Manual of standards LIST B DISEASES ..« ».. 3.7 ..« »» Chapter 3.7.3. ..« Table of content ? - Index -------------------------------------------------------------------------------- CHAPTER 3.7.3. VIRAL HAEMORRHAGIC DISEASE OF RABBITS -------------------------------------------------------------------------------- SUMMARY Viral haemorrhagic disease (VHD) of rabbits is a highly contagious and acute fatal disease of the European rabbit (Oryctolagus cuniculus), caused by a calicivirus. A similar disease, termed European brown hare syndrome (EBHS), has been described in the hare (Lepus europaeus); the aetiological agent is a different calicivirus, antigenically related to the VHD virus. VHD is characterised by high morbidity and high mortality (40-90%), and spreads very rapidly by direct and indirect transmission. Infection can occur by nasal, conjunctival or oral routes. Transmission of VHD is facilitated by the high stability of the virus in the environment. The incubation period varies from 1 to 3 days, and death usually occurs 12-36 hours after the onset of fever. The clinical manifestations have been described mainly in the acute infection (nervous and respiratory signs, apathy and anorexia). Clear and specific pathological lesions, both gross and microscopic, are present; these lesions are reminiscent of those seen in circulatory and degenerative disorders. The most severe lesions are in the liver, trachea and lungs. Petechial haemorrhages are evident in almost all organs and are accompanied by poor blood coagulation. A severe disseminated intravascular coagulation is evident in all organs and tissues. Identification of the agent: The liver contains the highest viral titre and is the most suitable organ for viral identification. As no satisfactory growth conditions or sensitive cell substrates have been established, in vitro isolation cannot be employed. The haemagglutination test using human type O red blood cells was the first test applied for routine laboratory diagnosis of VHD. However, other tests (negative staining immunoelectron microscopy, sandwich enzyme-linked immunosorbent assay (ELISA) and immunohistological staining) have shown a higher level of specificity and sensitivity. Serological tests: Characterisation and titration of specific antibodies arising from natural infection or from immunisation are performed using the haemagglutination inhibition test or the ELISA, either as an indirect or competitive reaction. The reagents used are prepared as follows: the antigen from infected rabbit liver, anti-VHD virus serum from convalescent or hyperimmunised rabbits after experimental infection, and negative serum from rabbits fully sensitive to VHD virus infection. Monoclonal antibodies have been produced in several laboratories. Requirements for vaccines and diagnostic biologicals: Indirect control of the disease is achieved by vaccination using a killed vaccine, prepared from clarified liver suspensions of experimentally infected rabbits, and subsequently inactivated and adjuvanted. Experimental data indicate that protection lasts for a long period (over 1 year). Moreover, vaccinated animals quickly produce solid immunity against VHD virus infection; vaccination is therefore considered an effective post-exposure treatment and its primary use is in rabbitries after an outbreak of the disease has been diagnosed. A. DIAGNOSTIC TECHNIQUES Viral haemorrhagic disease (VHD), a highly contagious and acute fatal disease of wild and domestic European rabbits, is also known as haemorrhagic septicaemia, infectious necrotic hepatitis, and rabbit haemorrhagic disease. VHD was first reported in 1984 in the People's Republic of China (9); currently it is endemic in east Asia and Europe. Outbreaks have also been recorded in Mexico, Reunion Island, Cuba, and parts of North Africa. The causative agent of VHD presents features of a calicivirus, including its size (32-35 nm in diameter), morphology as seen by electron microscopy (EM), the presence of a single major capsid polypeptide (Mr 60 kDa), a positively stranded RNA genome of approximately 7.5 kb and a subgenomic RNA detected in infected tissue and in purified virions (3, 5, 12, 13). The complete sequence of VHD virus has been reported (11). VHD is characterised by high morbidity and a mortality rate between 40% and 90% (1). Infection occurs in rabbits of all ages, but clinical disease is observed only in adults and young animals older than 40-50 days. The mechanism of resistance in young animals is still unclear and is probably correlated to the pathogenesis of the infection (3, 4). The European rabbit (Oryctolagus cuniculus) is the only species known to be affected by VHD. A similar disease, termed European brown hare syndrome (EBHS), has been described in the hare (Lepus europaeus), but its aetiological agent, which is also a calicivirus, is different from VHD virus, although it is related antigenically. VHD spreads very rapidly, and infection can occur by nasal, conjunctival or oral routes. The disease can be transmitted directly from infected animals to healthy ones, indirectly by contact with a carcass, or due to contamination of feedstuffs and water by excretions