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SUMMARY
Myxomatosis is a disease of the European rabbit caused by the myxoma virus, a member of the Poxviridae. The diagnosis of myxomatosis, regardless of its clinical form, depends on the isolation and identification of the virus or the demonstration of its antigens. The presence of a humoral immune response facilitates a retrospective diagnosis of a mild form of the disease, and can provide an indication of the prevalence of infection in a rabbit population. The disease is characterised by gross myxomatous skin lesions.
Identification of the agent: When skin lesions are present on a dead rabbit, the viral antigen may be demonstrated by immunodiffusion tests on lesion fragments. Monolayer cell cultures of rabbit kidney inoculated with lesion material will show the characteristic cytopathic effects of poxviruses. The presence of virus can be confirmed by immunofluorescence and negative-staining electron microscopy.
The inoculation of rabbits with suspect material takes longer to identify infection, but this will serve to confirm the presence of infective virus and indicate its pathogenicity.
Serological tests: Identification and titration of specific antibodies arising from natural infection or from immunisation are done by traditional complement fixation or by a recently developed and more sensitive enzyme-linked immunosorbent assay (ELISA), which is not affected by pro- or anti-complementary factors. The difficulty in obtaining blood samples from representative members of a population can be obviated by collecting blood dried on filter paper; this can later be extracted and examined by the indirect fluorescent antibody test or ELISA. Microcapillary blood sampling can also be used for the ELISA.
Qualitative agar gel immunodiffusion tests have the advantage of detecting both antigen and humoral antibodies.
Requirements for vaccines and diagnostic biologicals: Modified live virus vaccines prepared from fibroma virus or modified myxoma virus strains are available for immunisation of rabbits.
A. INTRODUCTION
Myxomatosis is a virus disease of the European rabbit (Oryctolagus cuniculus) caused by the myxoma virus, a member of the Poxviridae. The virus has a characteristic tropism for skin, causing a nodular skin form, or has an oculo-respiratory tract tropism, the nonmyxomatous form. It affects rabbits of all ages, and strains, which vary in virulence, give rise to inflammatory skin lesions, generalisation and, possibly, eventual death of the animal by secondary bacterial infections or through inanition. The virus is transmitted to susceptible rabbits by biting insects, such as fleas or mosquitoes. Limited transmission from rabbit to rabbit is possible if they are closely confined.
A primary myxoma lesion arises at the site of infection after 2-5 days, followed by conjunctivitis (which is a sign of systemic infection), inflammation of the anogenital region, and the formation of secondary skin lesions at various other sites. In the oculo-respiratory form, the primary lesion is usually an inflammatory pink macule followed by oculo-nasal catarrh. Virulent strains can kill a rabbit within 10-15 days. There is no health risk to humans working with the myxoma virus.
B. DIAGNOSTIC TECHNIQUES
As the signs of the disease become less distinct with the attenuation of virus strains, the submission of samples for laboratory diagnosis becomes more important. The different techniques available vary in their ability to detect myxoma virus in typical myxomatous lesions, oedema of the eyelids or genital oedema. With atypical lesions, these techniques distinguish myxoma virus infection from that due to Shope's fibroma virus.
| 1. | Identification of the agent
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A portion of lesion is excised with scissors and separated from the epidermis and superficial dermis. This is washed with phosphate buffered saline (PBS) with antibiotics as defined below and homogenised with ground glass at a dilution rate of 1 g tissue/4.5-9.0 ml of PBS. Cells are disrupted by two freeze-thaw cycles, or by ultrasonication to liberate virions and viral antigens. This suspension is centrifuged for 5-10 minutes at 1500 g. The supernatant fluid is used for the tests.
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| | a) | Culture
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| | | Isolation of the virus in cell culture is accomplished using primary cultures of rabbit kidney (RK) cells, or with established cell lines, such as RK-13, in minimal essential medium (MEM) containing 5% calf serum, 100 international units (IU)/ml penicillin; 100 µg/ml streptomycin; 100 µg/ml gentamycin; 50 IU/ml nystatin (mycostatin); and 5 µg/ml amphotericin (fungizone). The inoculum consists of the supernatant fluid from a homogenised lesion or oculo-respiratory discharge in Opti-MEM with 2% calf serum and antibiotics. This is removed from the cell layer after 2 hours. The cell layer is washed in a small volume of medium and then replenished with maintenance medium (Opti-MEM).
