Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 5th edition, 2004

1.   Identification of the agent

     Clinical observation may only lead to a suspicion of rabies because signs of the disease are not characteristic and may vary greatly from one animal to another (36). The only way to perform a reliable diagnosis of rabies is to identify the virus or some of its specific components using laboratory tests.

     As rabies virus is rapidly inactivated, refrigerated diagnostic specimens should be sent to the laboratory by the fastest means available. Shipment conditions must be considered to be part of the 'rabies diagnostic chain'.

     Several laboratory techniques may be used, and have been detailed and standardised in the fourth edition of the WHO's Laboratory Techniques in Rabies (37). The methods vary in their efficiency, specificity and reliability. They are classically applied to brain tissue, but they can also be applied, though less effectively, to other organs (e.g. salivary glands). In the brain, rabies virus is particularly abundant in the thalamus, pons and medulla. The hippocampus (Ammon's horn), cerebellum and different parts of the cerebrum have been reported to be negative in 3.9-11.1% of the positive brains. The structure of choice is the thalamus as it was positive in all cases. It is recommended that a pool of brain tissues that includes the brain stem should be collected and tested (12). To reach these parts of the brain, it is necessary to remove the entire organ after having opened the skull in a necropsy room. Under some conditions (e.g. in the field or when sampling for large epidemiological studies), a simplified method of sampling through the occipital foramen (11), or through the orbital cavity (26), can be used.

     a)   Shipment of samples

          During the shipment of suspect material for diagnosis (animal heads, brain or other tissue samples), no risk of human contamination should arise: brains must be placed in a leak-proof rigid container (animal heads will be wrapped in absorbent material) as prescribed in the International Air Transport Association (IATA) Dangerous Goods Regulations must be followed. These regulations are summarised in Chapter I.1.1. Sampling methods.

          When it is not possible to send refrigerated samples, other preservation techniques may be used. The choice of the preservative is closely linked to the tests to be used for diagnosis:

          .   Formalin inactivates the virus, thus the isolation tests cannot be used and diagnosis depends on using a modified and less sensitive direct fluorescent antibody test (FAT), immunohistochemistry or histology (33, 37);

          .   Infectivity at room temperature may be extended for several days if brain material is kept in a mixture of 50% glycerol in phosphate buffered saline (PBS). Glycerol/PBS slows bacterial action and therefore protects against the chemical and biological effects of putrefaction. It does not protect against titre decline due to thermal conditions and therefore, because rabies is thermo-labile, the virus titre will decline during glycerol/PBS storage. Under normal transport conditions in the tropics, this protection may only be effective for a matter of several days. Therefore, whenever possible samples in glycerol/saline should be kept refrigerated. As the virus is not inactivated by glycerol/PBS, all laboratory tests can be used on these samples.

     b)   Collection of samples

          Usually the brain is collected following the opening of the skull in a necropsy room, and the appropriate samples are collected. This step may be hazardous if laboratory technicians are not fully trained, or under field conditions. In such cases, there are two possible methods of collecting some brain samples without opening the skull:

          .   Occipital foramen route for brain sampling

          A 5 mm drinking straw (11) or a 2 ml disposable plastic pipette (16) is introduced into the occipital foramen in the direction of an eye. Samples can be collected from the rachidian bulb, the base of the cerebellum, hippocampus, cortex, and medulla oblongata. Bovine spongiform encephalopathy (BSE) should be considered in the differential diagnosis of most cattle that are considered to be 'rabies suspect'. Sampling of brain specimens for both diseases can be done using the 'brain scoop or tool' developed for BSE tissue sampling rather than a straw or pipette. The resulting samples are relatively easily recognised as to the area of brain sampled.

          .   Retro-orbital route for brain sampling

          In this technique (26), a trocar is used to make a hole in the posterior wall of the eye socket, and a plastic pipette is then introduced through this hole. The sampled parts of the brain are the same as in the former technique, but they are taken in the opposite direction.

     c)   Routine laboratory tests

          Laboratory diagnosis can be performed by using three kinds of procedure.

          .   Histological identification of characteristic cell lesions

          Negri bodies correspond to the aggregation of viral proteins, but the classical staining techniques detect only an affinity of these structures for acidophilic stains. Immunohistochemical tests are the only histological test specific to rabies.

          An unfixed tissue smear may be stained by the Seller's method, diagnosis is then obtained in under 1 hour. Generally, histological tests, such as Mann's test, are performed on fixed material after a paraffin-embedding step, and the result of the test is obtained within 3 days. These techniques have the advantage that the laboratory equipment needed to perform them is inexpensive and any need to keep specimens cold after fixation is avoided. Whichever staining method is used, the evidence of infection is provided by intracytoplasmic acidophilic bodies. These histological methods, especially the Seller's method, can no longer be recommended because they have very low sensitivity and should be abandoned.