and secretions of infected animals (3, 18). The possibility of indirect transmission by passive vectors, such as insects, birds and rodents, or contaminated utensils and vehicles, should be considered (1, 18). Humans seem to play an important role as a passive vector of the virus by transmitting the disease from one rabbitry to another (19). Dissemination by airborne particles is also not excluded. The hair from diseased rabbits has been shown to carry the virus (6). VHD virus is very stable and resistant in the environment; the viral infectivity is not reduced by treatment with ether or chloroform and trypsin, by exposure to pH 3.0, or by heating at 50°C for 1 hour. The virus survives at least 225 days in an organ suspension kept at 4°C, at least 105 days in the dried state on cloth at room temperature, and at least 2 days at 60°C, both in organ suspension and in the dried state (15). It also retains its infectivity at low temperatures, and remains remarkably stable during freezing and thawing. For this reason, frozen rabbit meat should be considered a potential source of infection. VHD virus is inactivated by 1% sodium hydroxide and by 1.0-1.4% formaldehyde. Even 0.2-0.5% beta-propiolactone (BPL) at 4°C produces the same effect. Such treatments do not alter the immunogenicity of the virus. The clinical evolution of the disease can be peracute, acute, subacute or chronic (10). Peracute VHD usually affects highly susceptible rabbits at first introduction of the disease into a disease free area. Rabbits die suddenly without any clinical signs. The acute form is prevalent in epidemic areas, whereas the subacute form usually occurs in few animals, in the later stages of an epidemic. The clinical manifestations have been described mainly in the acute infection, as the peracute form is usually symptomless and the subacute form is characterised by similar but milder signs. The incubation period varies between 1 and 3 days; death may occur 12-36 hours after the onset of fever (>40°C). During this phase, various signs can be observed, such as anorexia, apathy, dullness, prostration, congestion of the palpebral conjunctiva, nervous signs (convulsion, ataxia, paralysis, opisthotonos, paddling), groans and cries, respiratory signs (dyspnoea, frothy and bloody nasal discharge), and cyanosis of mucous membranes (10). During an outbreak, a limited number of rabbits (5-10%) may show a chronic or subclinical evolution of the disease, which is characterised by severe and generalised jaundice, loss of weight and lethargy. These animals often die one or a few weeks later, due to a liver dysfunction. However, in meat animals, the hepatic lesions (degeneration and necrosis) in sublinical and chronic cases may be evident only during meat inspection at slaughter. Gross pathological lesions are specific and appear as circulatory and degenerative disorders. The nose is often soiled by bloody discharge. At necropsy, the most severe lesions are in the liver, trachea and lungs. The liver appears yellowish-brown in colour, friable and degenerated, with a marked lobular pattern. The tracheal mucosa is hyperaemic and contains abundant frothy fluid, and the lungs are oedematous and congested. Petechial haemor-rhages are evident in almost all organs and on visceral serosa. These lesions are usually accompanied by poor blood coagulation. Catarrhal gastritis, haemorrhagic enteritis, splenomegaly, enlargement of mesenteric lymph nodes and discolouration of kidneys are detected occasionally. In rabbits affected by chronic disease, an icteric discolouration is clearly evident on the ears and subcutis (10). The microscopic hepatic lesions are of great diagnostic importance. The liver shows multifocal necrosis. Foci of disseminated necrosis may become confluent, forming extensive local areas mainly at the periphery of the lobules. Intra-sinusoidal microthrombi may be present within necrotic foci. The hepatocytes show acidophilic shrinkage or cytoplasmic lysis, occasionally with the formation of Councilman's bodies, hydropic rarefac-tion and cytoplasmic swelling, ischemic necrosis, bile pigment and iron pigment deposition, and dystrophic granular calcification. Inflammatory infiltrate is mild to moderate and consists of lymphocytes in portal spaces and sinusoids, and granulocytes in sinusoids. Tracheal and pulmonary lesions are mainly of the hyperaemic-oedematous type, often associated with haemorrhages and sometimes with microthrombi in alveolar capillaries. The most considerable microscopic lesions in other organs and tissues are karyorrhexis of lymphoid tissue, leading to depletion and lymphopaenia, and microthrombosis, which frequently occurs in the glomerular capillaries (10). The clinical symptoms and the gross and microscopic lesions observed in hares affected by EBHS are very similar to those described in VHD in rabbits. The behaviour of affected hares during disease has been observed in animals in captivity because wild hares are most often found dead due to the rapid course of the infection. Sick animals show abnormal behaviour or true neurological signs, such as loss of balance and coordination, paralysis and dullness. At necropsy, oedema and congestion of tracheal mucosa with foamy haemorrhagic content, liver degeneration, and enlargement of the spleen are the principal findings (3, 4). 