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| | | A cytopathic effect (CPE) typical of poxviruses (10) usually develops after 24-48 hours (37°C and 5% CO2), but with some strains, it may take up to 7 days for CPE to be observed. Groups of cells with a confluent cytoplasm form syncytia that vary in size from 2 to 50 or even 100 nuclei together, according to the strain of virus. The nuclei of some cells change, the chromatin forming basophilic aggregations that vary in size and number and give the culture a leopard-skin appearance. Eosinophilic intracytoplasmic inclusions remain discrete, if present at all. Affected cells round up, contract and become pyknotic. They then lyse and become detached from the glass or plastic support. Later, all cells are affected and the cell monolayer detaches completely.
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| | | Shope's fibroma virus at first produces well-defined voluminous masses of rounded cells, which proliferate and pile up (10). At the edge, cells just becoming infected show discrete nuclear changes and acidophilic cytoplasmic inclusions that are numerous at an early stage. The cell layer is destroyed after several days.
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| | b) | Immunological methods
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| | | Agar gel immunodiffusion (AGID) tests are simple and rapid to perform - results can be obtained within 24 hours. Agar plates are prepared with Noble agar (0.6 g), ethylene diamine tetra-acetic acid (EDTA) (2.5 g), sodium chloride (4.5 g), and distilled water (500 ml) containing thiomersal (merthiolate) at 1/100,000 dilution. Standard antiserum (see below), and the test sample are placed in opposing wells that are 6 mm in diameter and 5 mm apart. Another technique is to deposit a small portion of the lesion directly into the agar, 5 mm away from a filter paper disk impregnated with the antiserum. A number of lines of precipitation, usually up to three, appear within 48 hours, indicating the presence of myxoma viral antigens. Only one line forms in the presence of heterologous reactions with Shope's fibroma virus.
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| | | Indirect fluorescent antibody (IFA) tests can be applied to cultures from 24 hours onwards. IFA tests reveal intracytoplasmic multiplication of virus, without being able to distinguish myxoma virus from fibroma virus. The inoculation of chicken embryo cells (trypsinised at day 11 of egg incubation) does not result in CPE, but it is useful for detecting the viral antigens by IFA tests.
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| | c) | Electron microscopy
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| | | Negative-staining electron microscopy (EM) can be applied to a portion of skin lesion. The technique is simple and rapid to perform, giving results in 1 hour. About 1 mm3 of the tissue is laid in a watch glass and 3 drops of distilled water are added. After 1-2 minutes at room temperature an EM grid coated with formvar and carbon is laid over the liquid. After 1 minute any excess liquid is removed with filter paper and immediately a 2% aqueous solution of ammonium molybdate, pH 7.0, is dropped on to the grid. After 10 seconds the excess liquid is removed with filter paper and the grid is prepared for the electron microscope. In a positive case, typical poxvirus particles can be seen. Myxoma virus cannot be distinguished from fibroma virus using this method.
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| | d) | Inoculation tests
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| | | Rabbit intradermal inoculation also offers a means of identifying the virus through its special characteristics and pathogenicity, namely cutaneous tropism (nodular form) and oculo-respiratory tropism (nonmyxomatous form). It should be avoided if possible but has the advantage of being a gauge of virulence, from the type of inflammation in lesions (local or systemic infection) to the extent of lesions and survival time, and can distinguish fibroma virus (with its simple fibromatous local lesion) from myxoma virus (capable of causing generalised infection in adults). Rabbits should be of a domestic breed, weighing approximately 2 kg, unvaccinated and previously tested for the absence of antibodies (10).
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| | | The inoculum may be the supernatant fluid from a homogenised lesion (with antibiotics) or the product of a cell culture. Between 0.1 and 0.2 ml is administered intradermally behind the ear or into the dorso-lumbar region, which has previously been depilated. The inoculum may be assayed by injecting serial dilutions in saline buffer at one site for each dilution. A primary lesion will appear at the sites within 2-5 days, followed by conjunctivitis. Using five sites for each dilution allows a 50% infective dose (ID50) to be obtained. If the animal survives, the disease can be confirmed serologically after 15 days.