          .   Immunochemical identification of rabies virus antigen

          i)   Fluorescent antibody test

               The most widely used test for rabies diagnosis is the FAT, which is recommended by both WHO and OIE. This test may be used directly on a smear, and can also be used to confirm the presence of rabies antigen in cell culture or in brain tissue of mice that have been inoculated for diagnosis. The FAT gives reliable results on fresh specimens within a few hours in more than 95-99% of cases. The sensitivity of the FAT depends on the specimen (the degree of autolysis and how comprehensively the brain is sampled, see Section B.1.) (1, 9), on the type of lyssavirus and on the proficiency of the diagnostic staff. Sensitivity may be lower in samples from vaccinated animals due to localisation of antigen, which is confined to the brainstem. For direct rabies diagnosis, smears prepared from a composite sample of brain tissue, that includes the brain stem, are fixed in high-grade cold acetone and then stained with a drop of specific conjugate. Anti-rabies fluorescent conjugates may be prepared in the laboratory. Those available commercially are either polyclonal conjugates specific to the entire virus or specific to the rabies nucleocapisid protein, or they may be prepared from a mix of different MAbs. In the FAT, the specific aggregates of nucleocapsid protein are identified by their fluorescence. The specificity and sensitivity of these anti-rabies fluorescent conjugates for locally predominant virus variants should be checked before use.

               The FAT may be applied to glycerol-preserved specimens. If the specimen has been preserved in a formalin solution, the FAT may be used only after the specimen has been treated with a proteolytic enzyme (6, 7, 32, 33). However, the FAT on formalin-fixed and digested samples is always less reliable and more cumbersome than when performed on fresh tissue.

          ii)   Immunochemical tests

               The antibody may be conjugated to an enzyme such as peroxidase instead of fluorescein isothiocyanate (FITC). This conjugate may be used for direct diagnosis with the same sensitivity as FAT (22), but attention should be paid to the risk of nonspecific false-positive results. This risk is considerably reduced by the thorough training of the technicians. It must also be emphasised that this technique needs one incubation step more than the FAT.

               Peroxidase conjugate may be used on sections of formalin-fixed tissue for immunohistochemical tests.

               An enzyme-linked immunosorbent assay (ELISA) that detects rabies antigen is one variation of the immunochemical test. This rapid rabies enzyme immunodiagnosis test (RREID) is available commercially (28). The correlation between the FAT and the RREID ranges between 96% and 99% (8, 15). The 'routine' version of this test is not sensitive to rabies-related viruses as RREID only detects genotype 1 lyssaviruses.

     .   Detection of the replication of rabies virus after inoculation

          These tests detect the infectivity of a tissue suspension in cell cultures or in laboratory animals. They should be used if the FAT gives an uncertain result or when the FAT is negative in the case of known human exposure.

          i)   Mouse inoculation test

               Five-to-ten mice, 3-4 weeks old (12-14 g), or a litter of 2-day-old newborn mice, are inoculated intracerebrally. It is recommended, though not strictly essential, to use specific pathogen free (SPF) mice. The inoculum is the clarified supernatant of a 20% (w/v) homogenate of brain material (cortex, Ammon's horn, cerebellum, medulla oblongata) in an isotonic buffered solution containing antibiotics. To reduce animal pain, mice should be anaesthetised when inoculated. The young adult mice are observed daily for 28 days, and every dead mouse is examined for rabies using the FAT. For street fox rabies strains, deaths due to rabies generally begin 9 days post-inoculation. For faster results in newborn mice, it is possible to check one baby mouse by FAT on days 5, 7, 9 and 11 post-inoculation.

               This in-vivo test is quite expensive, particularly if SPF mice are used, and should be avoided where possible. It does not give rapid results (compared with in-vitro inoculation tests), but when the test is positive, a large amount of virus can be isolated from a single mouse brain for strain identification purposes. Another advantage of this low-tech test is that it can be easily and practicably be applied in situations where skills and facilities for other tests (e.g. cell culture) are not available.

          ii)   Cell culture test

               Neuroblastoma cell lines, e.g. CCL-131 in the American Type Culture Collection (ATCC: American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, United States of America), is used for routine diagnosis of rabies. The cells are grown in Dulbecco's modified Eagle's medium (DMEM) with 5% fetal calf serum (FCS), incubated at 36°C with 5% CO₂. Its sensitivity has been compared with that of baby hamster kidney (BHK-21) cells (29). This cell line is sensitive to street isolates without any adaptation step, but should be checked for susceptibility to locally predominant virus variants before use. Presence of rabies virus in the cells is revealed by the FAT. The result of the test is obtained after at least 18 hours (one replication cycle of virus in the cells); generally incubation continues for 48 hours (10) or in some laboratories up to 4 days.

               This test is as sensitive as the mouse inoculation test. Once a cell culture unit exists in the laboratory, this test should replace the mouse inoculation test as it avoids the use of live animals, is less expensive and gives more rapid results.

               It is often advisable to carry out more than one type of test on each sample, at least when there has been human exposure.

     d)   Other identification tests

          The tests above may be completed in specialised laboratories (such as OIE or WHO Reference Laboratories) using MAbs, nucleic acid probes, or the polymerase chain reaction (PCR), followed by DNA sequencing of genomic areas for typing the virus (16). This enables a distinction to be made between vaccine virus and a field strain of virus, and possibly the geographical origin of the latter.