1. Identification of the agent The liver contains the highest viral titre (from 103 LD50 [50% lethal dose] to 106.5 LD50) and is the most suitable organ to submit for viral identification both for VHD and EBHS viruses. The amount of virus present in other parts of the body is directly proportional to vascularisation, thus spleen and serum are quite rich in virus and can serve as alternative diagnostic material. The initial treatment of the diagnostic samples is identical irrespective of the diagnostic method to be applied, with the exception of immunostaining techniques. An organ fragment is mechanically homogenised in 5-20% (w/v) phosphate buffered saline solution (PBS), pH 7.2, and clarified by centrifugation at 5,000 g for 15 minutes. At this stage, the supernatants can be directly examined by haemagglutination (HA) test or enzyme-linked immunosorbent assay (ELISA). If the sample is to be observed by EM, it is advisable to perform a second centrifugation at 12,000 g for 15 minutes, before the final ultracentrifugation. Al-though the virus is quite stable at room temperature, it is preferable to perform all steps at 4°C. As no satisfactory growth condition and sensitive cell substrates have been established, in vitro isolation of VHD or EBHS viruses cannot be included among the virological methods. The possibility of propagating VHD virus in a mutant cell culture from primary rabbit kidney cells (DJRK strain) has been reported (8), but has not been confirmed in other laboratories and is not used at present as a diagnostic method. To date, viral isolation in vivo by experimental reproduction of VHD and EBHS remains of utmost importance. Large quantities of viral antigen are prepared for diagnostic reagents and to produce inactivated tissue-derived vaccines. Experimental infection is not practical as a routine diagnostic method, although it is still desirable in the case of samples that are unusual (HA negative / ELISA positive) or not clearly positive. Natural hosts are the only species in which the two diseases can be reproduced. Inoculations of tissue suspensions from infected rabbits failed to produce disease in guinea pigs, hamsters, rats, mice, chicken embryos, chinchillas, or piglets (15, 18). To perform successful experimental trials, the animals involved must be fully susceptible to the virus. Susceptibility depends both on the age of animals (which should be over 3 months), and on the absence of specific antibodies, even at low titres. VHD can be reproduced by using filtered and antibiotic-treated liver suspension, inoculated either by the intramuscular, intravenous or nasal route. When the disease is clinically evident, the symptoms and post-mortem lesions are similar to those described after natural infection. A rise in body temperature is registered between 18 and 24 hours post-inoculation (p.i.), followed, in 95% of cases, by death between 24 and 72 hours p.i. A few individuals may survive until the day 6 p.i. Animals that overcome the disease show only a transient hyperthermia, depression and anorexia, but present a striking seroconversion, that can be detected 5-6 days p.i. a) Haemagglutination test HA was the first test to be used for routine laboratory diagnosis of VHD (9). It should be performed with human type O red blood cells (RBCs), freshly collected, stored overnight in Alsever's solution, and washed in 0.85% PBS at pH 6.5 (range 6-7.2). HA is less evident, or nonexistent, when RBCs of other species are used. Washed RBCs are suspended at 0.75% in PBS; a two-fold dilution of the clarified supernatant of a 10% tissue homogenate of liver or spleen is incubated with an equal volume of washed RBCs in a sealed round-bottom microtitre plate at 4°C, 25°C or 37°C. After 1 hour (range from 20 minutes to 2 hours) of incubation, agglutination at an end-point dilution of >1/160 is considered positive. Lower titres should be regarded as suspicious, and should be checked using other methods. Positive results should be confirmed by ELISA, EM or immunostaining, particularly those showing a low titre. Around 10% of the positive samples in ELISA or EM give negative results in HA (HA false negative), mainly due to the presence of a proteolytic activity against the external part of the VHD virus capsid protein (4). Hare organs rarely give a significant titre when the same HA protocol employed for VHD virus is applied. To show the presence of HA activity in EBHS virus-positive organs, a modified procedure should be adopted: all steps are carried out at 4°C, the organ suspension is treated with an equal volume of chloroform, and RBCs are used at a pH not higher than 6.5 (4). Even using this method, only 50% of the samples usually give positive results. This is because of the degradation of the examined virus, which occurs much more frequently during EBHS virus infection (4). b) Electron microscopy Negative staining EM can be performed using the so called 'drop method'. A Formvar-carbon-coated grid is placed on a drop of organ suspension (prepared as described in Section A.1.a.), and