3. MAIN FINDINGS AND CONCLUSIONS
Annex 2 List of People Consulted
Annex 3 Equipment and Chemicals Supplied by LNERV-SIRA, Dakar
Annex 4 Equipment and Chemicals Available in Virology Department for ASF Diagnosis
Annex 5 Additional Equipment and Reagents Required
Annex 6 Standard Operating Procedure for ASF Antigen Detection by Direct Immunofluorescence
Immunofluorescence Antibody Test Worksheets for ASF Antigen Detection
Annex 7 Standard Operating Procedure for ASF Antibody Detection by Indirect ELISA
ELISA Worksheets for ASF Antibody Detection
Annex 8 Standard Operating Procedure for ASF Antibody Detection by Immunoblotting
Immunoblotting Worksheets for ASF Antibody Detection
Annex 9 IFAT, ELISA and Immunoblotting Results
Annex 12 Names and Addresses of Suppliers
ASF - African swine fever
ELISA - Enzyme linked immuno-sorbent assay
FAO - Food and Agriculture Organisation of the United Nations, Rome
FDL&PCS Federal Department of Livestock & Pest Control Services, Abuja
FMANR - Federal Ministry of Agriculture and Natural Resources, Abuja
GLP Good Laboratory Practice
GoN - Government of Nigeria
IAEA - International Atomic Energy Agency, Vienna
IAH - Institute for Animal Health, Pirbright
IFAT Immuno-fluorescence antibody test
INIA - Instituto Nacional de Investigaciones Agrarias, Madrid
NVRI - National Veterinary Research Institute, Vom
OD - Optical density
SOP - Standard Operating Procedure
This consultancy is part of the technical input of the first phase in the control of African swine fever (ASF) in Nigeria. It follows previous consultancies to Nigeria by Drs Davies (November 1997) and El Hicheri (June 1998), and also a technical workshop held in Togo (June 1998) that presented an overview of the current situation of ASF in West Africa.
The first reported outbreak of ASF in Nigeria was in 1973 at a commercial piggery in Ogun State (S.W. Nigeria) which caused 100% mortality. The second outbreak was reported in Lagos and Ogun States in September 1997. The total losses recorded for the two outbreaks in 1997 were 15,000 pigs. The disease was not confirmed by laboratory diagnosis.
Subsequently ASF was reported in December 1997 in Oyo State and in August/September 1998 in Benue, Kaduna, Oyun, Plateau and Rivers States. Four hundred pigs were reported to have died in Zonkwa, Kaduna State during 31 August and 10 September 1998. None of these outbreaks were confirmed as ASF by laboratory diagnosis. The aim of this consultancy was to provide NVRI, Vom with the capability to diagnose ASF using immunological techniques.
Three techniques (ELISA, IFAT and immunoblot) have been established at NVRI using reagents produced and quality controlled by INIA, Madrid and provided by LNERV, Daka. Technicians and veterinarians were trained in their use.
These techniques were applied to tissues and sera obtained from white pigs from Zonkwa and Jos, Plateau State in September 1998. The results on samples from Zonkwa indicated the presence of ASF virus/antigen in tissues from a dead pig exhibiting clinical ASF and the presence of ASF antibody in two sera from pigs that had recovered from ASF. The presence of ASF antibody was not detected in pig sera from Jos. The results of the laboratory tests together with clinical evidence and the high mortality rate indicate that ASF is present in Kaduna State.
On the basis of all the evidence the Ministry of Agriculture has arranged to issue a press release to the effect that ASF is widespread in Nigeria and all movement, sale, consumption, etc. of pigs and pig products should cease with immediate effect.
The ASF positive pig tissues from Zonkwa and any tissues that are subsequently found to have ASF should be forwarded to an FAO designated ASF reference laboratory so that attempts can be made to isolate and characterise the virus. Pigs with ASF antibody should be examined for the presence of ASF virus/antigen to determine if the animal is a carrier.
To enable the diagnostic techniques to be sustained it is important that NVRI provides facilities that allow the techniques to be performed under one roof. Disease security should be applied to the Pathology department where post mortem examinations are performed on samples from the field. In addition, NVRI should maintain a laboratory infra-structure that provides main services, such as water, electricity and disease secure waste disposal together with adequate supplies of basic chemicals, washed and sterilised glassware, pure water, plastic consumables (e.g. micro pipette tips and serum storage bottles) and appropriate disinfectant.
So that rapid diagnosis and sero-monitoring for ASF antibody can be performed quality assured diagnostic reagents should continue to be procured from INIA, Madrid. Assuming the standard operating procedures are followed accurately these reagents will ensure that the tests are performed in a quality-controlled manner thus ensuring reliable results.
African swine fever (ASF) is a highly contagious virus disease of pigs for, which there is no known treatment or vaccine. It is characterised by 85-100% mortality with any recovered pig potentially acting as a carrier of infection for life. The maintenance of the disease, as it occurs in East and South Africa, is through the African soft tick (Ornithodoros moubata porcinus) and Warthogs or domestic pigs. This cycle does not appear to occur in West Africa because the soft tick does not exist in this area. ASF virus and antibody has not been isolated or detected from Warthogs of West Africa. Once the pig is infected the virus can be found in high concentrations in all its tissues and secretions, and infection can occur rapidly. The virus is highly resistant to inactivation by chemicals (pH 4 to 10, trypsin and proteolytic enzymes) and temperature (-70oC, -20oC, 4oC, 37oC, 56oC and freeze/thawing). The virus can persist for long periods of time (typically several months) in organic matter originating from infected pigs, in tissues of dead pigs and in the environment. All infected carcasses and processed pig products can, therefore, contain the virus and whenever infected food wastes (e.g. ham. etc.) enter the animal food chain they can potentially contribute to the dissemination of the disease. To prevent disease dissemination dead pigs should be buried in lime pits.
ASF has been reported in various West Africa countries. Cape Verde reported ASF in the 1960's as being endemic with cycles of incidence occurring more strongly during the months of May to June and November to December. The disease was confirmed in Cape Verde in late 1997. ASF evolved into an epidemic in Cameroon in 1982 and has a history in Guinea Bissau and S�n�gal. In 1996 the disease was reported in C�te d'Ivoire, which resulted in the death and slaughter of more than 100,000 pigs. Another epidemic occurred in B�nin in August 1997, causing the death and slaughter of 65,000 pigs. It has now reached the borders with Togo and Nigeria. Occurrence of the disease has not been reported in Ghana, and C�te d'Ivoire has succeeded in eradicating the disease. Without exception ASF causes devastating losses to the meat/protein supply of a country and inflicts huge economic losses, to which could be added the costs of disease control and eradication programmes where these are envisaged.
The pig population in Nigeria is about 7 million most of which are raised on smallholder farms. They are concentrated mainly in the middle and southern States of the country due to religious, cultural and local taboo.
It has been reported (El Hicheri, 1998) that ASF is likely to have been endemic in Nigeria for the last 25 years. However, the first reported outbreak of ASF in Nigeria was in 1973, at Arakanga piggery, Abeokate, Ogun State (S.W. Nigeria). The disease caused 100% mortality killing approximately 3,000 pigs.
Since then the disease was not reported until September 1997 when clinical cases of ASF were seen in Lagos State (S.W. Nigeria) and again in Ogun State. The recent appearance of ASF, in neighbouring B�nin represents an important threat to Nigeria and it has been suggested that the recent outbreaks originated from B�nin. It is estimated that in Lagos State, Bodagary and environs, about 3,000 pigs died and in Ogun State about 12,000 pigs died.
Clinical disease, pathological signs and the high mortality rate, that in most cases reaches 100%, have only indicated the recent presence of ASF in Nigeria. The disease has not been confirmed by laboratory diagnosis.
For the presence of ASF to be determined and sero-monitoring to be conducted rapidly and efficiently in Nigeria the National Veterinary Research Institute (NVRI) requires the capacity to diagnose ASF virus/antigen and antibody. The purpose of this project is to assist the Government of Nigeria (GoN) to control ASF by improving the national capacity to handle ASF emergencies through an increased ASF diagnostic and control capacity. This consultancy addressed the diagnostic component.
Messrs A-Shami (FAOR) and Umam (OIC), briefed the consultant on the situation regarding the consultants visit. It was apparent from correspondence between FAO, Abuja and Dr. Ladan, Director, FDL&PCS that Dr. Ladan had tried to delay the consultant from visiting Nigeria. It was only through the persistence of the FAO office that final agreement to the mission was received from the Permanent Secretary.
A meeting, to which the consultant was invited, was held between FAOR, OIC, Drs Ladan, West (National Consultant), Oluokun (National Project Co-ordinator), Abegunde (Deputy Director) and Nwakonobi. Dr. Majiyagbe (Assistant Director Diagnostic Services) was unable to attend due to problems with transport from Vom.. After the meeting brief discussions were held between Dr. West and the consultant. Dr. West was informed of the consultant's TOR and his wish to work in close co-operation with Dr. West.
The following day the consultant travelled to Vom with Dr. Nwakonobi. On arrival at NVRI a meeting was held with Dr. Lamorde, Director of NVRI and promises of co-operation were given.
It should be noted that the consultant received full co-operation of NVRI staff and transport was arranged by NVRI to and from the hotel in Jos. Under the constraints that NVRI staff have to work it is a credit to the Institute that such help was provided.
.
Under the general supervision of the Chief, Special Relief Operations Service (TCOR), the technical guidance of the Chief, Animal Health Service (AGAH) and in close co-operation with the Retired Animal Disease Expert recruited under this project, the incumbent will:
Introduce laboratory methods for ASF diagnosis at the Federal laboratory, Vom.