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| 2. | Serological tests
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| | Antibodies develop within 8-13 days. In the nonlethal forms and in vaccinated rabbits, the titre is highest after 20-60 days; it declines thereafter, disappearing after 6-8 months in the absence of reinfection (serological response evaluated by use of the complement fixation [CF] test) (12).
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| | Various serological tests may be used, but CF, IFA and enzyme-linked immunosorbent assay (ELISA) (in order of increasing sensitivity) are the most appropriate tests for international trade and other applications. These tests require standard antigens and antisera. The antigen can be prepared from the Lausanne strain, or some antigenically related strain, propagated in rabbits or cell cultures.
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| | . | Preparation of standard reagents
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| | | Myxomatous lesions are removed from rabbits at 6-7 days after inoculation and homogenised in veronal buffer to a dilution of 1/5. The antigen is the supernatant fluid that is obtained following centrifugation. Any anticomplementary activity is abolished by adding 0.6% chloroform. The antigen fluid can be frozen at -30°C or -70°C for stock purposes or used directly in CF tests after titration against a standard antiserum.
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| | | Antigen is made from cell cultures using the RK-13 lines. The virus is harvested as a suspension of cells 48 hours after infection, and is centrifuged. The supernatant fluid is retained. The cell deposit is homogenised (gently) or ultrasonicated (for 3 minutes continuously or for 5 minutes, 50% pulse 100 watts) resuspended and recentrifuged, and the supernatant is added to the former fluid. The final supernatant fluid is the antigen, and is stored at -20°C or -70°C (for longer conservation). It is titrated in cell cultures before use in virus neutralisation (VN) tests.
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| | | For the standard antiserum, an adult serologically negative rabbit is vaccinated with an attenuated strain of myxoma virus, or with the Shope's fibroma virus. After 3-4 weeks, the rabbit is inoculated with myxoma lesion material derived from the Lausanne strain of virus. Serum is obtained 3 weeks later and titrated by the CF test. If the titre is 1/640 or more, the animal is bled out and the serum is stored at -20°C.
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| | . | Titration of standard reagents
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| | | i) | Inactivate standard antiserum for 30 minutes in a water bath at 56°C. No international standard serum for myxomatosis is available, but internal positive standards should be prepared and titrated in the appropriate range, using the CF test. After this, the following procedure is used to standardise batches of antigen.
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| | | ii) | Make doubling dilutions of standard serum in calcium/magnesium/veronal buffer (CMV), pH 7.2, from 1/2 to 1/4096, using a 96-well round-bottomed microtitre plate, one well per dilution and 25 µl volume in each line.
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| | | iii) | Using tubes, make doubling dilutions of antigen in CMV, from 1/10 to 1/1280.
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| | | iv) | Add 25 µl per well of the same dilution of antigen, (transferred from the tubes) to each dilution on one line of the plate, from 1/10 to 1/1280, vertically.
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| | | v) | Add 25 µl (6 H50 units [50% haemolysis]) per well of complement.
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| | | vi) | Incubate the plate, covered with a plastic film, for 1 hour at 37°C or 14 hours at 4°C.
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| | | vii) | Add 50 µl per well of the haemolytic system (2.5% sheep red blood cells [RBCs] and an equal volume of anti-sheep RBC serum 1/2000, both in CMV).
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| | | viii) | Cover the plate again and incubate for 30 minutes at 37°C.
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| | | ix) | Read the highest dilution of antigen giving complete haemolysis (H100) with the highest dilution of standard serum. There is 1 antigenic unit (AgU) in 25 µl of antigen of this dilution.
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| | | VN tests (12) can be carried out in flat-bottomed cell-culture grade microtitre plates using the constant virus/varying serum method with 100 TCID50 (50% tissue culture infective dose) of virus.
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| | | CF tests (12) are done in tubes or in microtitre plates (4) by conventional methods, recording 100% or 50% haemolysis. This is the standard method at the present time.
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| | a) | Complement fixation test
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| | | i) | Titrate the complement in haemolysis tubes, in the presence of 1 AgU, in order to determine the H50 unit.
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| | | ii) | Inactivate the positive and negative control sera in a water bath for 30 minutes at 56°C.