left for 5 minutes. After removing excess fluid with the edge of a torn piece of filter paper, the grid is put to float on a drop of 2% sodium platinum (NaPt), pH 6.8, for 1.5 minutes. Excess stain is removed and the grid is finally observed at x 25,000. Due to the lower sensitivity of the drop method, it is advisable to ultracentrifuge the sample in order to concentrate the viral particles. The pellet obtained after ultracentrifugation (at least 100,000 g for 30 minutes or, alternatively, using Beckman Airfuge at 21 psi for 15 minutes) is resuspended in PBS or distilled water, put onto a grid for a few minutes, and then stained as described. VHD virions are visible as uncoated particles, 32-35 nm in diameter, presenting electron-dense cores (25-27 nm in diameter), delineated by a rim from which radiate ten short, regularly distributed peripheral projections. Degraded viral particles are identified by the complete loss of external portions, becoming perfectly hexagonal and smaller, with only the capsid rim visible (4). For diagnostic purposes and especially when other methods give doubtful results, the best method is an immuno-EM technique (IEM). This method uses either a rabbit hyperimmune anti-VHD virus serum or specific monoclonal antibodies (MAbs), which are incubated with an equal volume of the sample for 1 hour at 37°C before ultracentrifugation. The immunological reaction induces the clumping of the viral particles into aggregates that are quickly and easily identified by EM . EM examination allows the identification of EBHS virus in diagnostic samples. Valid results can be obtained when performing the IEM method using convalescent anti-EBHS virus serum. By using heterologous antisera, it is possible to differentiate between EBHS and RHD viruses. c) Enzyme-linked immunosorbent assay Virus detection by ELISA relies on a 'sandwich' technique that uses the same reagents, solutions, times and temperature that are used in the competitive ELISA for serology (see Section A.2.b.), except that in the competitive ELISA, the Tween 20 concentration is two-fold (0.1% v/v). The liver homogenate is diluted 1/10 and 1/50, in duplicate, directly into the wells of a microplate previously coated with VHD virus-positive rabbit serum, as described for serology. Similar dilutions are also made in wells previously coated with a VHD virus-negative rabbit serum. Each ELISA plate contains positive and negative liver homogenate as controls. After the first incubation, anti-VHD virus horseradish peroxidase (HRP)-conjugated rabbit IgG are added. The final step is the addition of chromogenic substrate (orthophenylene-diamine [OPD]) for the detection of the peroxidase activity. Absorbance is read at 492 nm. Positive samples are those showing a difference in absorbance >0.3, between the wells coated with VHD virus-positive serum and wells coated with the negative serum. Usually, at the dilution 1/50, positive samples taken from rabbits showing the classical acute form of VHD give an absorbance value >0.8, while the absorbance value of the negative sample, at the dilution 1/10, ranges from 0.1 to 0.25. For diagnosis of EBHS virus, it is possible to use this VHD virus-specific sandwich ELISA, but there is a risk of obtaining false-negative results. Therefore, the adoption of an EBHS virus-specific sandwich ELISA technique with high-titre positive anti-EBHS virus hare serum instead of rabbit serum is highly recommended (4). d) Immunostaining Tissue fixed in 10% buffered formalin and embedded in paraffin can be immunostained using an avidin-biotin complex (ABC)-peroxidase method. The sections are first deparaffinised in xylene and alcohol, counter-stained with haematoxylin for 1 minute and rinsed in tap water. They are then put in a methanol bath containing 3% H2O2 and washed in PBS three times for 5 minutes. To limit background interference due to nonspecific antibody binding, the samples are incubated with normal rabbit serum for 1 hour at room temperature prior to the addition of biotin. The slides are incubated overnight in a humid chamber at room temperature with biotinylated rabbit anti-VHD virus serum or MAbs, washed as before and incubated again for 30 minutes at 37°C with an ABC-peroxidase. The slides are washed three times. Amino-ethyl-carbazole is used as substrate. Finally, the slides are rinsed in tap water and mounted (16). Intense nuclear staining and diffuse cytoplasmic staining of necrotic cells in the liver, mainly in the periportal areas, are characteristic and specific. Positive staining of macrophages and Kupffer's cells is also observed, as well as hepatocellular reactions. Positive reactions can also be detected in the macrophages of the lungs, spleen and lymph nodes, and in renal mesangial cells (16). Tissue cryosections fixed in methanol can be directly immunostained by incubation for 1 hour with fluorescein-conjugated rabbit anti-VHD virus serum or MAbs. Specific fluorescence can be detected in the liver, spleen, and renal glomeruli. 