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SUMMARY
Equine influenza is an acute, contagious respiratory disease caused by two distinct subtypes (H7N7, formerly equi-1, and H3N8, formerly equi-2) of influenza A viruses within the genus Influenzavirus A of the family Orthomyxoviridae. In fully susceptible animals, clinical signs include pyrexia, and a harsh dry cough followed by a mucopurulent nasal discharge. In partially immune vaccinated animals, one or more of these signs may be absent. Characteristically, influenza spreads rapidly in a susceptible population. Diagnosis of influenza virus infections is based on virus isolation from horses with acute respiratory illness, or on the demonstration of a serological response to infection. Ideally, both methods are used. Infection may also be demonstrated by detection of viral antigen in respiratory secretions using an enzyme-linked immunosorbent assay. Spread of infection and severity of disease may be reduced by the use of potent inactivated equine influenza vaccines containing epidemiologically relevant virus strains.
Identification of the agent: Embryonated hens' eggs and/or cell cultures can be used for virus isolation from nasopharyngeal swabs or nasal and tracheal washes. Viral growth is monitored by haemagglutination (HA) or, in cell cultures, by haemadsorption (HAD) using chicken or guinea-pig red blood cells. Isolates can be characterised by haemagglutination inhibition (HI) using strain-specific antisera. Isolates should always be sent immediately to an International Reference Laboratory (OIE or World Health Organization). Samples that yield negative results should be repassaged; up to five passages may be necessary to isolate viruses from vaccinated horses.
Serological tests: Diagnosis of influenza virus infections is usually only accomplished by tests on paired sera; the first sample should be taken as soon as possible after the onset of clinical signs and the second approximately 2 weeks later. Antibody titres are determined by HI or single radial haemolysis (SRH).
Requirements for vaccines and diagnostic biologicals: Inactivated equine influenza vaccines contain whole viruses or their subunits. The vaccine viruses are propagated in embryonated hens' eggs or tissue culture, concentrated, and purified before inactivation with agents such as formalin or beta-propiolactone. Vaccines provide protection by inducing humoral antibody to HA. Responses are generally short-lived and multiple doses are required to maintain protective levels of antibody. An adjuvant is usually required to stimulate durable protective levels of antibody. A live attenuated vaccine has recently been licensed in some countries.
Influenza vaccines are widely available and are routinely used in competition horses in Europe, the Americas, and Asia. In some countries, vaccination is mandatory for sport horses that are competing under rules of equestrian organisations. Vaccines are not widely used on the Indian subcontinent, China or Australia, the latter region, along with New Zealand, having remained free from infection. Following a primary course of three doses at intervals of 0, 1 and 6 months, an annual booster is the minimum requirement. In some regions, repeated vaccinations are given every 3-6 months. Foals should not be vaccinated in the presence of maternal antibody.
Vaccines have been highly successful in preventing infection with H7N7 viruses, but frequent reports of vaccine breakdown occur during outbreaks of H3N8 infections. This has been attributed to inadequate vaccine potency, inappropriate vaccination schedules, and outdated vaccine viruses that have become irrelevant as a result of antigenic drift. An improved in-vitro potency test (single radial diffusion) has been developed that can be used for in-process testing of the product before addition of an adjuvant. Levels of antibody required for protection of horses have been identified. A surveillance programme is underway to monitor antigenic drift among equine influenza viruses and to provide information on strain selection for vaccines.
A. INTRODUCTION
Equine influenza is caused by two subtypes: H7N7 (formerly subtype 1) and H3N8 (formerly subtype 2) of influenza A viruses (genus Influenzavirus A of the family Orthomyxoviridae). Although these are not genuine human pathogens, humans can become infected with equine influenza virus subtypes. Such infections are unusual and subclinical, but may represent a potential biohazard to laboratory personnel.
In fully susceptible animals, clinical signs include pyrexia, and a harsh dry cough followed by a mucopurulent nasal discharge. In partially immune vaccinated animals, one or more of these signs may be absent. Characteristically, influenza spreads rapidly in a susceptible population. Diagnosis of influenza virus infections is based on virus isolation from horses with acute respiratory illness, or on the demonstration of a serological response to infection. Ideally, both methods are used. Infection may also be demonstrated by detection of viral antigen in respiratory secretions using an enzyme-linked immunosorbent assay (ELISA).
B. DIAGNOSTIC TECHNIQUES
All influenza viruses are highly contagious for susceptible hosts, including embryonated hens' eggs and cell cultures. Care must therefore be taken during the handling of infected eggs or cultures to avoid accidental cross-contamination (21). Standard strains should not be propagated in the diagnostic laboratory, at least never at the same time or in the same place where diagnostic samples are being processed. All working areas must be efficiently disinfected before and after virus manipulations, which should preferably be conducted within biohazard containment.
It is important to obtain samples as soon as possible after the onset of clinical signs. These samples include nasopharyngeal swabs and nasal or tracheal washings, the latter taken by endoscopy. Swabs consist of absorbent cotton wool sponge/gauze on wire, and should be passed via the ventral meatus into the nasopharynx and held there for approximately 1 minute to absorb respiratory secretions. Swabs should be transferred to a vial containing transport medium immediately after use. This medium consists of phosphate buffered saline (PBS) containing 40% glycerol, or PBS containing 2% tryptose phosphate broth, 2% antibiotic solution (penicillin [10,000 units], streptomycin [10,000 units] in sterile distilled water [100 ml]), and 2% fungizone (250 mg/ml stock). If the samples are to be inoculated within 1-2 days they may be held at 4°C, but, if kept for longer, they should be stored at -70°C or below. Preferably, samples should also be transported on ice.