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| | | iii) | Make doubling dilutions of test and control sera in CMV, from 1/4 to 1/1024, using a 96-well round-bottomed microtitre plate and 25 µl per well. Use the first well for the initial 1/4 dilution and the second as a serum control (anticomplementary control at 1/4 dilution). Prepare antigen, complement, and RBC control wells (two wells of each).
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| | | iv) | Add 1 AgU in 25 µl per well (except to serum, complement and RBC control wells), then add 6 H50 of complement in 25 µl per well (except to RBC control wells).
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| | | v) | Incubate the plates, covered with plastic film, for 14 hours (overnight) at 4°C.
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| | | vi) | Add 50 µl haemolytic antiserum per well.
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| | | vii) | Cover the plates again and incubate for 30 minutes at 37°C.
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| | | viii) | Prepare H100, H75, H50, H25 haemolysis controls using complement controls (H100) and CMV.
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| | | ix) | Read after centrifugation or passive sedimentation at 4°C. The test sera results are determined as the highest dilution of serum that gives at least 50% haemolysis inhibition.
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| | | x) | A negative serum should give haemolysis inhibition <50% at 1/4 dilution.
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| | b) | Indirect fluorescent antibody test
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| | | IFA test (7) is carried out using chicken embryo cell cultures in flat-bottomed wells of microtitre plates: 200 µl of a cell suspension, diluted 1/1000 in medium, is distributed into all wells and a confluent cell sheet is formed within 24 hours. The medium is discarded and 100 µl of viral suspension containing 104 TCID50 is added to each well. After 2 hours, 100 µl of MEM containing 2% calf serum is added. After 48 hours of incubation, the plates are washed with PBS and fixed with acetone containing 15% water for 30 minutes at -20°C. The plates are then dried at 37°C for 15 minutes. The plates can be stored at -30°C or -70°C for 3 months. Sera are tested by IFA using anti-rabbit IgG conjugated to fluorescein isothiocyanate. The test results may be qualitative with sera diluted 1/10 or 1/20, or quantitative with serial dilutions of serum.
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| | c) | Enzyme-linked immunosorbent assay
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| | | A recently developed ELISA (6) uses a semi-purified myxomatosis virus (French hypervirulent T1 strain antigenically related to Lausanne strain) produced in RK-13. The virus is harvested as a suspension of cells 48 hours after infection, and is centrifuged. The cell deposit is homogenised in TL20 (20 mM Tris, pH 8.6, 150 mM NaCl, and 1 mM EDTA), disrupted in ground glass and centrifuged at 1200 g at 4°C for 10 minutes.
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| | | The supernatant fluid is laid down on an equal volume of a 36% sucrose cushion in TL20 and centrifuged at 200,000 g for 2 hours in an SW 41 rotor at 4°C. The deposit is homogenised in 4-12 ml TL20 and again laid down on a 36% sucrose cushion.
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| | | The new deposit is homogenised in 1 ml TL20 and run into a linear 30-65% saccharose gradient by centrifugation at 200,000 g for 3 hours. The viral disc is harvested and diluted in 10 ml TL20 and then centrifuged at 130,000 g for 1 hour to concentrate the virus.
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| | | The deposit is homogenised in 0.2 ml TL20 and quantified by the Bradford method (colorimetric reaction with Coomassie brilliant blue) (3) or spectrophotometry (viral proteins account for around three-fifths of total protein). It can be stored at -30°C before use.
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| | | i) | Coat probind assay plates for 16 hours (overnight at 37°C) with 1 µg per well viral proteins in 100 µl PBS, pH 7.6, leaving one column blank (i.e. coat with PBS only). Note that different batches of antigen vary in activity, and should be titrated against known standards to select antigen with high optical densities (OD).
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| | | ii) | After three washes in PBS, block free binding sites by incubation in 25 mg/ml gelatin in PBS for 1 hour at 37°C.
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| | | iii) | Wash the plate three times in PBS-0.01% Tween 20, and add 100 µl serial twofold dilutions of serum in PBS-Tween. Include positive and negative serum standards in each plate.
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| | | iv) | After 60 minutes' incubation at 37°C and three washes in PBS-Tween, add 100 µl of a dilution in PBS-Tween of goat anti-rabbit IgG serum (typically 1/3000) conjugated to alkaline phosphatase for 1 hour at 37°C.