2. Serological tests Infection by VHD virus can be diagnosed through detection of a specific antibody response. As the humoral response has great importance in protecting animals from VHD, determination of the specific antibody titre after vaccination or in convalescent animals is predictive of the ability of rabbits to resist VHD virus infection. It should be noted that some epidemiological data suggest the existence of an 'apathogenic' viral strain antigenically related to VHD virus. In fact, the presence of so called 'naturally acquired' antibodies, fully protective against virulent VHD virus infection, was shown in colonies where no previous outbreak had been reported or vaccination performed (15). Three basic techniques are applied for the serological diagnosis of VHD virus: haemagglu-ination inhibition (HI) (9), indirect ELISA and competitive ELISA (4). Each of these methods has advantages and disadvantages. With respect to the availability of reagents and technical complexity of carrying out the test, HI is the most convenient method, followed by the indirect ELISA and competitive ELISA, respectively. On the other hand, both ELISAs are more easily and quickly performed, particularly when a large number of samples are tested. The sensitivity and specificity of the competitive ELISA are markedly higher than those achievable with the other two methods (4). a) Haemagglutination inhibition Antigen: The antigen is prepared using infected rabbit liver collected freshly at death. The liver is homogenised in 10% (w/v) PBS, pH 6.4, and clarified by two consecutive low speed centrifugations (500 g for 20 minutes and 6,000 g for 30 minutes). The supernatant, drawn from the tube so as to avoid the superficial lipid layer, is filtered through a 0.22µm pore size mesh, titrated by HA, and divided into aliquots which are stored at –70°C. Serum samples: Before testing, sera are inactivated by incubation at 56°C for 30 minutes. The sera are then treated with 25% kaolin (serum final dilution: 1/10) at 25°C for 20 minutes and centrifuged. This is followed by another treatment, also at 25°C for 20 minutes, this time with 1/10 volume of approximately 50% packed human type O RBCs. These are freshly collected, stored overnight in Alsever's solution and washed in 0.85% PBS, pH 6.5. The sera are clarified by centrifugation. • Test procedure i) Dispense 0.05 ml of serum to the first well of a round-bottom microtitre plate and make double dilutions into wells 2-8 using PBS with 0.05% bovine serum albumin. ii) Add 0.025 ml of VHD virus antigen containing 8 HA units to each well and incubate the plate at 25°C for 30-60 minutes. iii) Add 0.025 ml of human type O RBCs at 2-3% concentration to each well and allow to settle at 25°C for 30-60 minutes. iv) Titrate the antigen with each test to ensure that 8 HA/0.025 ml were used, and include positive and negative serum controls. The serum titre is the end-point dilution showing inhibition of HA. The positive thres-hold of serum titres is correlated to the titre of the negative control sera; it usually is in the range 1/20-1/80. b) Competitive enzyme-linked immunosorbent assay Antigen: An international standard strain is not yet available; however, as only one serotype has been identified worldwide so far (indicative of the high antigenic stability of virulent VHD virus), reliable results can be obtained by different laboratories each using its own standard virus. The antigen can be prepared as described above for HI (Section A.2.a.), taking care to store it at –20°C in the presence of glycerol at 50% (v/v) to prevent freezing. If necessary, the virus can be inactivated before the addition of glycerol, using 0.1% for-maldheyde at 4°C for 24 hours. Antigen must be pretitrated in ELISA and then used as the limiting reagent: i.e. the dilution that corres-ponds to 60-70% of the plateau height (absor-bance value at 492 nm in the range 1.1-1.3). Anti-VHD virus serum: Specific rabbit polyclonal sera can be obtained in different ways. The two principal methods are as follows: i) Infection of seronegative rabbits with virulent VHD virus. At least ten rabbits are inoculated intramuscularly with a 1-ml dose of 1/10 (v/v) PBS dilution of the laboratory's own standard antigen prepared as for ELISA. (At least ten rabbits are needed in order to have a good chance of obtaining one or two convalescent sera.) Convalescent rabbit are killed 18-23 days post-infection. Using this method, it is possible to obtain a convalescent serum presenting a titre high enough to be used as an ELISA reagent. ii) Infection of seropositive rabbits showing a low titre of antibodies. In fact, if the concentration of anti-VHD virus anti-bodies is too high, viral replication will be largely hampered resulting in a poor immunological stimulus. Six seronegative rabbits are vaccinated, of which two are challenged with virulent VHD virus at 2, 4 and 6 days post-vaccination. In this way, sera with different titres are obtained from surviving rabbits. A serum is chosen which has a titre at least ten times higher than the average response to vaccination. Anti-VHD virus MAbs have been produced in several laboratories (4, 7, 14) and can be used instead of rabbit polyclonal sera. Purification of rabbit IgG and conjugation to HRP can be done following the standard protocol (17). The conjugated antibody is titrated in an ELISA