Only one sample is processed at a time. The liquid is expelled from the swab, which is then disposed of suitably. Further antibiotics may be added if samples appear to be heavily contaminated. The remainder of the fluid is stored at -70°C. Samples treated with antibiotics are allowed to stand on ice for 30 minutes and are then centrifuged at 1500 g for 15 minutes to remove bacteria and debris; the supernatant fluids are used for inoculation. Filtration of samples is not advised as influenza virus may adsorb on to the filter and be lost from the sample.
| 1. | Identification of the agent
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| | Isolation of infectious virus may be carried out in embryonated hens' eggs or cell cultures. Traditionally, eggs have been preferred for isolation of equine influenza. Comparison of H3N8 viruses isolated in eggs and Madin-Darby canine kidney (MDCK) cells indicated that MDCK cells are capable of selecting variant viruses that are not representative of the predominant virus in clinical specimens (6). However, in recent years some viruses have been successfully isolated in MDCK cells but not in eggs and selection of variants has occurred as a result of culture in eggs (18), therefore isolation should be attempted using both substrates. Recently, polymerase chain reaction techniques have been described for the identification of equine influenza virus from clinical specimens and for molecular epidemiology (4, 8, 17).
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| | a) | Embryonated hens' eggs
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| | | Fertile eggs are set in a humid incubator at (37-38°C) and turned twice daily; after 10-11 days, they are examined by candling. Only live embryonated eggs are selected for use. The area above the air sac is cleansed with alcohol and a small hole is drilled through the shell. Three eggs/sample are inoculated (0.1 ml) in the amniotic cavity. The syringe is withdrawn approximately 1 cm and a further 0.1 ml is inoculated into the allantoic cavity. Alternatively, the sample may be inoculated into the allantoic cavity alone. The hole is sealed with wax or Sellotape, and the eggs are incubated at
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35°C for 2-3 days.
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| | | The eggs are then transferred to 4°C for 4 hours or overnight to kill the embryos and to reduce bleeding at harvest. The shells are disinfected, and the allantoic and amniotic fluid is harvested by pipette, each harvest being kept separate. These are tested for haemagglutination (HA) activity by mixing in equal volumes (0.025 ml) with chicken red blood cells (RBCs) (0.5% [v/v] packed cells in PBS) in V- or U-bottomed microtitre plates or 0.4% guinea-pig RBCs (0.4% [v/v] packed cells in PBS) in V- or U-bottomed plates. If chicken RBCs are used, the plates may be read by tilting to 70( so that non-agglutinated cells 'stream' to the bottom of the well. Non-agglutinated guinea-pig cells appear as a button at the bottom of the well and may take longer to settle. If there is no HA activity, aliquots of each harvest are pooled and passaged into further eggs. All HA positive samples are divided into aliquots and stored at -70°C; one aliquot is titrated for HA immediately. If the HA titre is 1/16 or more, the isolate is characterised immediately. If titres are low, positive samples should be passaged. Care should be taken to avoid generation of defective interfering particles by prediluting the inoculum 1/10, 1/100, 1/1,000. Positive samples arising from the highest dilution should be selected as stocks for storage. It may be necessary to undertake as many as five passages to isolate the virus, particularly from vaccinated horses. If virus has not been recovered by the fifth passage, further passages are unlikely to be successful.
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| | b) | Cell cultures
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| | | Cultures of the MDCK cell line (MDCK, ATCC CCL34) may be used to isolate equine influenza viruses. The cells are grown to confluence in tubes and then infected in triplicate with 0.25-0.5 ml of each sample, processed as described above. The cultures are maintained with serum-free medium containing 0.5-2 µg/ml trypsin (treated with TPCK [L-1-tosylamine-2-phenylethyl chloromethyl ketone] to remove chymotrypsin, available pretreated, e.g. from Sigma), and examined daily for evidence of cytopathic effects (CPE). If positive, or after 7 days in any case, the supernatant fluids are tested for HA. Fluids with titres of =1/16 are characterised immediately. Negative fluids and those with titres <1/16 are repassaged up to five passages.
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| | | Alternatively, the cells are screened for evidence of haemadsorption (HAD). This procedure detects expression of viral antigens at the cell surface. The medium is removed from the cultures and the tubes are washed with PBS. One or two drops of a 50% suspension of chicken or guinea-pig RBCs are added, the tubes are rotated carefully, and kept at 22°C (±2°C) for 30 minutes. Unbound RBCs are washed off with PBS, and the cultures are examined microscopically for evidence of HAD.
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| | c) | Haemagglutinin
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| | | The HA subtype of new isolates of equine influenza viruses is best determined by haemagglutination inhibition (HI; Section B.2.a.) using H7N7- and H3N8-specific antisera. Isolates may first be treated with Tween 80/ether, which destroys viral infectivity and reduces the risk of cross-contamination. In the case of H3N8 viruses particularly, this treatment enhances the HA activity (7). However, treatment with Tween 80/ether may increase the variability of the results obtained. Standard antigens must be titrated in parallel with tests to identify viruses and should include H7N7 strains (e.g. A/eq/Prague/56, A/eq/Newmarket/77) and H3N8 strains (e.g. A/eq/Miami/63, A/eq/Fontainebleau/79, A/eq/Newmarket/2/93, and A/eq/Kentucky/ 94). Virus strains may be obtained from OIE Reference Laboratories (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm). Additionally, recent isolates from the same geographical area should be included if available. The standard antigens should be treated with Tween 80/ether to avoid cross-contamination. Test antigens and standard antigens are always back-titrated to confirm their antigen content.
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| | | New isolates of equine influenza viruses may be further characterised by HI using strain-specific antisera. The species in which antibodies are raised will influence the cross-reactivity of the antiserum, with ferrets providing the most strain-specific antibody (11).
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| | | All isolates should be sent immediately to an International Reference Laboratory designated by OIE or the World Health Organization (WHO) for inclusion in the strain surveillance programme to monitor antigenic drift and emergence of new viruses.
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| | d) | Neuraminidase
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| | | Typing of neuraminidase requires specific antisera and no routine technique is available. Such typing is best done, therefore, in reference laboratories.