2.   Serological tests

     Serological tests are rarely used in epidemiological surveys, due to late seroconversion and the low percentage of animals surviving the disease and therefore having post-infection antibodies. Oral immunisation of rabies reservoirs is the method of choice for wildlife rabies control. For follow-up investigations in oral vaccination campaigns, virus neutralisation (VN) tests in cell culture are preferred. However, if poor quality sera are submitted, the VN tests in cell culture are sensitive to cytotoxicity, which could lead to false-positive results. For such samples, the use of an indirect ELISA with rabies glycoprotein-coated plates has been shown to be as sensitive and specific as the VN test on cells (19).

     a)   Virus neutralisation test in cell culture: fluorescent antibody virus neutralisation test (a prescribed test for international trade)

          The principle of the fluorescent antibody virus neutralisation (FAVN) test (18) is the neutralisation in vitro of a constant amount of rabies virus ('challenge virus standard' [CVS] strain adapted to cell culture) before inoculating cells susceptible to rabies virus: BHK-21 C13 cells.

          The serum titre is the dilution at which 100% of the virus is neutralised in 50% of the wells. This titre is expressed in IU/ml by comparing it with the neutralising dilution of a standard serum under the same experimental conditions (OIE serum of dog origin or WHO standard for rabies immunoglobulin [human] No. 2, or both). An internal control calibrated against the international control may be used.

          This microplate method uses 96-well plates, and is an adaptation of the technique of Smith et al. (30), modified by Zalan et al. (38) and by Perrin et al. (27). Several publications (17, 18) have shown that the FAVN test and the rapid fluorescent focus inhibition test (RFFIT) give equivalent results.

          .   Essential equipment

          Humidified incubator at 37°C with 5% CO₂; dry incubator at 37°C; biocontainment cabinet; fluorescence microscope suitable for FITC fluorescence equipped with x10 eye-piece and x10 objective. The global magnification of the microscope ranges between x100 and x125 due to the extra magnification of some epi-fluorescence systems.

          .   Reagents and biologicals

          PBS buffer, pH 7.2, without Ca²⁺ and Mg²⁺, stored at 4°C;

          Trypsin ethylene diamine tetra-acetic acid (EDTA);

          High-grade acetone 80% (diluted with deionised water), stored at 4°C;

          Dulbecco's modified Eagle's medium (DMEM) + 10% heat-inactivated FCS;

          FITC anti-rabies conjugate;

          Cells: BHK-21 C13 (ATCC CCL-10);

          Virus: CVS-11 (ATCC VR 959) strain, which is available from the ATCC or the OIE Reference Laboratory for Rabies, Nancy, France (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm). Vials are stored at -80°C;

          WHO standard for rabies immunoglobulin (human) No. 2, 30 IU per ampoule (National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom) (reconstituted with 5 ml of sterile deionised or distilled water, stored at -20°C, and diluted to 0.5 IU/ml with deionised water before use), or preferably OIE Standard Serum of dog origin (OIE Reference Laboratory for Rabies, Nancy, France [please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm] stored at -20°C and diluted to 0.5 IU/ml with sterile deionised or distilled water according to the titre of the batch). It is advised that, for internal routine control, laboratories should use a positive serum or a pool of sera of dog origin that has been calibrated against the OIE International Standard Serum;

          Naive serum: This lyophilised pool of ten negative dog sera, is stored at 4°C, and reconstituted with 0.5 ml of sterile deionised or distilled water.

          .   CVS production

          i)   Cell growth: the BHK-21 C13 cells (ATCC CCL-10) used to produce the CVS virus (ATCC VR 959 CVS-11) are trypsinised during the rapid growth phase, i.e. cells are in the exponential phase of their kinetic growth. If the confluence of the layer is complete, a new passage should be made. The cells in the cell suspension should not be aggregated; 2 x 10⁷ cells are used for a 75 cm² cell culture flask. Cells are collected within a volume of 20-30 ml in cell culture medium with 10% heat-inactivated FCS.

          ii)   Infection of cells: the multiplicity of infection (number of infective particles per cell) is adjusted to between 0.1 and 0.5. The glass bottle containing the virus/cell suspension is incubated for 60 minutes at 35.5-37°C. The contents of the bottle are gently stirred every 10-15 minutes.

          iii)   Virus growth: the virus/cell suspension is then centrifuged at 800 g for 15 minutes and the cell pellet is resuspended in cell culture medium mixed with 10% heat-inactivated FCS. Virus is harvested 2 days later.

          iv)   Harvest and storage: the supernatant is centrifuged at 800 g for 15 minutes at 4°C. If several flasks have been used, the different centrifuged supernatants are mixed and then aliquoted and frozen at -80°C. The infective titre of the harvest is established at least 3 days after freezing.

          .   Titration of virus in TCID₅₀ (50% tissue culture infective dose)

          This titration method uses BHK-21 C13 cells (ATCC CCL-10) in microtitre plates.

