeta, C. 2010. Spread of canid rabies from Nkomazi,
Mpumalanga province (South Africa). Epub: Vector-borne and Zoonotic Diseases.
Sabeta, C.T., Lucille Blumberg, Debra Kgwana Mohale, Jacobeth Mmantshuruge Miyen, Shumba, W, and
Alexander Immanuel Wandeler. 2010. Mokola virus (MOKV) involved in a human contact (South Africa). FEMS
Medical Microbiology and Immunology. 58: 85-90.

Other communications
Presented progress at the review meeting of the OIE twinning project at NVRI (Nigeria), November 2010.
Submitted an interim and annual report on the OIE twinning project between the ARC and NVRI to the OIE (April
and December 2010).
13. Inscription of diagnostic kits on the OIE Register
i)
Did you participate in expert panels for the validation of candidate kits for inscription on the
OIE Register? If yes, for which kits?
No
Did you submit to the OIE candidate kits for inscription on the OIE Register? If yes, for which
kits?
No
________
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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010