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| | e) | Detection of viral antigen by enzyme-linked immunosorbent assay
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| | | In situations where laboratory facilities for virus isolation are unavailable, influenza virus antigen in nasal secretions may be detected directly by an antigen-capture ELISA for the H3N8 virus employing a monoclonal antibody (MAb) against the nucleoprotein (2, 9). Reagents may be obtained from the OIE Reference Laboratory, Newmarket. Commercial self-contained kits for detecting human influenza are available and have been shown to cross-react with equine influenza (1).
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| | | This approach provides a rapid result on which management decisions may be based. It should not be used in preference to virus isolation, as it is essential that new viruses be isolated and sent to reference laboratories for characterisation as part of the surveillance programme to monitor antigenic drift and emergence of new viruses and to provide isolates for inclusion in updated vaccines. Positive ELISA results are useful in the selection of samples if resources are limited or if samples must be sent to a reference laboratory for virus isolation attempts.
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| | | . | Test procedure
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| | | i) | Dispense 100 µl/well of nasal swab extract into triplicate wells of microstrips coated with polyclonal rabbit anti-H3N8, and incubate for 90 minutes at 22°C (±2°C). Wash the microstrips three times with PBS containing 0.05% (v/v) Tween 20 (PBST).
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| | | ii) | Add 100 µl/well of MAb-horseradish-peroxidase conjugate diluted 1/150 in dilution buffer then incubate for 1 hour at 37°C. Wash the microstrips six times with PBST.
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| | | iii) |
Add 100 µl/well of tetramethyl benzidine and incubate for 10 minutes at 22°C (±2°C). Stop the reaction by adding 100 µl/well of 0.18 M sulphuric acid, and measure the absorbance at 450 nm.
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| 2. | Serological tests
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| | Infections are detected by performing serological tests on paired sera to show a rise in antibody titre. These tests should be carried out whether virus isolation has been attempted or not. Two simple methods exist, HI and single radial haemolysis (SRH), each equally efficient and widely used. The complement fixation (CF) test can also be applied, but is not in general use. Both of the paired serum samples should be tested together at the same time to minimise variability. The standard antigens are described above (Section B.1.c.). If available, isolates from recent cases should be included. Freeze-dried post-infection equine antisera to A/eq/Newmarket/77 (H7N7), A/eq/Newmarket/1/93 ('American-like' H3N8) and A/eq/Newmarket/2/93 ('European-like' H3N8) and an influenza-negative equine serum, are available from the OIE Reference Laboratory, Newmarket. These sera have been assigned SRH values through an international collaborative study and can be used as primary reference sera for this assay.
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| | a) | Haemagglutination inhibition test
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| | | The antigen is first treated with Tween 80/ether in order to increase the sensitivity of the test, particularly for H3N8 viruses. The test is best done in microtitre plates using the appropriate dilution equipment. A macrotest may be used, for which antigen is diluted to a final HA titre of 1/8 per well and the volumes for PBS, sera and antigen are 0.5 ml. Sera are pretreated to remove nonspecific haemagglutinins, and inactivated at 56°C for 30 minutes. Pretreatments include the use of one of the following: (a) kaolin and RBCs absorption, (b) potassium periodate, or (c) Vibrio cholerae receptor-destroying enzyme. All three procedures yield similar results. The treated sera are diluted in PBS, a standard dose of antigen is added (HA titre of 1/4 per well for microtitration assay), and these are kept at 22°C (±2°C) for 30 minutes. After gentle mixing, RBCs are added and the test is read 30 minutes later. The HI titres are read as the highest dilution of serum giving complete inhibition of agglutination. Either chicken RBCs (1% [v/v] packed cells) in V-bottomed microtitre plates or guinea-pig RBCs (0.5% [v/v] packed cells) in V- or U-bottomed plates may be used. If chicken RBCs are used, the plates may read by tilting to 70( so that non-agglutinated cells 'stream' to the bottom of the well. Non-agglutinated guinea-pig cells appear as a 'button' in the bottom of the well and may take longer to settle. Titre increases of fourfold or more between paired sera indicate recent infection (21).
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| | | . | Tween 80/ether treatment of the virus
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| | | i) | To 40 ml of infective allantoic fluid, add 0.5 ml of a 10% (v/v) suspension of Tween 80 in PBS to give a 0.125% (v/v) concentration of Tween 80.
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| | | ii) | After mixing gently at room temperature for 5 minutes, add 20 ml of diethyl ether to give a final concentration of 33.3% by volume, and mix the suspension well at 4°C for 15 minutes.
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| | | iii) | After allowing the layers to separate by standing, remove the aqueous layer containing the disrupted virus particles to a glass bottle with a loose lid and allow the excess ether to evaporate off overnight (9).
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| | | iv) | Store treated virus in aliquots at -70°C.
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| | | . | Titration of haemagglutination
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| | | i) | Add 25 µl of PBS to all wells in a row of a microtitre plate.
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| | | ii) | Add 25 µl of virus to first well (dilution = 1/2) and titrate through, leaving the last well as a control.
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| | | iii) | Add an extra 25 µl of PBS to all wells.
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| | | iv) | Add 50 µl of RBCs to all wells. Leave at 22°C (±2°C) for 30 minutes. The HA titre is taken as the last virus dilution giving partial HA.
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| | | . | Pretreatment of sera
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| | | i) | Mix one volume (150 µl) of serum with two volumes (300 µl) of freshly prepared 0.016 M potassium periodate (0.38 g in 100 ml PBS), and leave at 22°C (±2°C) for 15 minutes.
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| | | ii) | Add a further one volume of 3% glycerol in PBS to neutralise any excess periodate solution, mix and leave at 22°C (±2°C) for 15 minutes.
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| | | iii) | Inactivate in a 56°C water bath for 30 minutes.