          Different steps in this procedure may be adapted according to the safety requirements and to the working practices of the laboratory, but the following should not be changed:

.   inoculation of a 24-hour cell layer,

. tenfold dilutions prepared using 0.9 ml of diluent and 0.1 ml of virus suspension,

. six 50 µl replicates per dilution,

. incubation for 72 hours,

. qualitative reading (i.e. the well is positive or negative),

. in every titration session, a vial of a control batch of virus is titrated and this titre is integrated in a control card to validate the titration process,

.   calculation according to neoprobit graphic or Spearman-Kärber methods.

          i)   Cell suspension: the day before titration, a cell suspension containing 10⁵ cells/ml is prepared in cell culture medium containing 10% heat-inactivated FCS, and is distributed, 200 µl per well, into 96-well microtitre plates. The plates are then incubated for 24 hours at 35.5°C-37°C with 5% CO₂.

          ii)   Dilution of the virus: the serial dilutions are performed in 5 ml tubes using a cell culture medium without FCS as diluent. Tenfold dilutions from 10^-1 to 10^-12 are prepared (0.9 ml of diluent with 0.1 ml of the previous dilution).

          iii)   Infection of the cells: the medium in the microtitre plates is discarded using an aspiration system. Fifty µl of each virus dilution is distributed per well. Six replicates are used per dilution. The microtitre plate is then incubated for 1 hour at 35.5-37°C with 5% CO₂. Then 200 µl of cell culture medium, containing 5% FCS, is added.

          iv)   Incubation: incubate for 3 days at 35.5-37°C in 5% CO₂.

          v)   Staining and calculation of titre: The cells are stained using the FAT, as detailed below. Reading is qualitative, every well that shows specific fluorescence is considered to be positive. The titre calculation is made using:

               .   the neoprobit graphic method (2) or

               .   the Spearman-Kärber formula:

               log₁₀ (end-point dilution) =

               x0 = ( (log₁₀ of the lowest dilution with all wells positive)                d = log₁₀ of the dilution step, one in this case                ni = number of replicates, six in this case                ri = number of positive wells

.    Test procedure

          i)   The microplates are used according to the pattern shown in Figure 1. Plate No. 1 is used for the titration of CVS (rows 1 to 4), and for the controls, standard sera and naive dog serum are used. Plate No. 2 and others are used for the sera to be tested.

          ii)   Medium is added to the wells as follows: plate 1, rows 1 to 4 and cells A9 to A12: add 150 µl per well; plate 2 and others, rows 6 and 12: add 200 µl per well; all other wells: add 100 µl.

          iii)   Sera to be tested are heat inactivated for 30 minutes at 56°C. As indicated in Figure 1, 50 µl of each undiluted serum to be tested is added to four adjacent wells.

          iv)   Dilutions of sera are conducted in the microplates as follows:

               OIE serum, the WHO serum, the internal control and the naive dog serum: with a 50-200 µl multichannel pipette, mix the first dilution wells by sucking in and out at least eight times, transfer 50 µl from one row to the next one, until the last one is reached. Discard 50 µl from the last row.

               Sera being tested (all plates): as above, transfer successively 50 µl from one row to the following one until rows 5 and 11 (dil. 10^-2.39). With a 5-50 µl multichannel pipette, transfer 10 µl from rows 5 and 11 to rows 6 and 12, respectively (from dil. 10^-2.39 to dil. 10^-4.23). Using a multichannel pipette adjusted to 100 µl, mix rows 6 and 12 and discard 180 µl. Then add 70 µl of medium to these rows. This final step does not lend itself to high throughput testing. To attain or exceed the recommended final dilution alternative procedures may be used. These may require modifications to the plate layout.

          .   Addition of challenge virus standard

          i)   Stock CVS is stored in 1 ml microtubes at -80°C. One tube is thawed rapidly under cold running water, and placed in melting ice.

          ii)   One dilution from this tube is prepared in order to obtain 100 TCID₅₀ in 50 µl. Of this dilution, 50 µl is added to each serum-filled well (see Figure 1). For virus titration, 50 µl is added to wells H1 to H4 (plate 1). Next, transfer 50 µl from row to row (plate 1, lines 1-4). Discard 50 µl from the last row (plate 1, wells A1 to A4). No virus is added to wells A9 to A12 of plate 1 (controls).

          iii)   Incubate the microplates at 37°C in a humid incubator with 5% CO₂ for 1 hour.

          iv)   Addition of cells: trypsinise a subconfluent culture of 3-day-old BHK-21 cells. Resuspend the cells to obtain a 4 x 10⁵ cells/ml suspension in DMEM supplemented with 10% heat-inactivated FCS. Add 50 µl of the cell suspension to each well.

          v)   Incubate the microplates for 48 hours at 37°C in a humid incubator with 5% CO₂.

          .   Fixation and staining

          i)   After the 48-hour incubation period, the medium is discarded, and the microplates are rinsed once in PBS, pH 7.2, and once in 80% acetone. The microplates are then fixed in 80% acetone at room temperature for 30 minutes (without a lid), and are dried at room temperature for at least 1 hour.

          ii)   Add 50 µl of the FITC anti-rabies conjugate, at the working dilution, to each well, gently rock the microplates and incubate at 37°C for 30 minutes. Discard the fluorescent conjugate and rinse the microplates twice with PBS. Excess PBS is removed by briefly inverting the microplates on absorbent paper.