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| | | v) | After four washes in PBS-Tween add, as substrate, 100 µl of disodium p-nitrophenyl phosphate at a concentration of 1 mg/ml in 10% diethanolamine.
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| | | vi) | After 12 minutes in the dark at room temperature, the enzymatic reaction is stopped by the addition of 50 µl of 2 N NaOH.
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| | | vii) | Read OD in a spectrophotometer at a wavelength of 405 nm.
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| | | viii) | Express the serum sample titre as the inverse of the highest dilution for which the OD is greater than three times the OD of the negative serum standard.
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| | | The detection of specific myxoma virus antibodies by ELISA has been shown to be a highly sensitive and specific method for kinetic studies in experimental infection (2). Evaluation of the test has shown its great value for diagnostic application in wild rabbit populations (6).
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| | | For epidemiological surveys, the IFA test and the indirect ELISA can also be carried out using blood dried on blotting or filter paper: discs are cut (paper punch size) and two discs are placed in each well, to which is added 100 µl PBS to extract the serum. The dilution is about 1/30 and this can be used in another well for testing (7). Blood samples collected in anticoagulant-coated capillary tubes can be used for the ELISA. The sample is washed in the diluting solution to obtain the required dilution (11).
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| | d) | Agar gel immunodiffusion test
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| | | Agar gel immunodiffusion (AGID) (15) is qualitative and can detect antigen or antibody. Agar is prepared as described previously (Section B.1.) using 6 ml per 10 cm Petri dish. Strips of filter paper containing the standard antigen and antiserum, and discs containing test sera are arranged on the surface of the agar (discs between the strips). The plates are incubated in a humid atmosphere at
37°C and read after 24-48 hours. Three precipitation lines should appear. If the test sera contain antibody, at least one of the three lines is bent towards the antigen band; otherwise it remains straight. If sera contain antigen, at least one of the lines is bent towards the standard serum strip. The test can also be carried out in a more conventional manner using liquid reagents in wells cut in the agar.
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C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
Two types of live vaccine are used for vaccination against myxomatosis: a heterologous vaccine prepared from Shope's fibroma virus (5, 9, 14), and a homologous vaccine prepared from an attenuated strain of myxoma virus (1, 8, 13, 16-18). They are administered subcutaneously or intradermally.
Recently a new recombinant myxoma virus expressing rabbit haemorrhagic disease virus (RHDV) capsid protein and conferring double protection against myxomatosis and RHDV (2) has been developed, but is not yet available commercially.
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.
A master seed virus (MSV) must be established and used according to a seed-lot system. A record must be kept of its origin, passage history and characteristics.
| 1. | Seed management
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| | a) | Characteristics of the seed
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| | | The viruses employed are fibroma virus or myxoma virus. The strains of fibroma virus are usually the original Shope's OA strain (1932), Boerlage's strain or various closely related strains. The strains of myxoma virus are modified by passaging in embryonated chicken eggs, rabbit kidney cells at decreasing temperatures, or chicken embryo cells. The strains usually result from having been cloned several times.
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| | b) | Method of culture
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| | | Fibroma strains are maintained by passage in specific pathogen free (SPF) rabbits or in unvaccinated rabbits from a stock known to be free from myxomatosis. Skin on the backs of healthy adult rabbits is shaved, and multiple sites are inoculated with a 1% suspension of virulent material. Fibromas are fully developed within 8-10 days, at which time the rabbits are killed and the tumours are removed aseptically and homogenised with distilled water. The suspension is stored at -30°C or -70°C in 50% buffered glycerol, or as a 5% dilution in a protein solution. The production of the fibroma virus is also possible in rabbit dermal cell line.
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| | | Myxoma virus is grown on chicken embryo cell culture obtained from flocks free from specified pathogens, or on suitable cell lines (rabbit dermal cell line).
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| | c) | Validation as a vaccine
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| | | i) | Identity
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| | | | Specific antigenic characteristics of the fibroma virus strains are verified by AGID using sera against fibroma and myxomatosis.
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| | | | The identity of myxoma virus is confirmed by neutralisation tests in RK-13 cells, or in a suitable cell line using a monospecific antiserum.
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| | | ii) | Purity
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| | | | The Master seed must be free from bacterial, fungal, mycoplasmal and viral contamination.