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| | | . | Test procedure
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| | | i) | Dispense 25 µl of PBS to all wells of a microtitre plate.
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| | | ii) | Add serum (25 µl) to the first well of a row of 12, and titrate through, leaving the last well as a control (1/8 to 1/512, allowing for dilution of 1/4 from treatment of serum).
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| | | iii) | Dilute the antigen to give a dose of 4 HA units (4 x minimum agglutinating dose, i.e. titre/4).
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| | | iv) | Add 25 µl to each well, and incubate at 22°C (±2°C) for 30 minutes.
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| | | v) | Add 50 µl of RBCs to each well. Leave at 22°C (±2°C) for 30 minutes.
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| | | vi) | The plates may be read by tilting to 70° so that non-agglutinated cells 'stream' to the bottom of the well. No agglutination is recorded as a positive result.
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| | b) | Single radial haemolysis
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| | | In this test, viral antigens are coupled to fixed RBCs that are suspended in agarose containing guinea-pig complement (C'). Wells are punched in the agarose and filled with test sera. Influenza antibodies and C' lyse the antigen-coated RBCs, resulting in a clear, haemolytic zone around the well; the size of this zone is directly proportional to the level of strain-specific antibody in the serum sample (10, 19, 20).
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| | | Special immunodiffusion plates (Hyland, Miles Scientific) may be used for the assay, but simple Petri dishes are also suitable. Sheep RBCs collected into Alsever's solution are washed three times. The C' can be obtained commercially, or normal guinea-pig serum can be used. The antigens are allantoic fluids or purified preparations; the strains used are the same as for the HI tests. The viruses are coupled to RBCs by potassium periodate or by chromic chloride. The coupled antigen/RBCs preparations are mixed with C', together with a 1% solution of agarose (low melting grade) in PBS. Care must be taken to ensure that the temperature is not allowed to rise above 42°C at any time. The mixture is poured into plates and left overnight at 4°C. Wells of 3 mm in diameter and 12 mm apart are punched in the solidified agarose, at least 6 mm from the edge of the plates. Such plates may be stored at 4°C for 12 weeks. Plates are prepared for each antigen and pretested with known positive and negative antisera.
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| | | Sera are inactivated at 56°C for 30 minutes, but no further treatment is necessary. Paired sera should be assayed in duplicate on the same plate. As a minimum, a subtype-specific antiserum should be included as a control serum in one well on each plate. All sera are tested in a control plate containing all components except virus to check for nonspecific lysis. Alternatively, an unrelated virus, such as A/PR/8/34 (H1N1), may be used in the control plate. Sera that show haemolytic activity for sheep RBCs must be pre-absorbed with sheep RBCs. Zones of lysis should be clear and not hazy or translucent. All clear zones should be measured and the area of haemolysis calculated.
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| | | . | Preparation of reagents
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| | | i) | Saline/HEPES: 0.85% NaCl (4.25 g/500 ml); 0.05 M HEPES (N-2-hydroxyethylpiperazine, N-2-ethanesulphonic acid; 5.95 g/500 ml); and 0.02% sodium azide. Make to pH 6.5 with NaOH.
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| | | ii) | Saline/HEPES/BSA: as saline/HEPES with 0.2% (w/v) bovine serum albumin (BSA).
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| | | iii) | CrCl3 stock solution (2.25 M) 6 g/10 ml: Make fresh 1/400 dilution in 0.85% NaCl for each assay.
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| | | iv) | PBS (London)/PBS 'A': NaCl (10.00 g); KCl (0.25 g); Na2PO4 (1.45 g); KH2PO4 (0.25 g); and Na azide (0.20 g). Make up to 1 litre with distilled water.
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| | | v) | Agarose in PBS: Place flask containing PBS 'A' on a stirrer. Slowly add 10 g agarose to the stirring solution. Liquefy in a pressure cooker. Dispense into glass bottles for storage at 22°C (±2°C).
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| | | vi) | Virus antigen: Allantoic fluid containing infectious virus is harvested and stored at -70°C. A short titration curve determines the optimum ratio of virus antigen to RBCs to be used when preparing sensitised sheep RBCs. The H7N7 influenza strains always produce clear zones; the H3N8 strains sometimes produce hazy zones, in which case it is necessary to concentrate the virus by centrifugation.
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| | | vii) | Sheep blood: Collect blood into an equal volume of Alsever's solution and store at 4°C. It may be necessary to test bleed several sheep, as characteristics of RBCs from individual sheep vary. Keep the blood for 2 days before use, it may then be usable for up to 3 weeks, providing sterility is maintained.
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| | | viii) | Complement: Use commercially available guinea-pig complement or collect serum from young guinea-pigs of 300-350 g body weight and store in small volumes at -70°C. For use, thaw in cold water and hold at 4°C prior to mixing.
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| | | ix) | Treatment of sera: Use undiluted sera heat inactivated at 56°C for 30 minutes. Avoid repeated freeze-thaw cycles.
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| | | . | Test procedure
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| | | i) | Wash sheep RBCs at least three times in saline/HEPES.
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Prepare an appropriate volume of 8% RBCs (v/v packed cells) in saline/HEPES, having first calculated the number of plates required and allowing 1 ml per 6 x 11 cm immunoplate and 1-2 ml extra.
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| | | iii) | Add a predetermined volume (prepare three plates by adding 0.6, 1.2 or 1.8 ml of virus antigen to 2 ml RBCs. Add 1.3, 1.6 and 1.9 ml CrCl3 respectively and resuspend to 2 ml in PBS 'A'; optimum volume of virus antigen is that which results in the largest and clearest zones with appropriate reference serum) of virus antigen to the 8% RBCs solution. Hold the mixture at 4°C for 10 minutes. Haemagglutination may be observed.
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| | | iv) | SLOWLY add CrCl3 (1/400 in 0.85% NaCl) at half the total volume of virus/RBCs suspension. Hold at 22°C (±2°C) for 5 minutes with occasional mixing.