          .   Reading and interpreting the results

          i)   The total surface of each well is observed. The reading evaluation is qualitative (plus or minus): no fluorescent cell - a minus score is recorded for the well; fluorescent cells (one cell or more) - a plus score is recorded for the well.

          ii)   Controls are read first. For control cells, titration of CVS, naïve serum, and standard sera (WHO standard and/or OIE Standard Serum), titres are calculated according to the Spearman-Kärber method or the neoprobit graphic method (2).

          iii)   Results of titration of CVS (TCID₅₀), naive serum (D₅₀ [median dose]) and positive standard (D₅₀) are reported on a control card for each of these three controls. The control results of the current test are compared with the accumulated control test results from previous tests using the same batch of control. The test is validated if the values obtained for the three controls in the current test are not statistically different from the mean of all the values obtained in the tests conducted previously according to this technique.

          iv)   The result of the test corresponds to the non-neutralised virus after incubation with the reference serum or with the serum to be tested. These titres are calculated with the neo-probit graphic method (2) or with the Spearman-Kärber formula (37).The comparison of the measured titre of the tested sera with that of the positive standard serum of a known neutralising titre allows determination of the neutralising titre of the tested sera in IU/ml.

     b)   The rapid fluorescent focus inhibition test (RFFIT) for determining rabies virus-neutralising antibody (a prescribed test for international trade)

     .   Standard procedure (from WHO Laboratory Techniques in Rabies, 1996; [ref. 37])

          .   Preparation of seed virus suspension

          i)   Trypsinise one 3-day-old 150 ml flask culture of mouse neuroblastoma (MNA) cells (available on request from the Rabies Laboratory, Division of Viral and Rickettsial Diseases, Centres for Disease Control and Prevention, Atlanta, Georgia, USA). These cells prefer an acidic medium, supplemented with vitamins (34). A similar cell line (CCL-131) may be obtained on request from the ATCC.

          ii)   Resuspend 3 x 10⁷ cells in a 50 ml conical centrifuge tube in 2.7 ml of Eagle's minimal essential medium supplemented with 10% fetal bovine serum (EMEM-10).

          iii)   Using standard rabies safety procedures, add 1 x 10⁷ infectious units of CVS-11 rabies virus (ATCC, VR959) and vortex/mix once. Incubate the cells and virus for 15 minutes at 37°C; vortex/mix the cells once during this time.

          iv)   Add 10 ml EMEM-10, vortex/mix, and centrifuge the cells at 500 g for 10 minutes.

          v)   Discard the supernatant. Resuspend the cells in 30 ml of growth medium and transfer to a 150 ml flask.

          vi)   Gently rock the flask to mix the cell suspension, and then prepare three eight-well tissue-culture chamber slides by pipetting 0.2 ml of the cell suspension into one well of each slide.

          vii)   Incubate the flask and slides at 37°C in a humidified incubator with 0.5% carbon dioxide (CO₂). The flask should be incubated as a closed culture (tighten the cap).

          viii)   At 20, 40 and 64 hours after infection, acetone fix and stain one slide using an immunofluorescence technique (23) to determine the virus infectivity. The supernatant should be harvested 24 hours after the cells reach 100% infectivity (typically 40 hours after infection).

          ix)   Transfer the supernatant to a 50 ml centrifuge tube and centrifuge at 4000 g for 10 minutes.

          x)   Distribute the supernatant into 0.5 ml aliquots and store at -70°C.

          .   Titration of seed virus suspension

          i)   Thaw one aliquot of the seed virus and prepare serial tenfold dilutions (from 10^-1 to 10^-8) in EMEM-10.

          ii)   Distribute 0.1 ml of each virus dilution into one well of an eight-well tissue-culture chamber slide. Add 0.2 ml of MNA cells suspended in EMEM-10 (concentration 5 x 10⁴ cells per 0.2 ml) to each well.

          iii)   Mix the cells and virus by gently rocking the slide, then incubate at 37°C in a humidified incubator with 0.5% CO₂ for 40 hours.

          iv)   Acetone fix and stain the slide using an immunofluorescence technique. Evidence of virus infection should be observed at the 10^-6 dilution of virus, indicating a virus stock suspension containing at least 1 x 10⁶ infectious units per 0.1 ml. Prepare sufficient seed virus so that frequent serial passage of the virus is unnecessary.

          .   Preparation of stock virus suspension

          i)   Infect 3 x 10⁷ MNA cells with 1 x 10⁷ infectious units of the seed virus preparation (see above).

          ii)   Harvest the supernatant 24 hours after the cells reach 100% infectivity (typically 40 hours after infection).

          iii)   Distribute the supernatant into 0.5 ml aliquots and store at -70°C.

          .  Titration of stock virus suspension

          i)   Thaw one aliquot of the stock virus and use this to prepare serial tenfold dilutions (from 10^-1 to 10^-6) in EMEM-10.

          ii)   Distribute 0.1 ml of each virus dilution into one well of an eight-well tissue-culture chamber slide. Add 0.2 ml of MNA cells suspended in EMEM-10 (concentration 1 x 10⁵ cells per 0.2 ml) to each well.

          iii)   Mix the cells and virus suspension by gently rocking the slide, then incubate at 37°C in a humidified incubator with 0.5% CO₂ for 20 hours.

          iv)   Acetone fix and stain the slide using an immunofluorescence technique.