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| | | iii) | Safety
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| | | | Samples for safety testing are taken from a batch produced according to the manufacturing process. The dose to be used shall contain the maximum titre or potency established by the manufacturer (release titre).
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| | | | Several tests are performed, at the Master Seed level, to demonstrate different aspects of safety. The safety of 10 times the normal dose must be demonstrated. Also, it is necessary to examine the dissemination of vaccine virus within the vaccinated animal, the ability of vaccine virus to spread from the vaccinated animal to in-contact animals and to test whether there is reversion-to-virulence of the vaccine virus, following serial passage in rabbits.
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| | | | The pathogenicity of the fibroma virus strains is tested by inoculating rabbits with serial dilutions of supernatant fluids obtained by centrifugation of tumour preparations. Macroscopic and histopathological features and the course of development of fibromas are tested in SPF rabbits periodically. (Numerous serial passages in rabbits may induce mutation to the inflammatory IA strain, which produces severe lesions that are more inflammatory than neoplastic.)
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| | | | The residual pathogenicity of the myxoma virus strains is tested by intradermal inoculation into SPF rabbits or unvaccinated rabbits free from myxomatosis. These rabbits should not develop more than a local reaction with perhaps small secondary lesions on the head that disappear within a few days.
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| | | | For both strains, the rectal temperature and the body weight should be recorded as additional parameters.
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| | | iv) | Efficacy
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| | | | Different trials must be undertaken from representative batches of final product containing the minimum titre or potency. The protective effect is demonstrated as follows:
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| | | | A minimum of ten adult rabbits are inoculated with a dose of fibroma vaccine, and three rabbits serve as unvaccinated controls. After 14 days, all rabbits are inoculated, intradermally into the eyelids, with a pathogenic strain of myxoma virus (example: 0.1 ml inoculum containing 103 ID50 [median infectious dose]). During the following 21 days, the controls will die from myxomatosis, and at least seven of the ten vaccinated rabbits must present no signs of generalised infection.
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| | | | Similarly, myxoma vaccine is tested in ten rabbits with three controls. After 14 days, all the rabbits are challenged with a sufficient quantity of virulent strain (example: 0.1 ml of the Lausanne virus strain containing 103 ID50). After 21 days, seven of ten rabbits must have survived, while controls must have died from myxomatosis.
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| | | | The manufacturer shall have established a minimum titre or potency taking into account loss in potency during the shelf life.
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| 2. | Method of manufacture
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| | Fibroma virus is produced by multiple intradermal inoculations of seed virus into the skin on the back of a number of rabbits. Production is also possible in rabbit dermal cell line. Only the second (and perhaps the third) passage can be used if modification of the virus is to be avoided. The product of fibroma homogenate can be stored by freezing or used immediately. After clarification by centrifugation, the supernatant fluid is mixed with a stabiliser containing antibiotics and is distributed into ampoules or bottles for Iyophilisation. Kaolin may be added as an adjuvant (40 mg/ml), in which case the vaccine is administered subcutaneously.
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| | Myxoma virus is produced in chicken embryo cells (derived from SPF eggs) or a suitable cell line, limiting the passage number to a maximum of five. Virus is harvested after 2-6 days. The viral suspension may be stored at -70°C. The vaccine is prepared by diluting in specified proportions the viral preparation with a stabiliser for Iyophilisation. After homogenisation, the product is distributed into bottles for Iyophilisation, the bottles being sealed under vacuum or in sterile nitrogen.
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| | Each virus can also be produced in RK-13 cells.
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| 3. | In-process control
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| | The fibroma virus titre is measured by calculating the ID50 after intradermal inoculation of serial dilutions of the clarified supernatant fluid into several sites (e.g. five) on up to six rabbits. A dilution of a standard preparation of fibroma virus is also inoculated into each rabbit to confirm the animal's correct response to inoculation. The titration can also be performed in a rabbit cell line. In each case the titre should correlate with the required potency as defined by the test for efficacy, see Section C.1.c.
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| | The identity of myxoma virus is checked in RK cells. Titration of each virus can also be done in RK-13 cells (TCID50).