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| | | v) | Sediment the sensitised RBCs by centrifugation at 1500 g for 5 minutes.
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| | | vi) | Gently resuspend in saline/HEPES/BSA and centrifuge at 1500 g for 5 minutes.
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| | | vii) | Resuspend RBCs to an 8% suspension in PBS 'A'.
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| | | During the sensitisation process, melt the agarose in a pressure cooker. Shortly before use, pipette 7.8 ml volumes to Universal bottles and retain at 42°C. Check that the agar has cooled to 42°C before use.
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| | | . | Preparation of plates
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| | | i) | Add 0.9 ml of virus-sensitised sheep RBCs to 7.8 ml of agarose (42°C). Mix quickly, but gently.
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| | | ii) | Add 0.3 ml of undiluted guinea-pig serum. Mix again and pour into immunoplates on a levelling table. Allow to set and air dry without a lid for 5 minutes.
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| | | iii) | Place lids on plates and store at 4°C in a humid box until used.
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| | | iv) | Prepare control plates with unsensitised cells or cells sensitised with an unrelated virus. Batches of prepared plates can be stored for several weeks.
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| | | v) | Punch 3 mm holes in the set gels to a prepared template, allow for 16 test sera and a positive control serum. On antigen control plates, prepare five rows of eight wells.
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| | | vi) | Pipette 10 µl of heat-inactivated (56°C for 30 minutes) test sera and a positive control serum to appropriate wells. Incubate at 34°C for 20 hours in a humid box.
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| | | vii) | Measure zone diameters, and calculate areas of haemolysis after the area of the well has been deducted.
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| | | . | Interpretation of the results
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| | | For results to be valid, positive and negative control sera should give results consistent with those expected on the basis of prior experience. Areas of haemolysis for the control sera should be clear and vary no more than 20% from the designated value for the control serum. Results may be expressed as mm2 or as a ratio of the control serum value. Sera giving positive results in the control plate should be adsorbed with sheep RBCs. For diagnostic purposes, acute and convalescent sera should be tested in duplicate on the same plate. Increase in zone areas produced by convalescent serum compared with acute serum is evidence of infection. The increase in area deemed to be significant depends on the reproducibility of the test within the laboratory, but should be equivalent to a twofold or more increase in antibody concentration. This area can be calculated from a standard curve generated from a dilution series of a standard antiserum.
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C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
Equine influenza virus vaccines consist of inactivated whole viruses or their subunits, with or without adjuvant. A live attenuated influenza vaccine for intranasal administration has recently become available commercially in some countries. Requirements for such a vaccine may be anticipated to differ in some particulars from the following, which are appropriate to inactivated vaccines.
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.
| 1. | Seed management
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| | a) | Characteristics of the seed
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| | | An ongoing surveillance programme by OIE and WHO Reference Laboratories aimed at providing information on suitable vaccine strains is being co-ordinated by the OIE Reference Laboratory (Newmarket) (15). Recommendations on vaccine strains made by the Expert Surveillance Panel will be published annually in the OIE Bulletin.
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| | | H7N7: Many vaccines still contain an H7N7 strain. However, the Expert Surveillance Panel has recommended that the H7N7 component should be omitted as no reports of infections with this subtype have been substantiated during the past 20 years.
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| | | H3N8: Antigenic variants of H3N8 viruses co-circulate (3). It is important to include recent isolates that are epidemiologically relevant to the region in which the vaccine is to be used. Typical European isolates include A/eq/Suffolk/89, A/eq/Borlange/91, and A/eq/Newmarket/2/93. A/eq/Kentucky/94 or A/eq/Newmarket/1/93 represent typical American isolates. However, it is recommended that vaccines contain representatives of both lineages (American-like and European-like), particularly where equine populations from different continents mix. Some vaccines also contain the original H3N8 prototype strain A/eq/Miami/63. However, this strain will not induce adequate protection against the predominant viruses currently circulating and should be omitted from vaccines.
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| | b) | Method of culture
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| | | Virus strains may be obtained from OIE Reference Laboratories (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm). Viruses selected as vaccine strains should be described in terms of origin and passage history. The strains are propagated in the allantoic cavity of 10-day-old embryonated hens' eggs or cell cultures, such as MDCK. All manipulations must be conducted separately for each strain. Viral growth is monitored by HA tests. Passaged virus is identified by serological tests, such as HI or SRH. If vaccine virus is grown in cell culture, antigenic studies with ferret sera and MAbs should be undertaken to ensure that variant viruses have not been selected during passage to prepare master and working seed viruses. Master and working seed viruses are divided into aliquots and stored in freeze-dried form at -20°C or at -70°C following testing for extraneous agents. Records of storage conditions should be maintained.
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| | | The master seed lot of each vaccine strain selected should be processed at one time to assure a uniform composition, tested for extraneous agents, and fully characterised. Antisera or MAbs for use in HI tests to characterise vaccine strains may be obtained from OIE and WHO Reference Laboratories.
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| | | Working seed lots are derived from a master seed lot and should be of uniform composition, free from extraneous agents, and fully characterised. Aliquots of the working seed are used for production of vaccine.
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| | | Master and working seed lots should be prepared in specific pathogen free eggs or, as a minimum, in eggs derived from a healthy flock.
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| | | If MDCK cells are used to propagate vaccine virus, master cell lines should be established and stored in liquid nitrogen, and should be tested for freedom from extraneous agents according to National Control Authority Guidelines.
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| | | Examination of seed viruses for extraneous agents including mycoplasmas and other equine viruses should be performed by appropriate techniques, including inoculation of susceptible tissue cultures and examination for cytopathic effect or application of fluorescent antibodies for antigen detection.
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| | | The presence of other common equine respiratory pathogens, e.g. equine herpesviruses 1, 2, 4, equine picornaviruses, equine viral arteritis, and equine adenoviruses, should be specifically excluded.