Introduce and demonstrate the use of ASF reagents for this purpose.
Introduce and demonstrate the use of serological tests for ASF surveillance and epidemiology.
Prepare a concise consultancy report according to applied FAO standards and submit it for clearance in Word 6.0 format (on diskette or as attachment by e-mail).
Perform any other duties as required.
Wednesday, 26 August to Monday, 14 September 1998
Refer to Annex 1 and 2, respectively.
My sincere gratitude goes to the staff of the FAO Offices in Daka and Abuja, and to the IITA in Lagos. I also wish to thank the staff of FDL&PCS and NVRI who, under many constraints, offered considerable support to the work programme. My thanks also go to Drs Sarr and Diop, LNERV-SIRA, Daka who kindly supplied reagents, chemicals and equipment that produced excellent results. I am also grateful to the staff of FAO, Rome who made arrangements for this consultancy.
Ian D. Gumm 18 September, 1998
The laboratory diagnosis of ASF can be achieved in three ways:
virus isolation;
antigen detection using blood (in anti-coagulant), tonsil, spleen, kidney or lymph nodes;
antibody detection using serum.
Pig inoculation: samples from ASF suspect infected pigs are inoculated into two groups of pigs, one group is vaccinated against classical swine fever (hog cholera) and the other remains unvaccinated. This method is mainly used to confirm the first outbreak in an ASF-free area and where classical swine fever and African swine fever can be confused such as in Europe. Where clear clinical signs of ASF are seen in both groups ASF should be confirmed by analysing tissues from infected pigs.
Autorosette/haemadsorption: white blood cells from a suspected ASF infected pig are mixed with red blood cells from the same animal and incubated for sometime at 37oC. A similar effect can be achieved more quickly by allowing whole blood, from an infected animal, to stand at room temperature for 1-2 hours and pipetting some of the plasma (containing white and red cells) onto a microscope slide adding a coverslip. In both cases the blood cells are examined microscopically for evidence of autorosetting and the result obtained with the test samples compared with ASF negative and positive controls.
- Haemadsorption: a suspension of tissue sample from suspected ASF infected pig is added to macrophage cultures of porcine origin. The macrophage cultures are examined microscopically for evidence of haemadsorption and cytopathic effect and the result obtained with the test samples compared with ASF negative and positive controls.
- Polymerase chain reaction (PCR): primers from a conserved region of the ASF virus genome are used to detect and identify ASF isolates. ASF positive, negative and test tissue samples are added to separate tubes containing PCR amplification buffer and Taq DNA polymerase. The tubes are placed in a DNA thermal-cycler and subjected to a programmed cycle of heating and cooling. Samples are taken from each tube and loaded into lanes in gel containing ethidium bromide, control lanes contain DNA markers and are electrophoresed. The gel is examined over UV light, if ASF virus is present in the sample a discrete band will be present that will co-migrate with the PCR product of the positive control. The size of the PCR products are compared with the DNA markers, the positive control should be 278 base pairs. No bands should be seen in the negative control.
- Direct immunofluorescence: specific ASF antibody conjugated to fluorescein dye is applied to cryostat sections or tissue smears from suspected ASF infected pigs. The smears are examined microscopically, using a fluorescence microscope, for evidence of fluorescence and the result obtained with the test samples compared with ASF negative and positive controls.
- Indirect immunofluorescence antibody test (IFAT): similar to direct immunofluorescence as given in 2.6.2 except pig serum, suspected of containing ASF antibody, is added to smears of suspensions of ASF infected pig cell cultures. The smears are examined microscopically, using a fluorescence microscope, for evidence of fluorescence and the result obtained with the test samples compared with ASF negative and positive controls.
- Counter immuno-osmo-electrophoresis (CIOEP): ASF antigen, and specific antibody and test serum are diffused through agar gel using an electric current. The gel is examined, using incident light, for evidence of precipitation lines and the result obtained with the test samples compared with ASF negative and positive controls.
- Immunoblotting: ASF viral proteins obtained from tissue cultures are separated in PAGE gels and transferred to nitrocellulose electrophoretically (Southern blotting). After blocking remaining protein binding sites ASF positive, negative and test sera are reacted with thin strips of impregnated nitrocellulose. A specific reaction is indicated by a colour change in the presence of chloronaphtol. This technique is suitable where equivocal ELISA results need to be confirmed.
- Indirect enzyme linked immunoassay (ELISA): ASF positive, negative and test sera are added to ASF antigen coated ELISA plates. A specific reaction is indicated by a colour change in the presence of the enzyme protein-A horseradish peroxidase, enzyme substrate and chromogen. IgM antibodies can be detected from 3-4 days post-infection and IgG antibodies can be detected from 6 days post-infection (Wilkinson, 1989).
- The use of pig macrophage cell cultures for the diagnosis of ASF virus/antigen is, at this stage, considered not possible as resources do not presently exist in the Virology department. Also, in the current situation, obtaining ASF free-pigs cannot be guaranteed.
- It is also considered that the use of PCR is not appropriate at this stage because although a PCR thermo-cycler is available it has not been set-up and reagents are not available.
- An adequate fluorescence microscope exists in the Dermatophilosis department and, therefore, the direct immunofluorescence technique was considered to be the method of choice for ASF antigen detection.
- It is considered that for antibody detection the CIOEP and indirect immunofluorescence tests have been superseded by the indirect ELISA test. The ASF ELISA has been used for several years by various internationally recognised laboratories and is considered to be the most reliable and reproducible technique for antibody determination (Sanchez- Vizcaino et al, 1983c). The test is capable of handling large numbers of sera and is therefore ideally suited to sero-monitoring and surveillance. A working ELISA reader that is connected to a computer is available in the Trypanosomosis department. Support is received by NVRI from IAEA, Vienna to perform assays for rinderpest, peste des petits ruminant and trypanosomosis.
- The immunoblot assay is also used to detect antibody. It is particularly useful for checking equivocal ELISA results and for testing haemolysed and/or contaminated serum samples. Specialised equipment is not required to perform this test.
NVRI consists of many different buildings that occupy the main site. The intention is to conduct ASF diagnosis in the Virology department that is currently undergoing refurbishment. This building is isolated from other departments and is therefore not considered a security risk.
The laboratory facilities of the Virology department at NVRI are old but adequate. The department is divided into rooms that will be used for glassware washing and sterilisation, cell culture and media preparation, and viral diagnosis. There is no fluorescence microscope and the ELISA reader is broken, probably beyond repair. However, a working fluorescence microscope is available in the Dermatophilosis department and a working ELISA reader is available in the Trypanosomosis department.
In general the equipment in the Virology department is old and in a state of disrepair.
Items in short supply were mainly glassware (e.g. bijoux, Universals, pipettes, micro-bottles and tubes with screw caps for preparing dilutions). This may cause problems when large numbers of samples are received during the sero-monitoring exercise.
Plasticware was also in short supply and may present a problem if large numbers of sera require storing.
In most cases air conditioning is not available, this may present a problem in the hot months. During the consultants visit the ambient temperature did not exceed 25oC.
The post mortem room and incinerators are located away from the main laboratory. The incinerator is not working and carcasses are disposed of by burial.
The use of correct disinfectants and disinfection procedures, and general concepts of disease security were not evident. However, it was possible to find Lysol that is active against ASF.
The NVRI site is supplied with water from the local main water supply. Sometimes this was not available. The pressure is very low, probably less than 1 bar.
Pure water is produced by each department. The Vaccine department was able to produce 75 litres in 24 hours from an old Manesty metal still. The pH of the tap water was 5.5 to 6.0 (chlorine could be smelt) and distilled water was 6.0 to 6.5. Despite several attempts by the Chemistry department it was not possible to obtain figures for hardness or conductivity.
Generally the mains electricity is good, but voltage fluctuations and power cuts occur occasionally. In the event of mains failure there is a diesel generator which appears to be well maintained and functions when mains electricity fails.
At present there are five staff in the Virology department, including Dr. Majiyagbe, who provides good leadership.
Several of the veterinarians and technicians have received external training, mainly through IAEA, in laboratory diagnosis.
The equipment, chemicals and reagents immediately required for project activities (Annex 3) were hand carried to NVRI by the consultant.
Equipment and chemicals available in the Virology department are listed (Annex 4). The ELISA reader (Titertek Multiskan Plus) was not functional. The fluorescent microscope (Zeiss Axioskop 20) in the Dematophilosis department was set-up by the consultant.
Suppliers' addresses are given in Annex 12.
A working pH meter could not be located, therefore, pH indicator strips were used.
The competence of the technical staff in basic laboratory procedures was good. However, there was a lack of an appreciation of disease security.
Advice was given on improving the washing and sterilisation procedures performed in the Virology department (e.g. use of detergents, rinsing of glassware with distilled water and use of autoclave tape instead of masking tape).
The method of washing micropipette tips was also reviewed. It is now generally accepted (IAEA, Vienna state in their ELISA manuals that tips should be discarded after use) that it is more economic to discard tips than to wash them. If one badly washed or rinsed tip is used it can affect the whole test thus requiring the test to be repeated. The method presently used to wash and rinse tips is unsatisfactory and will inevitably lead to problems.
During the consultancy 30 sera and one dead pig arrived at NVRI from the field.
Advice and instruction was given on the collection, packaging and transport of specimens within Nigeria and to International laboratories.