          Each well of an eight-well tissue-culture chamber slide contains 25-50 distinct microscopic fields when observed at x160-200 magnification. One unit of virus for the RFFIT is determined as the dilution at which 50% of the observed microscopic fields contain one or more foci of infected cells (the focus-forming dose, FFD₅₀). The stock virus suspension should contain at least 1 x 10⁴ FFD₅₀ per 0.1 ml (i.e. the well with cells infected with the 10^-4 dilution of the virus should contain at least one focus of infected cells in 50% of the observed microscopic fields). A stock virus suspension of this titre can then be diluted to 10^-2.3 to obtain a challenge virus containing 50 FFD₅₀.

          .   Reference sera

          A national or international reference serum standard diluted to a potency of 2.0 IU/ml should be included in each test. The reference serum used at the Centres for Disease Control and Prevention is the first international standard for rabies immunoglobulin (35), which may be obtained from the NIBSC. The reference serum should be maintained as frozen aliquots in amounts sufficient for 1 week of tests. A positive serum control standard diluted to a potency of 0.5 IU/ml and a negative serum control standard with a potency of <0.1 IU/ml should also be prepared by the laboratory and included in each test.

          .   Test sera

          Serum samples should be heated at 56°C for 30 minutes before testing in order to inactive complement. If sera are frozen, they should be reheated after thawing. Serial dilutions of test sera may be prepared in an eight-well tissue-culture chamber slide. Screening dilutions of 1/5 and 1/50 are sufficient for routine evaluation of vaccination efficacy and may be made as follows:

          i)   Prepare a 1/2.5 dilution by adding 0.1 ml of inactivated serum and 0.15 ml of EMEM-10 to one of the slides. Mix by gently rocking the slide.

          ii)   Transfer 0.05 ml of the 1/2.5 dilution to a second well containing 0.45 ml of EMEM-10. Discard all but 0.1 ml from the well containing the 1/2.5 dilution.

          iii)   Mix the second well and discard all but 0.1 ml.

          iv)   Add 0.1 ml of the challenge virus preparation (containing 32-100 FFD₅₀) to all serum dilutions.

          v)   Mix and incubate at 35°C in a humidified incubator with 0.5% CO₂ for 90 minutes.

          .   Addition of cells

          i)   During the incubation period, trypsinise a stock culture of 3-5-day-old MNA cells.

          ii)   Resuspend the cells in EMEM-10 to give a final concentration of 1 x 10⁵ cells per 0.2 ml.

          iii)   Distribute 0.2 ml of the cell suspension into each well of the slide and incubate at 35°C in a humidified incubator with 0.5% CO₂ for a further 20 hours.

          .   Acetone fixation and staining by immunofluorescence

          i)   After 20 hours, remove the slides from the incubator and pour off the medium into a virucidal solution.

          ii)   Rinse the slides once in PBS and then fix for 10 minutes at room temperature in cold acetone (-20°C).

          iii)   Leave the slides to dry for 10 minutes before adding FITC-conjugated anti-rabies serum. The conjugate may be prepared in EMEM-10 or PBS; there is no need to adsorb the conjugate with tissue or cells. The working dilution of the conjugate should be determined by titration. The slides should be stained for 20-30 minutes at 37°C and then rinsed in PBS and distilled water, respectively.

          iv)   Observe the slides under a fluorescence microscope.

          .   Calculation of virus-neutralising antibody titres

          Residual virus is detected using a standard fluorescence microscope. The serum neutralisation end-point titre is defined as the dilution factor of the highest serum dilution at which 50% of the observed microscopic fields contain one or more infected cells (i.e. a 97% reduction in the virus inoculum). This value may be obtained by mathematical interpolation. Alternatively, a 100% neutralisation titre may be determined by recording the highest serum dilution at which 100% of the challenge inoculum is neutralised and there are no infected cells in any of the observed fields. For both titration methods, the titre of antibody in the test serum (in IU/ml) can be obtained by comparison with the titre of the national reference standard included in each test. It should be noted that it is also valid to perform the RFFIT using BHK-21 cells instead of neuroblastoma cells. A modified protocol for this has been published (37).

     c)   Virus neutralisation in mice

          This method is no longer recommended by either OIE or WHO and should be discontinued.

     d)   Enzyme-linked immunosorbent assay (a prescribed test for international trade or movement)

           Commercial kits are available for indirect ELISA that allow a qualitative detection of rabies antibodies in individual dog and cat serum samples following vaccination. In accordance with the WHO recommendations (36), 0.5 IU per ml rabies antibodies is the minimum measurable antibody titre considered to represent a level of immunity that correlates with the ability to protect against rabies infection. The ELISA provides a rapid (~ 4 hours) test that does not require handling of live rabies virus, to determine if vaccinated dogs and cats have sero-converted. The sensitivity and specificity of any kit used should be determined by comparison with virus neutralisation methods. The ELISA is acceptable as a Prescribed Test for international movement of dogs or cats provided that a kit is used that has been validated and adopted on the OIE Register as fit for such purposes (see http://www.oie.int/vcda/eng/en_VCDA_registre.htm?e1d9)]. Virus neutralisation methods may be used as confirmatory tests if desired.

           ELISA methods are also useful for monitoring of vaccination campaigns in wildlife populations, provided the kit used has been validated for the wildlife species under study.