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| | | The absence of bacteria should be confirmed by standard sterility tests and toxicity tests in small animals.
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| | c) | Validation as a vaccine
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| | | For each vaccine strain, a prototype batch should be prepared to establish its suitability as a vaccine strain, i.e. purity and safety should be confirmed by standard techniques. The ability of seed-lot viruses to grow to high titre and generate sufficient antigenic mass to stimulate adequate antibody responses in the target species, should be confirmed.
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| | | Additionally, vaccine virus derived in MDCK cells should be fully characterised to ensure that antigenic variants have not arisen during the culture process, such that the vaccine virus is no longer representative of the original isolate.
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| 2. | Method of manufacture
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Production is based on a seed-lot system that has been validated with respect to the characteristics of the vaccine strains. Each strain of virus is inoculated separately into the allantoic cavity of 9-11-day-old embryonated hens' eggs from a healthy flock. The eggs are incubated at a suitable temperature for 2-3 days, and the allantoic fluid is collected. The viral suspensions of each strain are collected separately and inactivated. If necessary, they may be purified. Suitable adjuvants and antimicrobial preservatives may be added.
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| | Monovalent virus pools should be inactivated as soon as possible after their preparation, by a method approved by the National Control Authority. If formalin (40% formaldehyde) or beta-propiolactone (2-oxetanone) is used, the concentration by volume should not exceed 0.1%. Ideally, pools should be held at 4°C and should be inactivated within 5 days of harvest. Inactivation of the vaccine must be demonstrated. A suitable method consists of inoculating 0.2 ml of undiluted monovalent pool and 1/10 and 1/100 dilutions of the monovalent pool into the allantoic cavities of groups of fertile eggs (ten eggs in each group), and incubating the eggs at 33-37°C for 3 days. At least eight of the ten eggs should survive at each dosage level. A volume of 0.5 ml of allantoic fluid is harvested from each surviving egg. The fluid harvested from each group is pooled, and 0.2 ml of each of the three pools is inoculated, undiluted, into a further group of ten fertile eggs. Haemagglutinin activity should not be detected in these new groups of eggs. In some countries, the requirement that 80% of the eggs should survive during incubation may be impossible to satisfy, in which case the National Control Authority should then specify a modified requirement to be satisfied. Before inactivation, samples should be collected for bacterial and fungal sterility tests.
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| | Monovalent material may be concentrated and purified by high-speed centrifugation or other suitable methods approved by the National Control Authority, either before or after the inactivation procedure. It is important to concentrate and purify the virus under optimum conditions, e.g. temperatures that preserve its antigenic properties.
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| | The monovalent virus pool shall be shown not to contain viable influenza virus when tested by inoculation of embryonated hens' eggs, by a method approved by the National Control Authority.
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| 3. | In-process control
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| | Relevant in-process controls should be applied before and after inactivation and before and after concentration and purification.
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| | In-process controls include: (a) identity of virus strains (tested by HI); (b) sterility; (c) virus titre; (d) haemagglutinin content (tested by chicken RBCs agglutinating units, CCA [chick cell agglutination]); and (e) immunologically active HA (tested by single radial diffusion [SRD] or another suitable immunochemical method).
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| | . | Single radial diffusion test
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| | | SRD is a reliable method for measuring immunologically active HA in terms of µg HA, and is used routinely for potency testing human influenza vaccines (24).
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| | | The potency of inactivated equine influenza vaccine depends on the concentration of immunologically active haemagglutinin (16, 22, 23).
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| | | Assessment of the antigenic content of the vaccine by CCA alone may be misleading, as the sensitivity of this assay is a reflection of the ability of virus strains to agglutinate RBCs. Disruption of virus may lead to an apparent increase in HA as measured by CCA. The CCA assay does not provide a measure of the antigenic properties of the HA (HA may retain its properties to bind to RBCs while losing its ability to stimulate antibody).
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| | | The composition of some equine influenza vaccines is unusual in that products may contain more than one variant of the H3N8 subtype. In this situation, it is not possible to judge the potency of individual H3N8 components from serological tests performed on sera collected from horses or small animals vaccinated with the final product, because of cross-reactivity between the two isolates of the same subtype. Thus, it is important that a reliable method, such as SRD, be used to measure the potency of individual components before and after inactivation and prior to mixing and formulation with adjuvant.
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| | | In the SRD test, virus preparations are compared with a calibrated reference preparation of known HA content. Antigens are allowed to diffuse through a gel containing an antiserum specific for a particular HA. The distance diffused by the antigen before precipitation by the antibody incorporated in the gel is directly related to the concentration of haemagglutinin in the antigen preparations (13).
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| | | Standard reagents for SRD testing are available from the WHO International Laboratory for Biological Standards (National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK). Reagents for A/eq/Prague/56 (H7N7), and the H3N8 strains A/eq/Miami/63, A/eq/Kentucky/81, A/eq/Newmarket/1/93 (American-like), and A/eq//Newmarket/2/93 (European-like) are currently available.
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| | a) | Sterility
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| | | Tests for sterility and freedom from contamination of biological materials may be found in Chapter I.1.5.
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| | b) | Safety
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| | | i) | Using no fewer than three horses, each horse is inoculated intramuscularly (at two different sites) with the dose of vaccine specified by the manufacturer; these inoculations are repeated 2-4 weeks later. The animals are kept under observation for 10 days after the second set of injections. No abnormal local or systemic reaction should ensue.
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| | | ii) | If vaccine is to be used in mares, safety should be demonstrated by giving two doses of vaccine to no fewer than two pregnant mares at the prescribed interval within the trimester for which the vaccine is recommended.