Improved ASF diagnostic and serological capability has been achieved in Nigeria at NVRI by the establishment of three laboratory techniques: IFAT, ELISA and immunoblot. Descriptions of the tests are given in Section 2.6, and Annexes 6,7 and 8 give details.
Training in these techniques was provided to the relevant staff, particularly those from the Virology department, with emphasis given to quality control procedures. The diagnostic tests were performed by Dr. Shamaki and the consultant using pig tissue and sera obtained from the field. In addition, instruction was provided on collecting the correct type of tissue samples, how serum should be collected from whole blood, how best to store the samples and how samples should be transported.
The consultant, using the methods developed at INIA, Madrid and those recommended by OIE, wrote standard operating procedures (SOPs) for the IFAT, ELISA and immunoblot tests.
The consultant was invited by the Director, NVRI to lecture to staff. Lectures were, therefore, given to approximately 18 staff from various departments on good laboratory practice (GLP), the theory and practice of ELISA, IFAT and immunoblot tests, the role of mammalian cell culture to viral diagnosis and vaccine production, and general microscopy. Instruction was also given in the use and maintenance of micropipettes, pH meters, microscopes and balances.
Using reagents supplied by INIA the IFAT was applied to tissues from pigs code numbered Z01/98 (dead pig from Zonkwa exhibiting clinical ASF) and LID (piglet from NVRI farm) and examined for ASF specific fluorescence. The tissues from pig LID did not exhibit fluorescence, whereas the tissues from pig Z01/98 showed granular, cytoplasmic fluorescence characteristic of ASF infection. Details of the results are given in Annex 9.
The ELISA test was applied to pig sera taken from Jos abattoir on 5 September 1998 and in 1993, and Zonkwa on 7 September 1998. Using the test controls as reference the following results were obtained (details are given in Annex 9):
- two sera from Zonkwa, Z1 (G10) and Z8 exhibited a slightly elevated optical density (OD) reading, but below the positive threshold;
- one serum from Jos abattoir (P-89-LAF) exhibited a higher OD value than Z1 but just below the positive threshold;
- the OD's of all other sera were well below the negative threshold;
- the controls performed satisfactorily.
Confirmation of the ELISA results on two sera, Z1 (G10) and P-89-LAF was conducted using the immunoblot technique. The results are described below and in Annex 9.
Distilled water from the Vaccine department was used to prepare all buffers with satisfactory results.
The immunoblot test was established and applied to:
- two sera, Z1 and Z8, from Zonkwa of 7/9/98 that gave equivocal ELISA results;
- one sera from Jos abattoir of 5/9/98;
- four other sera from Zonkwa of 7/9/98.
Z1 and Z8 serum from Zonkwa reacted with 9 and 5 ASF viral proteins (molecular weight range 23K to 34K), respectively. All other sera did not react with any of the ASF viral proteins. The controls performed satisfactorily.
In addition, spleen tissue from Z01/98 was impregnated onto nitrocellulose giving a "dot-blot" and reacted in the same manner as for the immunoblot. The tissue exhibited a reaction with control ASF positive serum but not with ASF negative control serum. Details of the results are given in Annex 9.
Discussions were held with Dr. Majiyagbe on sample testing, documentation of results, providing the correct samples and mapping samples and results. Emphasis was given on feed-back of results to farmers and field staff, and on submission of fresh samples if an unsatisfactory laboratory result was obtained. Encouragement was given to communicate ASF results to FAO H.q., Rome.
Details of how and why serum banks should be set-up were discussed and reference papers on the subject were provided. The serum bank is fundamental to retrospective epidemiological NVRI investigations and all sera entering the laboratory should form part of the bank. Advice was also given on how to catalogue sera so that duplication of numbers can be avoided.
Copies on disc and hard copies of sample and test recording forms (see Annexes 6, 7 and 8) were given to the counterparts. These should be useful in providing an easy, efficient method of keeping track of diagnostic tests and the reagents used.
It is apparent that the little equipment that the Virology department has is old and in a state of disrepair. In comparison, other departments seem to be quite well supported and have equipment (e.g. fluorescent microscope, ELISA readers glassware and chemicals) that potentially could be shared. One of the problems encountered by the consultant was bringing all the equipment, chemicals, glassware, etc. required to establish and demonstrate the techniques under one roof. To enable ASF diagnosis and sero-monitoring to be conducted efficiently it is suggested that the work takes place where equipment that is common to diagnosis and serology, whether it be for trypanosomosis or virology, is housed together. This would allow more efficient use of resources.
Disease security is most important at the stage of receiving the carcasses and specimens and is, therefore, best handled in the Pathology department. Tissues and specimens can then be forwarded to the relevant department(s) in sealed containers. The containers should then opened in biological safety cabinets. To enable the Pathology department to perform its functions well it will require lighting, water to the taps in the room rather than via a hose-pipe from a tap outside the building, disinfectant, sharp knives, scissors, etc. Care should be taken to ensure carcasses are disposed of without risk to personnel, animals or the environment.
The removal of carcasses, etc. comes under waste disposal that in general requires addressing at NVRI. The incinerator next to the Pathology department is not working and if finances do not allow its rehabilitation then carcasses should ideally be autoclaved and/or disposed of in covered pits at least six feet deep. If ASF infected animals are placed in the pits lime should be added. Liquid waste (e.g. blood) should drain into a sewage system that allows its containment and disinfection. During post mortem it is important to use the correct disinfectant. To avoid problems with not knowing whether a disinfectant is going to work or not a broad spectrum disinfectant should be used,such as Virkon. The same disinfectant needs to be supplied to the field services. Sharp material (e.g. scalpel blades and syringe needles) should be disposed of in secure containers that can be autoclaved and then buried.
Provision of suitable pure water did not present a problem. However, the equipment is old and in disrepair and therefore it might be considered appropriate to purchase a small bench-top glass still. At present the mains water system at NVRI suffers from low pressure (=1 bar) which limits the type of pure water system that can be used. The minimum requirement would be deionised water produced from a two stage in situ regenerable deioniser giving water quality with a conductivity of <30 -Si/cm (<1.0 -Si/cm would be considered excellent). The minimum pressure required is 1.5 bar at 60l/hour. There is no guarantee, however, that the water is pyrogen free. Pyrogens are caused by Gram-negative organisms and are of detriment to pure water especially when used for the preparation of cell culture media and ELISA reagents.
Distillation is considered to almost totally remove dissolved ionised solids, organics, particulates, bacteria and pyrogens, therefore, the purest water is double, glass distilled.
A compromise system would be a two-stage in situ deioniser that can be regenerated followed by single, glass distillation. To avoid contamination with pyrogens, large quantities (10-20 litres) of water should not be stored at room temperature (25oC) for longer than a week. A practical method of pure water storage is in 1litre borosilicate glass vessels held at 4oC.
Voltage fluctuations occur regularly and any new electrical equipment should be provided with a voltage stabiliser.
A technical training programme is proposed in the early part of 1999 at Daka. It is important that at least two people from the Virology department attend this course.
Equipment and reagents have been identified for possible funding under the current project and are listed in Annex 5.
Generally it is considered to be uneconomic to re-use micropipette tips. However, if it is considered that for sustainable operations these tips should be washed, then a manifold that allows individual tips to be connected to the water supply should be obtained.
Constraints to the effective provision of samples to NVRI and, therefore, to the efficient diagnosis of ASF are lack of a quick means of communication and the national shortage of petrol.
If the proposed control of ASF is to be successful an effective survey for the presence of ASF antigen and antibody should be implemented. The diagnostic facilities, tests and expertise now exist in the Virology department, enabling such samples to be processed efficiently and competently.
Ideally, sero-monitoring for ASF antibody should be performed as soon as possible. This would allow pigs with ASF antibody to be identified and the existence of ASF carrier status determined. The presence of ASF antigen in pigs with antibody is determined by examining leukocytes from heparin blood samples using the autorosette technique or IFAT. However, before such a survey can be implemented, a method by which pigs, and therefore samples, can be obtained needs to be found and agreed upon.
It is recommended that NVRI is provided with suitable specimen collection packages allowing specimens to be transported safely to the ASF reference laboratory. Each package should consist of a sterile glass or plastic screw capped bottles, a metal screw capped tin (large enough for one to four bottles) containing absorbent paper and a paper/cardboard box (large enough for one tin). The box should be appropriately pre-labelled with stickers for addresses of sender and addressee, "biological specimens" and storage requirements. This type of system is the minimum requirement for IATA regulations. It is suggested that IAH, Pirbright is contacted for detailed information.
Although the technicians are able to perform the IFAT, ELISA and immunoblot tests well, confidence and proficiency in their use, and the use of related equipment is lacking. This can only be acquired by performing the test on a regular basis. It is, therefore, most important that samples are obtained from the field. Such practice will inevitably help develop pipetting skills and confidence.
The techniques are sustainable at NVRI if water, electricity and facilities for the production of pure water continue to be provided, and relevant equipment is maintained in working order. Biological reagents (e.g. ASF ELISA antigen, ASF positive and negative antibody, fluorescein conjugate, nitrocellulose strips impregnated with ASF viral proteins) should continue to be provided by INIA, Madrid. Except for control sera, the production of control reagents in-house should be resisted because, although most of the staff are technically competent, resources are not easily available.
It will be noted from the ELISA results (Annex 9) that the OD of the ASF control negative serum from Madrid and Plum Island were approximately 3 times higher than those obtained from Nigerian ASF negative pigs. It is, therefore, recommended that NVRI establish it's own ASF negative control sera and, if possible, ASF positive control sera.