sandwich assay in the presence and absence of VHD virus antigen (negative rabbit liver), and it is used at the highest dilution showing maximum (plateau height) absorbance (if the serum had a good anti-VHD virus titre, the value should range from 1/1,000 to 1/3,000). Control sera: Negative serum is taken from rabbits fully susceptible to VHD virus infection. Positive serum is either a convalescent serum diluted 1/100 in a negative serum or a serum taken from a vaccinated animal. • Test procedure i) Adsorb to an ELISA microplate of high adsorption capability (e.g. Nunc Maxisorb Immunoplate), diluting the rabbit anti-VHD virus serum 1/5,000 in 0.1 M carbonate-bicarbonate buffer, pH 9.6, at 4°C overnight. ii) Wash the plate three times for 3-5 minutes each time, in PBS, pH 7.4, with 0.05% Tween 80 (PBST). When plates are not immediately used, they can be stored, closed in a plastic bag, for at least 3 months at –20°C. iii) Distribute 0.025 ml/well of PBST with 1% of yeast extract (PBSTY) or 1% of bovine serum albumin (PBST-BSA) to all the wells needed on the plate (see below). Add 0.007 ml of the first serum sample to the first two wells (A1 and B1), 0.007 ml of the second serum to the second two wells (C1 and D1), and so on with the third (E1 and F1) and the fourth (G1 and H1) sera, thus completing the first column. If qualitative data (positive/ negative) are needed, repeat the operation in the second column with sera samples from 5 to 8, and in the third column with sera samples from 9 to 12, and so on. If the titre of the serum needs to be determined, the serum must be diluted further. Rock the plate and then use an eight-channel micropipette to transfer 0.007 ml from the wells in column 1 to the wells in column 2. This corresponds to a four-fold dilution of the sera. This last operation can be repeated once (titre 1/160), twice (titre 1/640), or four times (titre 1/10240). Complete each plate leaving 12 wells free for the control sera. Add 0.007 ml of positive sera to wells G6 and H6, and 0.007 ml of negative sera to wells G9 and H9, then dilute them twice (1/160). iv) Add 0.025 ml/well of antigen suspended in PBSTY to all the wells of the plate, at a dilution that is double the decided dilution, as described above in the antigen section (see the first part of this ELISA test description). v) Incubate the plate at 37°C on a rocking platform for 50 minutes. vi) Wash the plate as described (step ii). vii) Add 0.05 ml/well of rabbit IgG anti-VHD virus conjugated with HRP at the decided dilution, as described above in the 'anti-VHD virus serum' section (see the first part of this ELISA test description). viii) Incubate the plate at 37°C on a rocking platform for 50 minutes, and wash as described (step ii), adding a fourth wash of 3 minutes duration. ix) Use 0.05 ml/well of OPD as hydrogen donor under the following conditions: 0.5 mg/ml OPD in 0.1 M phosphate-citrate buffer, pH 5, + 0.02% H202. Stop the reaction after 5 minutes by addition of 0.05 ml/well of 1 M H2SO4. x) Read the plate on a spectrophotometer using the filter at 492 nm. The serum titre corresponds to the dilution giving an absorbance value equal to 50% (± 0.1) of the value of the negative serum at dilution 1/160 (reference value). The serum is classified as negative when the absorbance value of the first dilution (1/10) decreases by less than 20% of the reference value, while it is positive when the absorbance value decreases by 50% or more. A wide range of titres will be found, depending on the origin of the sample. Positive sera range from 1/2,000 to 1/16,000 in convalescent rabbits, from 1/80 to 1/1,000 in vaccinated rabbits and from 1/10 to 1/160 in 'nonpathogenic' infection. Knowledge of the origin of the sample allows a choice between testing one or more dilutions. Testing only the first dilution gives a positive or negative result. Testing all dilutions, up to the sixth one, will indicate the titre. Due to the significant antigenic differences existing between VHD and EBHS viruses (19), the serological techniques described above, which use VHD virus as antigen, are not recommended for the serological diagnosis of EBHS. However, a direct ELISA method could be employed for the detection of positive and negative EBHS virus hare sera; in fact, the adsorption of VHD virus onto the solid phase of an ELISA microplate leads to the exposure of cross-reactive antigenic determinants. B. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS In all the countries where VHD is endemic, indirect control of the disease is achieved by vaccination using the same type of vaccine, prepared by using clarified liver suspension of experimentally infected rabbits, subsequently inactivated and adjuvanted. The methods of inactivation (formaldehyde, BPL or other substances) and the adjuvants used (incomplete mineral oil or aluminium hydroxide) can vary according to the protocol adopted by the different manufacturers. The usual programme is to administer the inactivated vaccine twice with an interval of at least 2 weeks. Normally, a 1-ml dose is inoculated subcutaneously in the neck region. In those units where the anamnesis for VHD is negative, it is advisable to vaccinate only