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| | c) | Potency
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| | | Following mixing of viral antigens and adjuvants, aliquots should be potency tested in vivo using horses and guinea-pigs or a suitable alternative immunochemical assay. Adjuvants cause interference in quantitative in-vitro tests, such as CCA and SRD, although SRD may be used on the final product as a qualitative assay to demonstrate the presence of antigen for each vaccine strain. For repeated batch tests, only guinea-pigs or a suitable alternative immunochemical assay are used, subject to agreement of the National Control Authority.
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| | | i) | Serological responses in horses
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| | | | For a valid in-vivo potency test, naive seronegative horses must be selected for vaccination. Young horses or ponies (not less than 6 months old) should be screened for the presence of antibody using recently isolated viruses relevant to the area in which the horses were reared. If HI tests are used for screening, H3N8 viruses should be treated with Tween 80/ether to maximise the sensitivity of the test. Alternatively, SRH may be used to establish the seronegative status of animals to H7N7 and H3N8 viruses.
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| | | | To test a vaccine for efficacy in horses, inject a volume corresponding to one vaccine dose by the recommended route into each of five susceptible seronegative horses. After the period recommended between the first and second doses, as stated on the label, a volume of vaccine corresponding to the second dose of vaccine is injected into each horse.
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| | | | Three blood samples are collected from each animal, the first at the time of the first vaccination, the second 1 week after the first vaccination, and the third 2 weeks after the second vaccination.
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| | | | The serological assay used to measure the antibody response to the viruses contained in the vaccine must be standardised for a valid in-vivo potency test, therefore the SRH assay (see Section B.2.b.) is preferred. Standard sera for the quality control of equine influenza vaccines are available from the European Pharmacopoeia (serum to A/eq/Newmarket/1/77 [Catalogue number E0850010], A/eq/Newmarket/1/93 [E0850021] and A/eq/Newmarket/2/93 [E0850022]). These sera should be tested in parallel with the test sera to ensure that the test is valid with respect to sensitivity; the values obtained should not vary by more than 20% from the SRH values assigned in an international collaborative study (12). Due to poor repeatability and reproducibility of the HI test, no HI titre could be assigned to these sera.
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| | | | The antibody value measured by SRH should not be less than 150 mm2. This is higher than the titre required in the European Pharmacopoeia Monograph for inactivated equine influenza vaccines (85 mm2) as this value is not considered to be protective. If the titre found for any horse after the first vaccination indicates that there has been an anamnestic response, the result is not taken into account. A supplementary test is carried out, as described above, replacing the horses that showed an anamnestic response with an equal number of new animals.
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| | | | If the HI test is used, the antibody titre of each serum taken after the second vaccination in each test should not be less than 1/64 (calculated for the original serum, taking into account the predilution of 1/8). Alternatively, the antibody levels stimulated by the vaccine under test should be shown to be at least equal to the antibody levels stimulated by a standard vaccine tested in parallel that has been shown previously to protect horses against challenge infection.
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| | | ii) | Challenge studies in horses
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| | | | It may be desirable in certain cases to undertake challenge studies in horses to demonstrate potency, particularly if vaccines are being assessed for their ability to protect against antigenically dissimilar viruses. Challenge studies may be carried out by exposing six vaccinated horses/ponies to an aerosol of virulent influenza virus no fewer than 2 weeks after the second dose of vaccine. Comparisons of clinical signs, virus excretion and serological responses are made with a group of no fewer than four unvaccinated control animals challenged at the same time (13, 14). The timing of the challenge procedure will reflect the claims to be made on the data sheet regarding duration of immunity.
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| | | | If tests for potency in horses have been carried out with satisfactory results on a representative batch of vaccine, these tests may be omitted as a routine control on other batches of vaccine prepared using the same seed-lot system, subject to agreement by the National Control Authority.
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| | | iii) | Serological responses of guinea-pigs
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| | | | Inject each of no fewer than five guinea-pigs free from specific antibodies with one vaccine dose. Collect blood samples 21 days later, and test the serum by SRH or HI (see Sections B.2.a. and B.2.b.). Perform the tests of each serum using, respectively, the antigen(s) prepared from the strain(s) used in the production of the vaccine. The antibody titre of each serum in each test should not be less than the titre stimulated by a standard vaccine that has been shown to stimulate protective levels of antibody in horses.
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| | d) | Duration of immunity
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| | | Where claims for duration of immunity are made on the data sheet, these should be supported with data on the duration of protective levels of antibody maintained in horses vaccinated according to the recommended schedule. Antibody levels quoted as protective should be validated in challenge studies (see Section C.4.c.ii.) or by comparison with published reports.
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| | e) | Stability
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| | | Vaccines should be stored at 5±3°C and protected from light. The shelf life quoted on the data sheet should be demonstrated by testing the potency of aliquots over time using the guinea-pig potency test (see Section C.4.c.iii.).
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| | f) | Preservatives
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| | | Preservatives are not normally included.
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| | g) | Precautions (hazards)
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| | | The contents of each opened vial should be used within 1 hour of opening. Aseptic precautions should be observed during administration, and only healthy horses should be vaccinated. Occasionally, transient local and or general reactions may occur, and rest may be advisable for 24-48 hours after vaccination.
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| 5. | Tests on the final product
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| | a) | Safety
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| | | Tests are performed as described in Section C.4.b.i. Once safety has been demonstrated on a prototype batch, safety testing in pregnant mares may be omitted for routine testing of subsequent batches of the final product.
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| | b) | Potency
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| | | See Section C.4.c.iii. As a minimum, serological testing in guinea-pigs should be performed on each batch of the final product.
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| | c) | Maintaining epidemiologically relevant strains in vaccines
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| | | To enable vaccine manufacturers to respond quickly to recommendations from the Expert Surveillance Panel to update vaccine strains, the Committee for Veterinary Medicinal Products for the European Agency for the Evaluation of Medicinal Products has developed a fast-track licensing system to be used when vaccine strains are updated (5).