To ensure conformity of ASF diagnosis in the West African region it is important to adhere as closely as possible to the SOPs. If major changes to the methods are required the changes should be agreed with INIA, Madrid before they are implemented. If problems arise with the tests, either through lack of reagents or through the test not working correctly back-stopping should be sought from INIA, Madrid or LNERV, Daka.
It is recommended that the SOPs for diagnosis of ASF antigen by IFAT (Annex 6) and ASF antibody by ELISA (Annex 7) and immunoblotting (Annex 8) be followed as closely as possible. If it is found that changes to the SOPs are required, INIA, Madrid should be informed so that the changes can be agreed to before they are implemented.
ASF positive tissues should be forwarded to a FAO internationally recognised laboratory that can perform molecular characterisation. The results will be helpful in performing comparative studies to establish the relationships between different ASF isolates and their possible origin.
If reports from Zonkwa of biting flies engorging blood from ASF infected pigs can be confirmed it would be interesting to examine these flies, and if necessary ticks, for the presence of ASF virus/antigen.
There are various scenarios of how the pig industry in Nigeria may control and recover from the devastating effects of the ASF epidemic. In Nigeria's present circumstances probably the most important measure is to ensure that ASF does not infect commercial piggeries. This can only be achieved by mounting a public information and awareness campaign. The control measure that is thought by authorities to be most effective is slaughtering infected pigs but for this to be conducted effectively the farmers will require compensation, either financial or in-kind. To avoid the re-occurrence of ASF in restocked pigs it is imperative that sound epidemiological data be produced from sero-surveillance studies. This can only be provided from diagnostic tests performed correctly and under strict quality controlled conditions. The situation is further complicated by the fact that a carrier status can exist in pigs that have recovered from the effects of ASF. If the slaughter policy is not adopted then these pigs become a potential threat to any newly introduced pigs, as would be the case if re-stocking occurred. It is, therefore, important to identify those pigs with ASF antibody and determine their antigenic status. On the basis of these studies and allowing adequate time for infection to disappear (it may be necessary to place susceptible pigs onto sites designated for re-stocking to ensure the infection has gone) particular areas or farms free of ASF could be considered for re-stocking.
1. Alcaraz, C., de Diego, M., Pastor, M.J. and Escribano, J.M. (1990). Comparison of a radioimmunoprecipitation assay to immunoblotting and ELISA for detection of antibody to African swine fever virus. J. Vet. Diagn Invest., 2,191-196.
2. Davies, F.G. (1997). African Swine Fever in Nigeria. FAO, Rome.
3. El Hicheri, K. (1998). Report of FAO mission to Nigeria. FAO, Rome.
4. FAO, Rome (1998). Technical meeting in Togo for the control/containment of ASF. Appendix III.
5. Office International des Epizooties (OIE) (1996). African swine fever. Manual of Standards for Diagnostic Tests and Vaccines, Chapter 2.1.12, OIE, Paris.
6. Pastor, M.J., Laviada, M.D., Sanchez-Vizcaino, J.M. and Escribano, J.M. (1989). Detection of African swine fever virus antibodies by immunoblotting assay. Can. J. Vet. Res., 53, 105-107.
7. Sanchez Vizcaino et al, 1983c. Comparative study of two antigens for use in the indirect ELISA test for the detection of African swine fever antibodies. In: Wilkinson, P.J. (Ed.) African swine fever EUR 8466 EN, Proc. CEC/FAO Research Seminar, Sardinia, September, 1981, 101-106.
8. Wilkinson, P.J., 1989. African Swine Fever. Virus Infections of Porcines, Chapter 2, 17-35. Ed. Pensaert, M.B. Elsevier Science Publishers.
Wednesday, 26 August:
- by air to Dakar from UK.
Thursday, 27 August:
- briefing at INIA meetings with Drs Sarr and Diop, and reagents collected. Briefing with Mr. Tapsoba, FAOR.
Friday, 28 August:
- by air to Lagos via Bamako and Abidjan. Collected at airport by IITA.
Saturday, 29 August:
- by air to Abuja.
Monday, 31 August:
- briefing at FAO with Messrs. A-Shami (FAOR), Umar (NPO), Esso (PS) and Hallini (PS), and Drs Ladan (Director, FDL&PCS), Abegunde (Deputy Director, FMANR), Saidu and Oloko (Assistant Directors, FDL&PCS), Oluokun (Project National Co-ordinator, FDL&PCS), Nwakonobi (Principal Veterinary Officer, FDL&PCS) and Dr. West (National Consultant). Meeting at FDL&PCS with Dr. Abegunde. Unable to commence journey to Jos due to meetings.
Tuesday, 01 September:
- by road to Jos. Discussions and programme for consultant established with Drs. Lamorde (Director, NVRI, Vom), Makinde (Head Dermatophilosis Division and Shamaki (Veterinarian). Handed over ELISA reagents and equipment brought from LNERV-SIRA, Daka.
Wednesday, 02 September:
- lectured on GLP. Located fluorescent microscope, installed bulb and set-up microscope. Instructed Drs. Shamaki, Joannis and Makinde in use of microscope. Discussions with Dr. Majiyagbe.
Thursday, 03 September:
- continue lecture on GLP and begin immunoassays. Reviewed laboratories where ASF diagnosis will take place. Also reviewed condition and availability of equipment in these laboratories.
Friday, 04 September:
- continue lecture on immunoassays. Print draft SOPs for ELISA, IFAT and immunoblotting. Assisted at post mortem of ASF negative pig and suspect ASF pig from the field. Set-up equipment and reagents for demonstration of IFAT on pig samples on 5 September.
Saturday, 05 September:
- lecture on tissue culture. Performed IFAT on normal and suspect ASF pig samples. Fluorescent microscope working.
Monday, 07 September:
- print final version of SOPs and worksheets for ELISA, IFAT and immunoblotting techniques. Prepared buffers, etc. for ELISA and immunoblotting techniques. Identified sera and tissues for diagnosis. Checked ELISA reader and manual. Coated ELISA plates with ASF antigen.
Tuesday, 08 September:
- ELISA on all available pig sera.
Wednesday, 09 September:
- immunoblotting on all ELISA doubtful sera plus tissue sample from ASF suspect pig from Zonkwa. Discussed results. Lectured on microscopy, met Zeiss representative for Nigeria, discussed availability and cost of spare bulbs and accessories, e.g. camera.
Thursday, 10 September:
- discussions with Drs. Lamorde, Majiyagbe and Makinde. Informed Dr. Lamorde that results of tests indicate that pig from Zonkwe is ASF positive. Discussions with other staff relating to techniques and general laboratory management. By road to Abuja.
Friday, 11 September:
- debriefing at FAO with Mr. A-Shami, and at FDL&PCS with Alhaji Alfa Walis, Honourable Minister for Agriculture, the Permanent Secretary, Dr. Alkaleri, and Drs. Abegunde, Nwakonobi, Oluokun and Majiyagbe. Discussed implementation of three diagnostic tests, technology transfer, test application to field samples, results and training of staff.
Saturday, 12 September:
- by air to Lagos
Sunday, 13 September:
- by air to Rome
Monday, 14 September:
- debriefed at FAO H.q.