Rabies vaccines prepared from Pasteur's original 1885 strain and its derivative strains (Pasteur Virus, Challenge Virus Standard, Pitman-Moore, etc.), and strains isolated more recently (Flury, Street-Alabama-Dufferin [SAD], Vnukovo and Kelev), protect against all strains of genotype 1 isolated so far. Conventional rabies virus vaccines may not provide adequate cross-protection against other lyssaviruses; there is no protection provided against Mokola virus (31). The principles governing the preparation of inactivated rabies vaccines are identical whether they are to be used in humans or animals, although an adjuvant may be added to vaccines for animal use.

For animals, live vaccines are also effective by the oral route and can be distributed in baits in order to immunise wild (or domestic) animals. Live recombinant vaccine (e.g. vaccinia rabies-glycoprotein recombinant) has also proved to be effective (25).

Guidelines for the production of veterinary vaccines are given in Chapter I.1.7. Principles of veterinary vaccine production. The guidelines given here and in Chapter I.1.7 are intended to be general in nature and may be supplemented by national and regional requirements.

Different standards apply to vaccines containing live virus modified by passage in animals, eggs or cell cultures to reduce its virulence for the target animal, and to vaccines prepared from inactivated virus. Both types of vaccine have their advantages and disadvantages (5), but they can both be used to immunise animals for periods of between 1 and 3 years. Live attenuated rabies vaccines are not accepted in some countries. They are not to be relied on to protect previously unvaccinated animals that have been exposed to infection (13). Only in humans has the efficiency of post-exposure treatment with vaccine alone been proven and even in these cases there is an additional strong recommendation to administer anti-rabies immunoglobulin.

All handling of the virus during manufacture and testing of vaccines must conform to the strict safety precautions specified by WHO (36, 37), the OIE (Chapter I.1.6.) and to national guidelines and regulations.

1.   Seed management

     a)   Characteristics of the seed

          Any strain belonging to serotype 1, which has been proved to protect against field rabies viruses (currently found in the country where the vaccine is to be used), is suitable. The strain of virus used should have well-known biological (e.g. pathogenicity) and antigenic properties (typing by MAbs). If it is to be used as a live vaccine, the master seed virus must be shown not to cause clinical rabies. At least two animals (preferably five to six per group) of each of the species for which the vaccine is intended and, so far as possible, any species that might be in contact with vaccine or vaccinated animals, should be tested. This can be done by inoculating in or adjacent to a major nerve, a dose equivalent to ten times the intended viral titre in one dose of the proposed final product. Animals should be observed for at least 90 days for any adverse effect attributable to the master seed.

     b)   Method of culture

          A master cell stock of the seed virus should be prepared and kept at or below -70°C. Subculture from this stock will be used for vaccine production. Virus multiplication is verified by titration during growth of the seed virus.

     c)   Validation as a vaccine

          Before a vaccine is licensed, evidence of efficacy should be established by the challenge of vaccinated and control animals of each target species. The challenge should be performed at the end of the period after vaccination for which the manufacturer claims maintenance of immunity. Antibody kinetics should also be determined in order to establish the correlation between antibody titre and resistance to challenge.

          The efficacy of the produced vaccine is assessed by studies on every target species previously vaccinated as recommended. Protection at the end of the period of immunity is monitored by a measurement of specific neutralising antibodies and by challenge with rabies virus. The experimental conditions of this challenge should mimic the natural conditions of infection, but, from a practical point of view, it could be easier to obtain 100% mortality of the control animals with a well-known rabies virus strain than with a locally isolated one. In animals vaccinated with inactivated vaccines, the percentage of seroconversion and the mean level of antibody allow a good prognosis for survival to challenge (3).

          The correlation between potency in the target species and antigenic value as estimated in mice should be established (see Section C.4.c. below).

          For the purposes of licensing a vaccine, safety tests should be conducted in the target species. In the case of live virus vaccines (including recombinant vaccines) used in oral vaccination campaigns, safety tests should also be carried out on those other species that live in the area of vaccination and could become exposed to the vaccine (5).

          Vaccine stability is ascertained by testing batches after prolonged storage, usually 1-2 years. A process of accelerated ageing, by storage at 37°C for 1 week, is sometimes used. The storage life claimed by the manufacturer is checked by the national licensing authority. In general, it is 12-18 months for fluid vaccines, and possibly 24 months for lyophilised vaccines.

2.   Method of manufacture

     Whatever method is adopted, close attention should be paid to the quality of the substrate. Both animals and eggs should be of SPF origin, and the cell cultures, such as BHK cell lines, should conform to international standards of sterility and innocuity.

     a)   In animals

          The virus is inoculated intracerebrally and the nervous tissue is harvested when the animal has been killed in the terminal stages of rabies. The virus is inactivated by physical methods, such as irradiation with ultraviolet light, or by chemical methods, such as the addition of phenol or beta-propiolactone. Vaccines should be prepared in young animals (mice, lambs, etc.) for high yield of virus and to reduce the myelin content of vaccine and associated adverse events. In certain cases, the virus is not inactivated entirely, as for example in the phenol-treated vaccines of the Fermi type, but such vaccines are no longer recommended.

     b)   In eggs

          A modified egg-adapted strain of virus is inoculated into SPF-embryonated chicken eggs, which are then incubated at 38°C for 5-6 days. The virus is harvested in the form of infective embryo tissues, and is usually lyophilised and used as a live vaccine. Examples of such vaccines include those that contain the Flury low egg passage (LEP), or the more desirable high egg passage (HEP) variant strain, which is safer for some animal species such as the cat.

     c)   In cell cultures

          Cultures are infected with cell-culture-adapted strains of rabies virus and incubated at 35-36°C. These may then be used as live virus vaccines (as in Flury and SAD vaccines), or as inactivated vaccines after the addition of phenol (Semple vaccine) or some other chemical, such as beta-propiolactone.