the breeding stock; the first injection should be done at 3 months of age. Annual revaccination is strongly recommended to ensure a good level of protection, although experimental data indicate that protection usually lasts for a long time (over 1 year) (2). The vaccination of meat animals is not necessary if disease has not occurred on the farm. Following an outbreak of VHD, even if strict hygienic and sanitary measures are adopted, including cleaning and disinfection, safe disposal of carcasses and an interval before repopulation, it is strongly recommended to also vaccinate meat animals at the age of 40 days, because the incidence of re-infection is very high. Only after several production cycles is it advisable to stop vaccination of meat animals. In order to verify the persistence of infective VHD inside the unit, a variable number of rabbits, starting with a small sentinel group, should not be vaccinated. Vaccinated animals quickly produce strong immunity against VHD virus infection, therefore vaccination is considered an effective post-exposure treatment and its primary use is in rabbitries after an outbreak of the disease has been diagnosed. Vaccine should be stored at 2-8°C and it should not be frozen, or exposed to bright light or high temperature. 1. Seed management a) Characteristics of the seed The source of seed virus for the production of inactivated tissue vaccines is infected liver homogenates obtained by serial passages in rabbits, that have been inoculated with a partially purified VHD viral suspension. The latter is obtained by centrifuging the 1/5 liver suspension (w/v) in PBS at 10,000 g for 20 minutes at 4°C. The resulting supernatant is treated with 8% (v/v) polyethylene glycol (PEG) 6,000 for 1 night at 4°C. The pellet is resuspended at a dilution of 1/10 in PBS, and subsequently centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant is ultra-centrifuged at 80,000 g for 2 hours at 4°C through a 20% cushion of sucrose. The pellet is resuspended in PBS (1/100 of the starting volume). This viral suspension is then charac-terised by negative-staining EM examination, determination of reactivity in ELISA, and capability of HA at room temperature with slow elution (HA titre against human group O erythrocytes higher than 1/1,280). Seed virus is titrated before use and should contain at least 105 LD50. It should be stored frozen (–70°C) or freeze-dried. b) Method of culture At the moment, VHD virus replication can be obtained exclusively in susceptible animals (2). The rabbits used for inoculation are selected from colonies shown to be susceptible to the disease by periodic serological testing. The animals (at least 4 months old) must be kept in strict quarantine upon arrival, located in separated areas and reared under satisfactory health conditions (see 'Laboratory animal facilities' in Chapter I.5). Seed virus propa-gation and production of vaccine batches rely on the same protocol of experimental infection, involving intramuscular injection of a dose of at least 100 LD50. c) Validation as a vaccine The seed virus used for vaccine production must be shown to be free from other viruses, bacteria, mycoplasma and fungi. Seed virus is controlled by direct inoculation into susceptible rabbits followed by evaluation of the clinical symptoms in the course of the experimental infection. Suitable seed virus should cause the death of 70-80% of the rabbits within 24-72 hours post-inoculation (p.i.), accompanied by the characteristic lesions of internal organs due to VHD. To validate the test, other adverse effects or different clinical manifestations, as well as the signs of intercurrent diseases, must be absent. 2. Method of manufacture Following inoculation of susceptible rabbits, the liver, spleen and sometimes kidney of those rabbits which die between 24 and 72 hours p.i. are col-lected. The organs are minced in the presence of sterile PBS, pH 7.2-7.4, in the proportion 1/10 (w/v), and the mixture is homogenised for 10 minutes in a blender operating in a refrigerated environment. Treatment with 2% chloroform is then performed (18 hours at 4°C), followed by centrifugation at 6,000 g for 1 hour at 4°C. The supernatant is collected by high pressure continuous pumping and is subsequently inactivated. The viral suspension is assayed by HA test and ELISA (see Section B.3.) and, once the number of HA units from the initial titration is known, more sterile PBS is added in sufficient volume to provide, after inactivation and adsorption on adjuvant, a concentration of 640-1,280 HA units/dose in the commercial product (2). Various agents have proved effective at abolishing viral infectivity. The most frequently used are formaldheyde and BPL, which can be used at different concentrations, temperatures, for a variable period of time and also in combination (2, 7). During inactivation, it is advisable to keep the fluid in continuous agitation. Either aluminium hydroxide, Freund's incomplete adjuvant or another oil emulsion are then incorporated in the vaccine as adjuvant. A preservative, thiomersal (merthiolate), is finally added at a dilution of 1/10,000 (v/v) before distribution into bottles. 