Monday, 14 September:
- by air to London
FAO, Dakar
Mr Tapsoba, FAOR
LNERV-SIRA, Daka
Dr. Joseph Sarr, Head Virology and Regional Reference Laboratory
Dr. Mariam Diop, Research Scientist
FAO, Abuja
Mr. Hashim A-Shami, FAOR
Mr. Imam Umam, NPO
FDL&PCS, Abuja
Dr. J. Ladan, Director
Dr. T.O. Abegunde, Deputy Director
Dr. S.N.A. Saidu, Assistant Director
Dr. O.A. Oloko, Assistant Director
Dr. S.B. Oluokun, Project National Co-ordinator
Dr. I.Z. Nwakonobi, Principal Veterinary Officer
Consultant
Dr. David West, National Consultant
NVRI, Vom
Dr. A.G. Lamorde, Director
Dr. K.A.A. Majiyagbe, Assistant Director of Diagnostic Services
Dr. A.A. Makinde, Head Dermatophilosis Research Centre
Dr. D. Shamaki, Veterinarian
Dr. T. Joannis, Veterinarian
Dr. P.A. Okewole, Veterinarian/Pathologist
Dr. M.C. Ohaeri, Head of Large Animal Viral Vaccine Production Division
Dr. J. Luther, Veterinarian
Mr. Elalayo, Technologist
Mr. T. Udeani, Technologist
Field Staff
Dr. Salami, Veterinarian, Kaduna State
Dr. A. Bahago, Veterinarian, Zonal Veterinary Officer for Zonkwa region
FAO H.q., Rome
Dr. Kris Wojciechowski, Animal Health Officer (Virology)
Dr. Cheneau, Head AGAH
Mr. Angus Graham, Operations Officer
Dr. Timothy Obi, Visiting Scientist
Dr Roger Paskin, Visiting Scientist
Item Unit Qty
Equipment:
Micropipette (BDSL), single channel, 5-50-l Ea 1
Micropipette (BDSL), single channel, 50-250-l Ea 1
Micropipette (BDSL), four channel, 50-250-l Ea 1
Plastic micropipette tips (clear) Pkt 500
Reagent reservoir Ea 1
ELISA microplates (NUNC Maxisorp) Ea 10
Microscope slides Ea 4
Custom made trough to hold nitrocellulose strips 1
pH indicator strips (range 4.5 - 10.0) Ea. 50
Plastic tubes Ea 8
Marker pen Ea 1
Thermoflask Ea 1
Biological reagents:
ASF positive pig serum (batch A204) 2ml 1
ASF negative pig serum 2ml 1
ASF direct fluorescence conjugate 25-l 1
Protein-A peroxidase conjugate 5ml 1
Nitrocellulose strips Ea. 10
Chemicals:
Tween 20 50ml 1
Glycerol 10ml 1
Hydrogen peroxide Tab. 1
Orthophenylene diamine (OPD) Tab. 5
Carbonate/bicarbonate Tab. 6
Citric acid Sachet. 1
Skimmed milk powder 100g 1
Phosphate buffered saline (powder, BDSL) 10g 3
4-chloro-1-naphthol 8mg/tab. 6
Item Qty Condition
Orbital shaker 1 Erratic
ELISA, Maxisorp, plates Supplied by IAEA
Refrigerators, -20oC 3 All working
Fridges, +4oC 2 All working
Balances 3 All working
Incubator, 20 to 70oC 2 One not working
ELISA reader 1 Not Working
Microscope, fluorescence 1 Working
Single channel micropipettes:
5 - 50-l 1 Working
50 - 200-l 1 Working
200 - 1000-l 1 Working
Multichannel pipettes:
8-channel, 5 - 200-l 1 One tip broken
12-channel, 5 - 50-l 1 Handle cracked
ELISA "Maxisorp" plates, PBS, powder, OPD, tablets and H2O2, tablets are available through the IAEA TCP for rinderpest and PPR diagnosis
| Item | Qty |
Approx. Cost $ |
| 1. Titertek Multiskan EX ELISA reader Spares are available through local agent, see addresses |
1 | 8,800 |
| 2. Photomicrography attachment for Zeiss Axioskop
fluorescence microscope and plastic UV filter for operator protection |
1 1 |
Prices can be obtained from local Zeiss representative |
| 3. Voltage stabilisers (for ELISA reader, microscope and incubator/shaker) | 3 | 300 |
| 4. Glass distillation unit (4 litres per hour) | 1 | 1,072 |
| 5. pH meter with accuracy of 0.01 pH unit | 1 | 250 |
| 6. Microplate incubator/shaker, Insel IS89 | 1 | 2,438 |
| 7. Timer + spare batteries | 3 | 16 |
| 8. Automatic pipettor, mains operated, 240V | 1 | 300 |
| 9. Automatic pipettor, rechargable battery | 1 | 350 |
| 10. To support the introduction of a serum bank and store
approximately 5000 sera (purchase from Nalgene or similar): - metal racks - plastic boxes |
4 30 |
864 451 |
| 11. Cryovials, 1ml | 2000 | 486 |
| 12. Water and freezer-proof marker pens | 10 | 33 |
| 13. Magnetic bar retrievers | 2 | 32 |
| 14. Microscope slides, 50 slides per box | 12 boxes | 48 |
| 15. Coverslips, glass (refractive index 1.515) | 1000 | 15 |
| 16. VIRKON disinfectant, Antec International, UK (use at 25g/5l) | 25kg | 1,152 |
| 17. Methanol, absolute | 2.5l | 16 |
| 18. Ethanol, absolute | 2.5l | 104 |
| 19. ELISA plates 8-well strips. Microwell frame for 8-well strips Maxisorp F8 |
50 60 strips |
165 12 |
| 20. Microplates, 96-U shaped wells | 100 | 512 |
| 21. Individually wrapped serological pipettes, plastic 25ml 10ml 5ml 2ml 1ml |
50 50 50 50 50 |
32 25 14 32 35 |
| 22. Micropipettes, multichannel 8 channel: 0 - 50-l 50 - 250-l or 12 channel: 0 - 50-l 50 - 250-l |
1 1 1 1 |
600 600 736 736 |
| 23. Micropipettes, single channel 0 - 50-l 50 - 250-l 250 - 1000-l |
1 1 1 |
270 270 270 |
| 24. Micropipette tips, to fit 0 - 250-l single and multichannel pipettes | 5 x 1000 | 60 |
| 25. Micropipette tips, to fit 250 - 1000-l single | 5 x 1000 | 85 |
| 26. Cellulose nitrate paper (Scheider & Scholl or equivalent), 0.1-m and 0.05-m | 5 pkts each | 700 |
| 27. Tissue culture medium: MEM M199 |
40 litres each | 64 48 |
| 28. Foetal calf serum | 500ml | 88 to 173 depending upon quality |
| 29. Antibiotics, powder form: Penicillin & Streptomycin, 10ml Gentomycin,10ml Mycostatin, per vial Neomycin |
10 10 10 25g |
240 400 250 35 |
| 30. Tissue culture flasks 25cm2 75cm2 |
20 flasks 5 flasks |
25 10 |
| 31. Autoclave tape, rolls | 10 | 110 |
| 32. Control reagents from INIA, Madrid for ELISA, IFAT and immunoblot | Proforma requested from INIA 13/9/98 | |
| ASF infected mammalian cells fixed in 4% buffered formalin (cells should be infected with ASF at high-multiplicity of infection for approx. 12 hours prior to harvesting). INIA, Madrid should be approached in the first instance. | Unknown | |
| 33. Negative control cells (same type of cells as used in) | Unknown | |
| 34. Plastic coverslips (NUNC plastic), cat. no. 154453K, 16 per box | 1box | 74 |
| 38. Containers for specimen collection and transport | 100 | 336 |
(based on methods used at INIA, Madrid1 and OIE Manual of Standards for Diagnostic Tests and Vaccines 2)
Equipment required:
1. Fluorescence microscope fitted with 50 or 200W UV high pressure mercury lamp, oil immersion objective (x100 magnification), x10 magnification eyepieces, condenser and barrier filters. Provision of a camera is useful for recording results
2. Slide staining jars with lids
3. Microscope slides
4. Coverslips
5. Humidified chamber, plastic sandwich box
6. Timer: any type, preferably count-down type with audible alarm
7. Diamond pen
8. Sterile petri dishes
9. Absorbent paper: disposable, lint-free and non-abrasive.
10. Worksheets
11. Forceps and scissors
12. Bunsen burner
13. pH meter with accuracy of 0.1 pH unit, or pH indicator strips, range 6.0 to 8.0
14. Wax pencil or coloured nail varnish
15. If acetone is not available a desk lamp fitted with 60 or 100 watt light bulb will be required
16. Hair drier, optional
17. Incubator, any type of radiant, warm wall incubator or hot room, giving a temperature range 35oC to 39oC
18. Micropipettes, 50 - 200-l plus tips
19. Glassware/plasticware: one litre cylinder, one litre bottle or flask, storage bottles capacity 2ml, 5ml and 25ml
20. Waterproof marker pens
21. Water purification system: minimum requirement glass distilled or deionised pyrogen-free water
22. Refrigerator: any type that gives a temperature range of +2oC to +6oC
23. Freezer: any type that gives a temperature range of -15oC to -30oC
24. Wash bottles
25. Magnetic stirrer and stir bars
26. Balance, electronic
Chemicals required:
1. Acetone
2. Glycerol
3. Sodium chloride (NaCl)
Potassium chloride (KCl)
Potassium dihydrogen orthophosphate (KH2PO4)
Disodium hydrogen phosphate dodecahydrate (Na2HPO4.12H2O)
or
Phosphate buffered saline tablets
4. Ethyl alcohol, absolute
5. Lens oil immersion fluid
6. Disinfectant, trade name VIRKON3 or suitable alternative that will inactivate ASF virus
7. pH standards, pH 4.0, 7.0 and 9.0
Biological reagents required and supplied by INIA:
1. ASF virus antibodies conjugated to fluorescein
2. If available, fluorescein conjugated pig antibodies negative for ASF
3. ASF positive control tissues
4. ASF negative control tissues
Preparation of Chemicals and Reagents
1. PBS
Sodium chloride (NaCl) ................................................................................. 8.0g
Potassium chloride (KCl) ............................................................................... 0.2g
Potassium dihydrogen orthophosphate (KH2PO4) ........................................ 0.2g
*Disodium hydrogen phosphate dodecahydrate (Na2HPO4.12H2O) ............. 2.9g
Distilled water ............................................................................................... 1.0 litre
Dissolve completely and adjust pH to 7.2 - 7.4, as necessary.
or
Dissolve one PBS tablet in one litre of distilled water, dissolve completely and adjust pH to 7.2 - 7.4, as necessary.
Store at 4oC or for long term storage at -20oC.
*If disodium hydrogen phosphate dodecahydrate (Na2HPO4.12H2O) is not available disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O) can be substituted as follows:
Disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O) ........................... 1.44g
Distilled water ................................................................................................ 1.46ml
Making the total volume 1,001.46ml
2. PBS-glycerol (10%)
Glycerol .......................................................................................................... 1.0ml
PBS, pH 7.2 to 7.4 .......................................................................................... 9.0ml
3. Microscope slides: place approximately 50 slides into 100% ethyl alcohol, leave to de-grease.
4. Disinfectant solution
VIRKON powder .................................................................................. 25.0g
Tap water ...................................................................................................... 5 litres
TEST METHOD
In brief:
1. Prepare disinfectant
2. Remove slides from alcohol and wipe clean with lint-free tissue
3. Make impression smears of ASF positive, negative and sample tissues onto clean slides
4. Air dry
5. Fix smears by placing slides in acetone at room temperature or by using heat from lamp
6. Wash slides in PBS
7. Air dry
8. Encircle smears with wax pencil or nail varnish
9. Add ASF antibodies conjugated to fluorescein and incubate at 37oC
10. Wash slides in PBS
11. Air dry
12. Mount coverslips
14. Examine smears using fluorescence microscope
In Detail:
1. Prepare two, 5 litre, buckets of "VIRKON" (see suppliers, Antec) disinfectant at 1/200 (25g in 5l). One bucket should be kept near the sink and the other at the bench.