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REFERENCES
| 1. | Chambers T.M., Shortridge K.F., Li P.H., Powell D.G. & Watkins K.L. (1994). Rapid diagnosis of equine influenza by the Directigen FLU-A enzyme immunoassay. Vet. Rec., 135, 275-279.
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| 2. | Cook R.F., Sinclair R. & Mumford J.A. (1988). Detection of influenza nucleoprotein antigen in nasal secretions from horses infected with A/equine influenza (H3N8) viruses. J. Virol. Methods., 20, 1-12.
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| 3. | Daly J.M., Lai A.C.K., Binns M.M., Chambers T.M., Barrandeguy M. & Mumford J.A. (1996). Antigenic and genetic evolution of equine H3N8 influenza A viruses. J. Gen. Virol., 77, 661-671.
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| 4. | Donofrio J.C., Coonrod J.D. & Chambers T.M. (1994). Diagnosis of equine influenza by the polymerase chain reaction. J. Vet. Diagn. Invest., 6, 39-43.
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| 5. | European Agency for the Evaluation of Medicinal Products (EMEA), Committee for Veterinary Medicinal Products (CVMP) (1998). Note for Guidance: Harmonisation of Requirements for Equine Influenza Vaccines Specific Requirements for Substitution or Addition of a Strain or Strains. Document EMEA/CVMP/112/98-FINAL, EMEA, London, UK.
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| 6. | Ilobi C.P., Henfrey R., Robertson J.S., Mumford J.A., Erasmus B.J. & Wood J.M. (1994). Antigenic and molecular characterisation of host-cell mediated variants of equine H3N8 influenza viruses. J. Gen. Virol., 75, 669-673.
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| 7. | John T.J. & Fulginiti V.A. (1966). Parainfluenza 2 virus: increase in haemagglutinin titre on treatment with Tween-80 and ether. Proc. Soc. Exp. Biol. Med., 121, 109-111.
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| 8. | Lai A.C., Chambers T.M., Holland R.E. Jr, Morley P.S., Haines D.M., Townsend H.G. & Barrandeguy M. (2001). Diverged evolution of recent equine-2 influenza (H3N8) virurs in the Western Hemisphere. Arch. Virol., 149, 1063-1074.
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| 9. | Livesay G.J., O'Neill T., Hannant D, Yadav M.P. & Mumford J.A. (1993). The outbreak of equine influenza (H3N8) in the United Kingdom in 1989; diagnostic use of an antigen capture ELISA. Vet. Rec., 133, 515-519.
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| 10. | Morley P.S., Bogdan J.R., Townsend H.G.G. & Haines D.M. (1995). The effect of changing single radial haemolysis assay method when quantifying influenza A antibodies in serum. Vet. Microbiol., 44, 101-110.
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| 11. | Mumford J.A. (1992). Progress in the control of equine influenza. In: Equine Infectious Disease VI: Proceedings of the Sixth International Conference, Plowright W., Rossdale P.D. & Wade J.F., eds. Newmarket, R & W Publications, UK, 207-217.
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| 12. | Mumford J. (2000). Collaborative study for the establishment of three European Pharmacopoeia biological reference preparations for equine influenza horse antiserum. PHARMEUROPA Special Issue, Bio 2000-1, 5-21.
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| 13. | Mumford J.A., Hannant D. & Jessett D.M. (1990). Experimental infection of ponies with equine influenza (H3N8) viruses by intranasal inoculation or exposure to aerosols. Equine Vet. J., 22, 93-98.
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| 14. | Mumford J.A. & Wood J. (1993). Establishing an acceptability threshold for equine influenza vaccines. Dev. Biol. Stand., 79, 137-146.
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| 15. | Mumford J.A. & Wood J. (1993). WHO/OIE Meeting: Consultation on Newly Emerging Strains of Equine Influenza. Vaccine, 11, 1172-1175.
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| 16. | Mumford J., Wood J.M., Scott A.M., Folkers C. & Schild G.C. (1983). Studies with inactivated equine influenza vaccine 2. Protection against experimental infection with influenza virus A/equine/Newmarket/79 (H3N8). J. Hyg. (Camb.), 90, 385-395.
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| 17. | Oxburgh L. & Hagstrom A.A. (1999). A PCR based method for the identification of equine influenza virus from clinical samples. Vet. Microbiol., 67, 161-174.
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| 18. | Oxburgh L. & Klingborn B. (1999). Cocirculation of two distinct lineages of equine influenza virus subtype H3N8. J. Clin. Microbiol., 37, 3005-3009.
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| 19. | Plateau E. & Cruciere C. (1983). Study on radial haemolysis method for the detection of anti influenza equiantibodies in equine sera: reliability and expression of the results. Zentralbl. Veterinarmed [B], 30, 512-520.
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| 20. | Schild G.C., Pereira, M.S. & Chakraverty P. (1975). Single radial haemolysis: a new method for the assay of antibody to influenza haemagglutinin. Bull. WHO, 52, 43-50.
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| 21. | United States Department of Health and Human Services (1982). Concepts and Procedures for Laboratory-Based Influenza Surveillance. Centers for Disease Control, Atlanta, Georgia, USA, 81-835.
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| 22. | Wood J.M., Mumford J., Folkers C., Scott A.M. & Schild G.C. (1983). Studies with inactivated equine influenza vaccine 1. Serological responses of ponies to graded doses of vaccine. J. Hyg. (Camb.), 90, 371-384.
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| 23. | Wood J.M., Schild G.C., Folkers C., Mumford J. & Newman R.W. (1983). The standardisation of inactivated equine influenza vaccines by single-radial immunodiffusion. J. Biol. Stand., 11, 133-136.
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| 24. | Wood J.M., Schild G.C., Newman R.W. & Seagroatt V. (1977). An improved single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen. Application for potency determinations of inactivated whole virus and subunit vaccines. J. Biol. Stand., 5, 237-247.
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*
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NB: There are OIE Reference Laboratories for Equine influenza (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm).
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