3. In-process control Antigen content: The VHD titre is determined before inactivation by calculating the HA titre, which should be higher than 1/1,280, and the ELISA reactivity. Both values are again determined after inactivation and adsorption on adjuvant. The identity of VHD is confirmed by negative-staining EM. Sterility: The organs are tested for the presence of viable bacteria, viruses, fungi or mycoplasma according to the protocol used for testing master seed virus. PBS solution and aluminium hydroxide gel are sterilised by autoclaving; oil emulsion is sterilised by heating at 160°C for 1 hour. Inactivation: Before incorporation of the adjuvant, the inactivating agent and the inactivation process must be shown to inactivate the vaccine virus under the conditions of manufacture. Thus, a test is carried out on each batch of the bulk harvest as well as on the final product. Five rabbits are inoculated with a 2-ml dose of the suspension and five unvac-cinated rabbits are kept as controls. After 10 days, adequate inactivation and absence of undesirable side-effects are demonstrated by the absence of clinical signs and by similar weight increments in the two groups. At the end of the trials, the animals are slaughtered and liver extracts are tested by HA, ELISA and EM. 4. Batch control Of the following, sterility, safety and potency tests should be carried out on each batch of final vaccine; tests for duration of immunity should be carried out once using a typical batch of vaccine; and stability tests should be carried out on three batches. a) Sterility Each batch of vaccine must be tested for the presence of viable bacteria, viruses, fungi or mycoplasma according to the same protocol recommended for testing master seed virus. b) Safety Ten rabbits should be inoculated by the recommended routes with three times the vaccinal dose. The rabbits are observed for 3 weeks. No abnormal local or systemic reaction should develop. c) Potency Ten seronegative adult rabbits that are at least 4 months old, are vaccinated with one full dose of vaccine given by the recommended route. Two other groups of five animals each are vaccinated respectively with 1/4 and 1/16 of the full dose. A fourth group of ten unvaccinated rabbits is maintained as controls. All animals are challenged at 4 weeks post-vaccination by intramuscular inoculation of a dose of VHD virus containing at least 100 LD50 or presenting an HA titre higher than 1/2,560. No vaccinated rabbits should show signs of infection, while the mortality rate among control animals should be higher than 70%. The antibody response of each vaccinated animal is then determined with reference to titrated standard antisera; the mean antibody level should not be significantly less than the level recorded in the protection test performed using as vaccine the inactivated seed virus. d) Duration of immunity The data reported in the literature (2) indicate a long-term duration of immunity induced by a single vaccination (up to 15 months). However, it is advisable to carry out the following test: 20 rabbits vaccinated once are divided into four groups and are serologically tested at monthly intervals over a period of 1 year. Each group is inoculated with virulent VHD virus at 3, 6, 9 months and 1 year, respectively, post-vaccination (see Section B.4.c.). Challenge infection should produce increasing sero-conversion, which is directly related to the time that has elapsed since the vaccination. The absence of clinical signs and of mortality prove that the VHD virus has not multiplied. e) Stability Evidence should be provided to show that the vaccine passes the batch potency test at 3 months beyond the suggested shelf life. f) Preservatives A suitable preservative is normally required for vaccine in multidose containers (see Section B.2.). Its persistence throughout shelf life should be checked. g) Precautions (hazards) When oil emulsion vaccines are prepared, vaccinators should be warned against the risk and consequences of accidental self-injection. 5. Tests on the final product The tests for safety, potency and sterility of the final product must be performed following the same indications outlined above (Sections B.4.a.-c.), the only difference being in the use of the vaccine after bottling and packaging. Thus, it is important that these two last manufacturing steps are performed following standardised good manufacturing proce-dures. Safety, potency and sterility tests are done using doses of vaccine taken from randomly chosen multidose containers (20 or 100 doses). The number of vaccine bottles checked is statistically determined (p<0.01) on the basis of the consistency of each batch. REFERENCES 1. Allegranza G., Vanzetti T., Lavazza A., Capucci L. & Scicluna M.T. (1990). Malattia emorragica virale del coniglio: indagine epidemiologica nel Canton Ticino, Svizzera. Sel. Vet., 31, 847-858. 2. Arguello Villares J.L. (1991). Viral haemorrhagic disease of rabbits: vaccination and immune response. Rev. sci. tech. Off. int. 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Med., 10, 12-13. * * * -------------------------------------------------------------------------------- Other languages Table of content | »»