2. Remove slides from alcohol and using lint free cloth or paper towel carefully wipe clean. This should remove all traces of grease. The clean slides should be carefully handled using the edges of the slide. Finger prints will leave greasy marks that may cause the smears to detach from the slide. Using a diamond pen mark the slides so that the position and whether the smears are ASF positive or negative controls or test samples can be identified.
3. Remove ASF positive, negative and test samples from +4oC. If tissues have been stored at -20oC thaw quickly. Starting with the ASF negative tissue, using forceps and scissors, remove from container and cut the tissue into a small cube. Touch the tissue onto absorbent tissue (repeat several times if necessary) to remove excess moisture then press gently onto clean slide to make impression smear in the required position. Carry out the same process with the test sample and then the ASF positive sample. The forceps and scissors should be dipped in alcohol and flamed to sterilise between tissue samples.
4. Air dry smears at room temperature or use cool air from hair drier.
5. Fix smears by placing slides in staining jar filled with 100% acetone. Leave at room temperature for 10 minutes. Alternatively smears can be fixed using heat from a lamp for 30 minutes.
6. Wash slides by gentle irrigation using wash bottle containing PBS. Discard washings in disinfectant. Then place slides in staining jar filled with PBS and leave for 3 minutes. After use discard PBS into disinfectant.
7. Air dry smears at room temperature or use cool air from hair drier.
8. Using wax pencil or nail varnish draw circle around smears, this will limit the spreading of the conjugate when it is applied to the smear.
9. Dilute ASF virus antibodies conjugated to fluorescein, supplied by INIA-CISA, Madrid, 1 : 50 in PBS (4-l of conjugate in 200-l of PBS). If available fluorescein conjugated normal pig serum antibodies can be used as a control at the same dilution.
10. Add 100-l - 400-l (depending upon the size of the smear) to each smear. Incubate at 37oC in humidified container for 1 hour.
11. Wash slides by gentle irrigation using wash bottle containing PBS. Discard washings in disinfectant. Then place slides in staining jar filled with PBS and leave for 3 minutes. After use discard PBS into disinfectant.
12. Air dry at room temperature or use cool air from hair drier.
13. Mount clean coverslips over each smear in 10% PBS-glycerol.
14. Examine smears using fluorescence microscope.
15. The ASF positive and negative controls should be examined first. When assured that the test has worked examine the test samples and compare with the controls. Record result accurately and/or photograph the smear.
Tissues are positive if specific granular, cytoplasmic fluorescence is observed.
1Drs. Sanchez-Viscaino and Arias, INIA-CISA, 28130 Valdeolmos, Madrid, Spain.
Tel. +34-91-6202300; Fax. +34-91-6202247; e-mail. [email protected]
2Office International des Epizooties (OIE), 12, Rue de Prony, 75017 Paris, France.
Tel. +33-44-15-1888; Fax. +33-42-670987
3VIRKON. Antec International Ltd, Chilton Industrial Estate, Sudbury CO10 6XD, UK.
Tel. +44-1787-377035, Fax. +44-1787-310846
Date:...................................... Operator:..........................................................
Fixation:............................................................................................................
ASF negative tissue:..........................................................................................
ASF positive tissue:.........................................................................................
Test tissue:...................................................................................................
Conjugate:................................ Diluent:.............................. Dilution:...............
Wash buffer sol'n:...............................................
Incubation: Conjugate at 37oC for 1 hour. Washing in PBS for 5 minutes at room temperature.
| Date submitted | Tissue number | Location | Tissue type | Storage | Description of fluorescence | Result |
(based on the methods used at INIA, Madrid1 and OIE Manual of Standards for Diagnostic Tests and Vaccines 2)
Equipment required:
1. Photometer, Titertek Multiskan microplate reader (or equivalent) with a 492nm filter and internal printer. Should include spare bulbs and paper rolls
2. Microplate washer or a container (5 - 10 litres) with tap connected to suitable
tubing and clamp
3. Pipettes: multichannel (8 or 12 channel) micropipettes 50-200-l single channel micropipettes, 5 -50-l, 50-200-l, and 200-1000-l
4. Plastic tips
5. Reagent troughs
6. Water Purification System: minimum requirement is glass distilled or deionised
pryogen-free water
7. Microplates: NUNC Immuno I - Maxisorp (Cat. no. 439454), flat bottom 96-well
microplates (substitution may alter diagnostic performance). The manufacturers
recommend that the plates should be held at between +4oC and 15oC
8. Refrigerator: any type that gives a temperature range of +2oC to 6oC
9. Freezer: any type that gives a temperature range of -15oC to -30oC
10. Incubator: any type of radiant, warm wall incubator or hot room giving a
temperature range of +35oC to +39oC, or incubator/ orbital shaker
11. pH meter: any type with an accuracy of 0.01 pH unit
12. Glassware/plasticware: a selection of beakers (20-4000ml), flasks (50-1000ml),
graduated cylinders (10-2000ml), graduated pipettes (1-25ml), storage bottles
with caps (1-100ml) and dilution tubes (2-4ml) and suitable racks
13. Bottles: 5ml, 10ml and 25ml bottles
14. Timer: any type, preferably count-down type with audible alarm
15. Absorbent towels: disposable, lint-free and non-abrasive
16. Waterproof marker pens
17. Adhesive labels
18. Humidified chamber (plastic sandwich boxes).
19. Worksheets
20. Magnetic stirrer and stir bars
21. Balance, electronic
Chemicals required:
1. Sodium hydrogen carbonate (NaHCO3)
2. Sodium carbonate, anhydrous (Na2CO3)
or
Carbonate/bicarbonate tablets
3. Sodium chloride (NaCl)
4. Potassium chloride (KCl)
5. Potassium dihydrogen orthophosphate (KH2PO4)
6. Disodium hydrogen phosphate dodecahydrate (Na2HPO4.12H2O)
or
Phosphate buffered saline tablets
7. Tween 20 (polyoxyethylene sorbitan monolaurate)
8. Ortho-phenylenediamine (OPD)
9. Sulphuric acid (H2SO4)
10. Hydrogen peroxide (H2O2)
11. Sodium hydroxide (NaOH)
12. Hydrochloric acid (HCl)
13. Disinfectant, trade name VIRKON3, or suitable alternative that will inactivate ASF
Biological reagents required and supplied by INIA, Madrid;
- African swine fever positive antigen
- African swine fever negative antigen
- African swine fever positive antibody
- African swine fever negative antibody
- Protein-A horseradish peroxidase conjugate
Preparation of Buffers
1. Coating Buffer for ASF antigen (0.05M carbonate/bicarbonate buffer), pH 9.6
Sodium hydrogen carbonate (NaHCO3) ........................................................ 4.2g
Sodium carbonate, anhydrous (Na2CO3) ....................................................... 5.3g
Distilled water ................................................................................................ 1.0 litre
Dissolve completely and adjust pH to 9.6, as necessary.
or
One carbonate/bicarbonate tablet dissolved in one litre distilled water. Adjust pH to 9.6, as necessary.
Store at +4oC
2. Wash Buffer (Saline/Tween 20), pH 7.2 - 7.4
Sodium chloride (NaCl), optional.................................................................... 8.5g
Tween 20 ...................................................................................... 0.5ml
Distilled water ............................................................................................... 1.0 litre
Store at +4oC.
3. Diluent for Sera and Conjugate (PBS/Tween 20 )
Sodium chloride (NaCl) ................................................................................. 8.0g
Potassium chloride (KCl) ............................................................................... 0.2g
Potassium dihydrogen orthophosphate (KH2PO4) ........................................ 0.2g
*Disodium hydrogen phosphate dodecahydrate (Na2HPO4.12H2O) ............. 2.9g
Tween 20 ..................................................................................... 0.5ml
Distilled water ............................................................................................... 1.0 litre
Dissolve completely and adjust pH to 7.2 - 7.4, as necessary.
or
Dissolve one PBS tablet in one litre of distilled water and add 0.5ml Tween 20. Dissolve completely and adjust pH to 7.2 - 7.4, as necessary.
Store at 4oC or for long term storage at -20oC.
*If disodium hydrogen phosphate dodecahydrate (Na2HPO4.12H2O) is not available disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O) can be substituted as follows:
Disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O) .......................... 1.44g
Distilled water ................................................................................................ 1.46ml
Making the total volume 1,001.46ml
4. Preparation of Chromogen - OPD
Ortho-phenylenediamine (OPD) ................................................................ one tablet Distilled water .............................................................................................. 75ml
Dissolve completely and store in 20ml - 25ml aliquots at -20oC.
5. Preparation of substrate - H2O2 (3%)
Hydrogen peroxide (H2O2) .................................................................... one tablet
Distilled water ................................................................................. 10ml
Store at +4oC in the dark.
Just before the addition of OPD to the ELISA plate, add hydrogen peroxide at 4-l per ml of OPD (i.e. 100-l per 25ml OPD), mix thoroughly and use immediately.
6. Stopping Solution (1M sulphuric acid)
Sulphuric acid, concentrated (H2SO4) ........................................................... 16.1ml
Distilled water ................................................................................................ 200ml
Measure the water first and add sulphuric acid slowly.