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| | Testing for contaminating viruses is done by inoculating a confluent cell sheet of Vero cells. Vaccine, adjusted to the equivalent of 20 doses/ml, is neutralised with an equal volume of monospecific hyperimmune serum for 30 minutes at 37°C. The mixture is filtered through a 0.22 µm membrane filter, and 1 ml volumes are inoculated into five 25 ml bottles of cell cultures. These are kept under observation for 7 days. After harvesting, the cells are suspended in medium and subjected to several freeze-thaw cycles, followed by centrifugation and filtration, and the material is inoculated into fresh cultures and observed for 7 days. There should be no evidence of CPE or haemadsorption to chicken RBCs.
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| 4. | Batch control
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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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| | | After rehydration, ten doses of the lyophilised fibroma vaccine are injected subcutaneously into each of three susceptible rabbits, which are then observed for 21 days. Local reactions should be slight, with no generalisation and no effect on general health.
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| | | Myxoma vaccine is tested using ten doses injected intradermally into the ears of three susceptible rabbits, which are then observed for 21 days. The primary myxoma lesion should remain mild.
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| | c) | Potency
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| | | The batch potency is determined by measurement of virus content. Serial dilutions of the vaccine are inoculated into suitable cell cultures. One dose of vaccine shall contain not less than the minimum titre established in Section C.1.c.
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| | | If the vaccine strain is not adapted to cultures, an efficacy test in rabbits shall be carried out (see Section C.1.c.).
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| | d) | Duration of immunity
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| | | Several batches of ten susceptible rabbits are vaccinated. One batch is tested by challenge infection (as in the batch potency test), at 1, 2, 3, etc., months post-vaccination for fibroma virus, and at 1, 3, 6, and 9 months for myxoma virus. The duration of immunity is deduced from the time during which at least seven of the ten rabbits prove to be resistant to infection.
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| | e) | Stability
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| | | Titrations of vaccine virus are carried out at intervals until 3 months beyond the requested shelf life on at least three batches of vaccine.
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| 5. | Tests on the final product
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| | a) | Safety
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| | | See Section C.4.b.
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| | b) | Potency
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| | | See Section C.4.c.
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REFERENCES
| 1. | Arguello Villares J.L. (1986). Contribución a la profilaxis de la mixomatosis del conejo mediante el uso de una ceba homologa. Medicina Veterinaria, 3, 91-103.
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| 2. | Bertagnoli S., Gelfi J., Legall G., Boilletot E., Vautherot J.F., Rasschaert D., Laurent S., Petit F., Boucraut-Baralon C. & Milon A. (1996). Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma virus expressing rabbit hemorrhagic disease virus capsid protein. J. Virol., 70, 5061-5066.
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| 3. | Bradford M. (1976). A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254.
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| 4. | Chantal J., Boucraut-Baralon C., Ganiere J.P., Petit F., Py R. & Picavet D.P. (1993). Réaction de fixation du complément en plaques de microtitration: application ŕ la sérologie de la myxomatose. Etude comparative des résultats avec la réaction d'immunofluorescence indirecte. Rev. sci. tech. Off. int. Epiz., 12, 895-907.
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| 5. | Fenner F. & Woodroofe G.M. (1954). Protection of laboratory rabbits against myxomatosis by vaccination with fibroma virus. Aust. J. Exp. Biol., 32, 653-668.
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| 6. | Gelfi J., Chantal J., Phong T.T., Py R. & Boucraut-Baralon C. (1999). Development of an ELISA for detection of myxoma virus-specific rabbit antibodies; test evaluation for diagnostic applications on vaccinated and wild rabbit sera. J. Vet. Diagn. Invest., 11, 240-245.
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| 7. | Gilbert Y., Picavet D.P. & Chantal J. (1989). Diagnostic de la myxomatose: mise au point d'une technique d'immunofluorescence indirecte. Utilisation de prélčvements sanguins sur papier buvard pour la recherche d'anticorps. Rev. sci. tech. Off. int. Epiz. 8, 209-220.
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| 8. | Gorski J. & Mizak B. (1985). Polish vaccine against myxomatosis in rabbits. Med. Weter., 41, 113-116.
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| 9. | Jobert R. (1983). Contribution ŕ l'étude de la vaccination contre la myxomatose, vaccination ŕ l'aide du virus fibromateux. Thčse Doctorat Vétérinaire Toulouse n° 82.
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