          Cell culture can also be used to grow the vector viruses (e.g. vaccinia virus) harbouring the gene coding for the expression of rabies virus glycoprotein (25).

          During manufacture, the multiplication of the virus in one of the substrates mentioned above is monitored, followed by harvesting at the most appropriate time, usually 4-6 days after inoculation of animals, eggs or cell cultures. The virus harvest is suspended in a buffer solution at a dilution that will provide an optimum antigenicity of the end-product. If required, the suspension is either inactivated or lyophilised. An adjuvant is recommended for vaccines prepared from inactivated virus, as well as for other vaccine antigens that may be incorporated in polyvalent vaccines.

3.   In-process control

     This consists of monitoring virus growth to provide an optimum titre and ensure the absence of undesirable microbial contamination.

     In live virus vaccines, kinetics of virus growth should be established in order to ensure a final titre of virus correlated to the desired protection in target species.

     In inactivated virus vaccines, immunogenic properties of the final product may be evaluated by in-vitro techniques (e.g. ELISA, agar gel immunodiffusion, antibody-binding tests or infected cell staining). These evaluations will indicate the best time for harvesting the virus in cell cultures.

4.   Batch control

     a)   Sterility

          Tests for sterility and freedom from contamination of biological materials may be found in Chapter I.1.5.

     b)   Safety

          Safety tests for batches of inactivated virus vaccines are carried out by inoculation of cell culture or intracerebrally into mice to detect viable virus. A suitable safety test for live rabies vaccines should be carried out on each lot of vaccine, in the intended host species. At least three, preferably five to six animals of the intended host species should be given a dose equivalent to ten times the recommended field dose, by the recommended route of administration. The animals should be observed for 90 days for adverse reactions attributable to the vaccine.

     c)   Potency

          The amount of virus present in live attenuated and recombinant vaccines is determined by titration. Once a correlation has been established between the activity of the vaccine in the target species and virus titres, virus titrations become reliable indicators of vaccine efficacy. This is carried out using cell cultures or by the intracerebral inoculation of unweaned mice (in mice it is only possible with a few attenuated viruses). Recombinant vaccines should be monitored for the expressed rabies protein until assured that expression stablilty is maintained in the manufacturing process. Titre of the vector can then be used as a reliable indicator of vaccine efficacy.

          For inactivated virus vaccines, correlation between potency in the target species and antigenic value as estimated in mice provides a reliable indicator of vaccine activity. The potency of the vaccine is established in the USA by the National Institutes of Health (NIH) test. Elsewhere, the European Pharmacopoeia test is widely adopted.

          Groups of at least ten mice, aged 3-4 weeks, are inoculated with single, decreasing doses of vaccine in accordance with the European Pharmacopoeia (20), or with two doses, 1-week apart, according to the NIH test (37). A sufficient number of dilutions of vaccine are compared to estimate the dilution at which 50% of the mice are protected against intracerebral challenge 14 days later (20, 37).

          A WHO international standard vaccine is available for calibration of national standards, so that the results of testing for antigenicity can be expressed in IUs. The test is not valid unless:

          i)   For both the vaccine to be examined and the standard preparation, the PD₅₀(50% protective dose) lies between the largest and smallest doses given to the mice.

          ii)   The titration of the challenge virus suspension shows that 0.03 ml of the suspension contained at least 10 LD₅₀. The challenge dose should be in the range 12-50 LD₅₀ for a valid test.

          iii)   The confidence interval (p = 0.95) for the test should not be less than 25% and not more than 400% of the estimated potency: statistical analysis should show a significant slope and no significant deviations from linearity or parallelism of the dose-response lines.

          The vaccine passes the test if the estimated potency is not less than 1 IU per dose, or the potency demonstrated in the duration of immunity test used to licence the product, in the smallest prescribed dose.

          A simplified test can also be used for the purpose of anticipating which vaccines are likely to be of an antigenic value greater than or equal to 1 IU per dose (4). This test used as a screening test is a good way to reduce the number of mice used in vaccine potency control tests.

     d)   Duration of immunity

          Duration of immunity must be established for the product licence in the target species with a defined vaccination protocol. After that, it is not tested for every batch (see Section C.4.c. above).

     e)   Stability

          The proposed shelf life must be verified by appropriate tests. These experiments include biological and physico-chemical stability tests, and should be performed on a sufficient number of batches of vaccine stored under recommended conditions.

          The thermostability of live virus vaccines in liquid form is generally poor. For freeze-dried inactivated virus vaccines, stability is generally granted for 2 years at 4°C.

     f)   Preservatives

          Inactivated virus vaccines may contain preservatives (formalin, merthiolate). The nature and quantity of these preservatives should comply with national control regulations.

5.   Tests on the final product

     a)   Safety

          See Section C.4.b.

     b)   Potency

          See Section C.4.c.

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