7. Disinfectant Solution
VIRKON powder .................................................................................. 25.0g
Tap water ...................................................................................................... 5 litres
TEST METHOD
In brief:
1. Prepare disinfectant.
2. Dilute ASF positive antigen in coating buffer. Add to each well of ELISA plate(s) and leave overnight at +4oC.
3. Wash plate(s) with wash buffer.
4. Dilute serum samples and ASF positive and negative sera. Add to plate(s) and incubate at 37oC for 1 hour.
5. Wash plate(s) with washing buffer.
6. Dilute Protein-A horseradish peroxidase conjugate. Add to plate(s) and incubate at 37oC for 1 hour.
7. Wash plate(s) with wash buffer.
8. Prepare chromogen/substrate solution. Add to plate(s) and incubate at room temperature for 10-20 minutes.
9. Stop reaction with addition of sulphuric acid.
10. Read the O.D. of the colour produced using ELISA reader.
In Detail:
1. Prepare two, 5 litre, buckets of "VIRKON" disinfectant at 1/200 (25g in 5l). One bucket should be kept near the sink and the other at the workbench.
2. Dilute ASF positive antigen (PPA, batch number A 204 supplied by INIA) 1in 1,600 in coating buffer (10-l of antigen plus16ml of coating buffer). Add to NUNC Maxisorp ELISA microplates, 100-l per well. Leave overnight at +4oC in sealed containers (plastic bags are suitable).
3. Record the sera to be tested on the ASF ELISA worksheets.
4. The next day discard contents of plates into disinfectant and wash plate(s) at least 6 times in wash buffer. After each wash the contents of the plate(s) must be discarded into disinfectant. Blot plates dry onto paper towelling and discard into disinfectant. At this stage the plates can be used immediately or stored at -20oC, in sealed humidified containers, for several months. If the plates are stored at -20oC they must be washed at least twice with wash buffer before use.
5. Dilute the serum samples, and ASF positive (C++, batch number 69, supplied by INIA) and ASF negative sera (C-, batch number 23, supplied by INIA) 1 in 30 in PBS/Tween 20 (20-l of serum plus 600-l PBS/Tween 20) in clean plastic or glass tubes or bottles. Alternatively the sera can be diluted in round bottom well microplates (haemagglutination plates) 10-l of sera plus 290-l PBS/Tween 20. The diluted sera can be used immediately or stored at -20oC until required.
6. Using the ASF ELISA worksheets as a reference add sera to coated plates, 100-l per well. All sera are tested in duplicate. ASF positive serum is added to wells A1 and B1, C4 and D4, E9 and F9, G12 and H12. ASF negative serum is added to wells G1 and H1, E4 and F4, C9 and D9, A12 and B12. Incubate plates at 37oC for 1 hour on incubator shaker or in sealed humidified containers in normal incubator. 40 test sera can be tested in each plate. If sera have pre-diluted (see 5. above) in microplates with round bottom wells the sera can be easily transferred to the ELISA plate(s) using a multi-channel micropipette.
7. Discard contents of plate(s) into disinfectant and wash plates at least 6 times in wash buffer. After each wash the contents of the plates must be discarded into disinfectant. Blot plates dry on paper towelling.
8. Prepare a stock solution of Protein-A horseradish peroxidase conjugate (supplied by INIA) 1 in 2,000 in PBS/Tween 20 (8-l conjugate in 16ml PBS/Tween 20). To determine the optimum dilution, the conjugate may require titrating against negative and positive sera. Add 100-l per well and incubate at 37oC for 1 hour in an incubator/shaker or in sealed humidified containers in normal incubator. Keep any diluted conjugate remaining as this can be used later to check that there is a colour reaction with the chromogen/substrate solution.
9. Discard plate contents into disinfectant and wash plates at least 6 times in wash buffer. After each wash the contents of the plate(s) must be discarded into disinfectant. Blot plates dry on paper towelling.
10. Switch on the ELISA reader, ensure a paper roll is fitted and using the numerical key pad enter date, select mode 1 (ABS1) and select filter 4 (492nm). The reader must be allowed to warm up for at least 30 minutes before plates are read.
11. Remove one bottle containing 20ml of OPD from -20oC and allow to thaw. Thawing can be quickened by gently warming in 25oC water bath. Just before use add 100-l (4-l of H2O2 per ml OPD, that is 80-l H2O2 per 20ml OPD) ) of 3% hydrogen peroxide and mix thoroughly. Add 100-l to each well and incubate at room temperature in the dark for 10 to 20 minutes. The time taken for the colour to develop will depend on both the temperature of the substrate when added to the wells, and the room temperature. If more than one plate is used the addition of the chromogen/substrate should be staggered at 1 minute intervals.
At the same time prepare a new ELISA plate as a "blanking" plate. Add 100-l per well of the chromogen/substrate solution to column 1, wells A to H.
To ensure development of colour add a small quantity of the substrate solution to the conjugate that was kept in 7, above, and check that colour develops, usually within 10 minutes. If there is no colour development, see "Trouble Shooting", below.
12. Stop the reaction by the addition of 1M sulphuric acid, 100-l per well and repeat for column 1 in the "blanking" plate.
13. Before the plates can be read the ELISA reader must be "blanked" using the "blanking" plate prepared above. For a Titertek Multiskan MCC* perform the following:
Place the "blanking" plate on the carriage of the ELISA reader and at the "In" message press the "STEP" key. the plate will be automatically drawn into the machine under the detectors and stopped when column 1 is in position. The "blank" message will be printed out. Press the "BLANK" key and the LED display shows the blanking proceeding message. If blanking is accepted the message "Blank OK Col. 1 Ready" is printed. If "Blank Error" message occurs refer to section 7.9 of the instruction manual. The plate is ejected or removed from the machine by pressing the "STOP" key. The "In" message will be displayed indicating that the reader is ready to read the test plates.
*If another type of ELISA reader is used refer to the instructions for that particular model.
14. Place the first plate, containing the test sera, on the carriage and press the "START" key. The plate will automatically enter the machine and the optical density (OD) of each well will be measured. Correction for the OD of chromogen/substrate is automatically calculated from the "blanking" plate and the corrected figure is printed.
15. The colour intensity is proportional to the amount of specific antibody bound to the antigen. ELISA results should always be evaluated in relation to the controls.
A serum is considered ASF antibody positive if it has an OD value greater than twice the mean OD value of the control ASF negative serum.
Any serum that gives a doubtful result should be re-tested and/or tested using the immunoblotting technique.
ELISA Trouble Shooting
If the test is not correctly carried out or the reagents are faulty the following problems may arise.
1. No colour on plate, possible causes:
- Omission of one of the reagents.
- Lack of hydrogen peroxide in the substrate solution.
2. Weak colour, especially in positive control sera, possible causes:
- Too little antigen on plate. Check antigen concentration by titration.
- Weak conjugate. Re-titrate the conjugate.
- Check the PBS/Tween 20 diluent buffer.
- Check water quality.
- Incubation too short.
- Incorrect plates used, e.g. tissue culture grade plates.
3. Strong colour in all sera including the negative control sera, possible causes:
- Conjugate too concentrated.
- Laboratory temperature significantly higher than that in the laboratory when the test was standardised.
4. Large variation between OD values of control sera, possible causes:
- Incorrect pipetting.
- Poor batch of microtitre plates.
1Drs. Sanchez-Viscaino and Arias, INIA-CISA, 28130 Valdeolmos, Madrid, Spain.
Tel. +34-91-6202300; Fax. +34-91-6202247; e-mail. [email protected]
2Office International des Epizooties (OIE), 12, Rue de Prony, 75017 Paris, France.
Tel. +33-44-15-1888; Fax. +33-42-670987
3VIRKON. Antec International Ltd, Chilton Industrial Estate, Sudbury CO10 6XD, UK.
Tel. +44-1787-377035, Fax. +44-1787-310846.
Date:....................... Plate No:........... Operator:..................................................
Antigen:............................................. Diluent:.......................................................
Neg. serum:....................................... Pos. serum:................................................
Test sera:......................................... Sera Diluent:................................................
Conjugate:............................................ Diluent:....................................................
Chromogen/Substrate:............................................. Diluent:...................................
Stopping sol'n:........................... Washing sol'n:...................................................
Incubation: Antigen, antibody and conjugate stages at 37oC for 1 hour on shaker. Antigen may be incubated at 4oC overnight. Chromogen/substrate at room temperature for approximately 10 minutes. All sera diluted 1 in 30.
(-) = ASF negative pig serum; (+) = ASF positive pig serum; ( ) = Test sera
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | - | + | ||||||||||
| B | - | + | ||||||||||
| C | - | + | ||||||||||
| D | - | + | ||||||||||
| E | - | + | ||||||||||
| F | - | + | ||||||||||
| G | + | - | ||||||||||
| H | + | - |
Mean O.D. Values of Duplicate Wells
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
A positive result is indicated if the mean O.D. of duplicate wells is greater then twice the mean O.D. of the negative serum.
RESULTS:
Mean O.D. of negative serum =
Twice the mean O.D. of negative serum =
| Sample No. | Mean OD | Result | Sample No. | Mean OD | Result |
(based on the methods used at INIA, Madrid1 and OIE Manual of Standards for Diagnostic Tests and Vaccines2)
Equipment required:
1. Electrophoresis equipment enabling proteins to be electrophoresed through acrylamide gel and transferred to nitrocellulose
Note: If pre-prepared nitrocellulose strips are obtained from INIA, Madrid the equipment in 1. above is not required.
2. Plastic lid holding small plastic rectangular troughs (home-made, supplied by LNERV-SIRA, Daka)
3. pH meter and/or indicator strips
4. Single channel micropipettes, 5 -50-l, 50-200-l, and 200-1000-l
5. Plastic tips
6. Water Purification System: minimum requirement is glass distilled or deionised
pryogen-free water
7. Refrigerator: any type that gives a temperature range of +2oC to 6oC
8. Freezer: any type that gives a temperature range of -15oC to -30oC
9. Incubator: any type of radiant, warm wall incubator or hot room giving a
10. temperature range of +35oC to +39oC, or incubator/orbital shaker
11. pH meter: any type with an accuracy of 0.01 pH unit
12. Glassware/plasticware: a selection of beakers (20-4000ml), flasks (50-1000ml),
13. graduated cylinders (10-2000ml), graduated pipettes (1-25ml), storage bottles
14. with caps (1-100ml) and dilution tubes (2-4ml) and suitable racks
15. Bottles: 5ml, 10ml and 25ml bottles
16. Timer: any type, preferably count-down type with audible alarm
17. Absorbent towels: disposable, lint-free and non-abrasive
18. Waterproof marker pens
19. Adhesive labels
20. Humidified chamber (plastic sandwich boxes).
21. Worksheets
22. Magnetic stirrer and stir bars
23. Balance, electronic
Chemicals required:
1. Sodium chloride (NaCl)
2. Potassium chloride (KCl)
3. Potassium dihydrogen orthophosphate (KH2PO4)
4. Disodium hydrogen phosphate dodecahydrate (Na2HPO4.12H2O)
or
Phosphate buffered saline tablets
5. Skimmed (fat free) milk powder
6. Protein-A horse radish peroxidase conjugate
7. 4-chloro-1-naphthol
8. Methanol
9. Hydrogen peroxide (H2O2)
10. Sodium hydroxide (NaOH)
11. Hydrochloric acid (HCl)
12. Disinfectant, trade name VIRKON3, or suitable alternative that will inactivate ASF
Biological reagents required and supplied by INIA, Madrid;
- African swine fever positive antibody
- African swine fever negative antibody
- Nitrocellulose strips pre-prepared with ASF viral proteins
Preparation of Buffers
1. Blocking Buffer (PBS/Milk), pH 7.2
Sodium chloride (NaCl) ................................................................................. 8.0g
Potassium chloride (KCl) ............................................................................... 0.2g
Potassium dihydrogen orthophosphate (KH2PO4) ........................................ 0.2g
*Disodium hydrogen phosphate dodecahydrate (Na2HPO4.12H2O) ............. 2.9g
Distilled water ............................................................................................... 1.0 litre
Dissolve completely and adjust pH to 7.2, as necessary using NaOH or HCl.
Add 2g skimmed milk powder to 100ml PBS. Dissolve completely.
or
Dissolve one PBS tablet in one litre of distilled water, dissolve completely and adjust pH to 7.2, as necessary. Add 2g skimmed milk powder to 100ml PBS. Dissolve completely.
Store at 4oC or for long term storage at -20oC.
*If disodium hydrogen phosphate dodecahydrate (Na2HPO4.12H2O) is not available disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O) can be substituted as follows:
Disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O) .......................... 1.44g
Distilled water ................................................................................................ 1.46ml
Making the total volume 1,001.46ml
2. Preparation of Chromogen/Substrate (4-chloro-1-naphthol/H2O2) prepare just before use, do not store
4-chloro-1-naphthol ................................................................................... 8mg
Methanol ........................................................................................... 2.6ml
Add 4-chloro-1-naphthol/ethanol solution slowly to 8.9ml PBS while stirring.
Remove white precipitate that is formed by filtration through Whatman no. 1 filter paper.
Just before use add substrate (H2O2), 7-l of 30% H2O2 or 70-l of 3% H2O2 (3% H2O2 is prepared by adding 1 tablet to 10ml distilled water, store at 4oC in the dark).
3. Disinfectant Solution
VIRKON powder .................................................................................. 25.0g
Tap water ...................................................................................................... 5 litres
TEST METHOD
In brief:
1. Prepare disinfectant
2. Add pre-prepared nitrocellulose strips to plastic troughs
3. Add blocking buffer and incubate at 37oC for 30 minutes
4. Discard buffer
5. Dilute serum in blocking buffer
6. Add dilutions of serum to strips, one serum sample per strip, incubate at 37oC for 45 minutes
7. Wash strips with blocking buffer
8. Dilute protein-A HRPO conjugate and add to strips. Incubate at 37oC for 45 minutes
9. Wash strips with blocking buffer
10. Prepare chromogen/substrate solution and add to strips, incubate at room temperature for 5-15 minutes
11. Stop reaction with addition tap water
10. Examine each strip and count the number of protein bands that are observed. Compare the test sera with the positive and negative sera
In Detail:
1. Prepare two, 5 litre, buckets of "VIRKON" disinfectant at 1/200 (25g in 5l). One bucket should be kept near the sink and the other at the workbench.
2. Using clean, plastic forceps remove nitrocellose strips from tube and place with line upper-most into plastic trough. One strip per trough per sample. Add 0.5ml blocking buffer (PBS/Milk) per strip and incubate at 37oC for 30 minutes with continuous agitation.
3. Prepare 1 in 30 (20-l of serum plus 580-l) dilutions of test sera, and ASF positive control serum (C++, no. 69 ex INIA) and ASF negative control serum (C-, no. 23 ex INIA).
4. Discard blocking buffer from troughs into disinfectant and add 0.5ml of diluted serum to each strip. Incubate at 37oC for 45 minutes with continuous agitation.
5. Wash strips four times in blocking buffer each for 5 minutes at room temperature with agitation. Discard washings into disinfectant.
6. Prepare stock solution of protein-A HRPO at 1 in 500 (4-l protein-A plus 2ml blocking buffer, sufficient for 3-4 strips). Add 0.5ml per strip and incubate at 37oC for 45 minutes with continuous agitation.
7. Discard protein-A into disinfectant and wash strips as in 5. Above.
8. Prepare fresh solution of chromogen/substrate (see 2., in Preparation of Buffers, above), add 0.5ml per strip and incubate at 37oC for no longer than 15 - 20minutes. Stop the reaction with an equal volume of tap water.
Note: If grey/blue bands of colour do not develop on the strip that receives the ASF positive control, do not add tap water, instead incubate overnight at +4OC.
9. Test serum is considered positive if it reacts with the same number or more ASF viral protein bands as the positive control. The positive control should react with at least 5 ASF viral proteins. The negative serum should not react with the proteins, therefore, the strip should remain white.
1Drs. Sanchez-Viscaino and Arias, INIA-CISA, 28130 Valdeolmos, Madrid, Spain.
Tel. +34-91-6202300; Fax. +34-91-6202247; e-mail. [email protected]
2Office International des Epizooties (OIE), 12, Rue de Prony, 75017 Paris, France.
Tel. +33-44-15-1888; Fax. +33-42-670987
3VIRKON. Antec International Ltd, Chilton Industrial Estate, Sudbury CO10 6XD, UK.
Tel. +44-1787-377035, Fax. +44-1787-310846.
Date:...................................... Operator:..........................................................
Antigen nitrocellulose strips:..................................................................................
Neg. serum:....................................... Pos. serum:................................................
Test sera:......................................... Sera Diluent:................................................
Conjugate:............................................ Diluent:....................................................
Chromogen/Substrate:............................................. Diluent:...................................
Stopping sol'n:........................... Wash buffer sol'n:...............................................
Incubation: First washing of strips at 37oC for 30 minutes. Serum and conjugate at 37oC for 45 minutes. Chromogen/substrate at room temperature for 15 minutes. All sera diluted 1 in 30.
| Date submitted | Serum number | Location | No. of ASF viral protein bands reacting with serum | Result |
Methods:
- Mammalian cell culture
- Immunoassays, general principles
- Microscopy
- Collection of Specimens for Virus Diagnosis
- General Procedures for the Cell Culture Laboratory
- Diagnostic methods for African swine fever
- SOPs for ELISA, IFAT and immunoblotting
References:
- Wilkinson, 1989. African Swine Fever
- Wilkinson, 1986. Epidemiology of African Swine Fever
- Pastor et al, 1987. Detection of African swine fever virus antibodies by immunoblotting
- Alcaraz et al, 1990. Comparison of a radioimmunoprecipitation assay to immunoblotting and ELISA for detection of antibody to African swine fever virus
- Labsystems Oy, Pulttitie 8, P.O. Box 8, 00881 Helsinki, Finland.
Tel: +358-0-75821, FAX: +358-0-789732
- Life Sciences International (UK) Ltd, Unit 5, The Ringway Centre, Edison Road, Basingstoke, Hants RG21 2YH, UK.
Tel: +44-1256-817282, Fax: +44-1256-817292
- Biological Diagnostic Supplies (BDSL) Ltd, Riverbank Business Centre, 5 New Mill Road, Kilmarnock, Ayrshire KA1 3JB, UK.
Tel: +44-1563-543755, FAX: +44-1563-543723
- Techmate (Nalgene) Ltd, 10 Bridgeturn Avenue, Old Wolverton, Milton Keynes
MK12 5QL, UK.
Tel: +44-1908-322222. FAX: +44-1908-319941
- Antec International Ltd, Chilton Industrial Estate, Sudbury, Suffolk CO10 6XD, UK
Tel: +44-1787-377035. Fax: +44-1787-310848
- Antec Healthcare Africa PTY Ltd, P.O. Box 1229, Hilton 3245, Natal, SA
Tel: +27-331-461411/13. Fax: +27-331-461412
