VN October 2023

Vetnuus | October 2023 36 • Western blotting: used when HA or ELISA are inconclusive. • RT-PCR: ideal rapid diagnostic test for RHD because of its high sensitivity. This method is performed on organ specimens (optimally liver), urine, faeces and sera. Different sets of primers may be used for RT-PCR, of which some allow the identification of all RHDV viruses. Other protocols were set up employing specific primers for the diagnosis of RHDV2. Similar RT-PCR methods have been used to identify the nonpathogenic RCV and EBHSV. RT-PCR is not strictly necessary for routine diagnosis, but it is more sensitive (105-fold more sensitive than ELISA), convenient and rapid than other tests. Considering its high sensitivity and the complicated epidemiological picture of rabbit caliciviruses, PCR results require a careful interpretation. A best choice is real-time PCR that, allowing a viral quantification (in a 10% liver homogenate there are between 106 and 109 copies of genome per ml), permits an easy diagnosis and the identification of acute cases. • In-situ hybridisation. highly sensitive and can detect RHDV as early as 6–8 hours after infection, but this technique is mainly used in research. • Never grown in cell cultures. Rabbit inoculation remains the only way of isolating, propagating and titrating the infectivity of RHDV. Not a practical method for the routine diagnosis of RHD, and should be considered only when all other methods give inconclusive results.When this occurs, the rabbits involved must be fully susceptible to the virus, i.e. they should be over 2 months old and have no RHDV antibodies (see serological methods). Serological tests Characterisation and titration of specific antibodies arising from natural infection or from immunisation are performed using the haemagglutination inhibition test or indirect or competitive ELISAs. Antibodies may be detected experimentally 4–6 days post- inoculation. Humoral response has great importance in protecting animals from RHD. At least three basic techniques are applied for the serological diagnosis of RHDV: • Haemagglutination inhibition (HI). • Indirect ELISA (I-ELISA) • Competitive ELISA (C-ELISA) Each of these methods has advantages and disadvantages. With respect to the availability of reagents HI is the most convenient method, followed by the I-ELISA and C-ELISA, respectively. However, both ELISAs are quicker and easier than HI, particularly when a large number of samples are tested. The specificity of the C-ELISA is markedly higher than the other two methods. However, considering the antigenic difference existing between the “classical” RHDV/RHDVa strains and RHDV2, two different antibody responses following homologous vaccination or infection could be qualitatively and quantitatively detected using protocols employing specific sets of MAbs recognising the respective viruses. Indirect ELISA (RHDV directly adsorbed onto the solid phase of the plate) is the test of choice for detecting cross-reactive lagovirus antibodies induced in rabbit by non-pathogenic calicivirus and as well in hares by EBHSV. The isotype-specific ELISAs (detecting IgM, IgA and IgG) have been very useful for epidemiological studies both in commercial rabbits and wild populations. Similarly to C-ELISA, two sets of anti-isotype- specific ELISAs were developed and can be used to determine specific Ig response to RHDV/RHDVa and RHDV2, respectively. For more detailed information regarding laboratory diagnostic methodologies please refer to Chapter 2.6.2 Rabbit haemorrhagic disease in the latest edition of the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals under the heading“Diagnostic Techniques”. PREVENTION AND CONTROL Sanitary prophylaxis • In uninfected countries prevention of introduction is the optimal control method. Restrictions are placed on the importation of rabbits, meat and angora wool from endemic areas. • In an outbreak, strict quarantine is necessary. • In those regions where wild rabbits belong to non-susceptible species (i.e. Sylvilagus spp. and Romerolagus spp.), control through stamping out is possible. • RHDV is extremely contagious; it can be transmitted on fomites and by insects, birds and scavenging mammals. Eradication can therefore be accomplished by depopulation, disinfection, surveillance and quarantines. • Sentinel seronegative rabbits can be used on treated premises to monitor for persistence of viral circulation. • In regions where RHDV circulates in wild rabbits ( Oryctolagus cuniculus ), eradication is not feasible. Instead, this disease is controlled in domesticated rabbits with biosecurity measures including sanitation and disinfection, the maintenance of closed colonies, and vaccination. • Vaccination may be limited to breeding animals if RHD has not been reported on a farm, but all animals should be vaccinated if an outbreak has occurred. Even with strict sanitation and other control measures, the likelihood of reinfection is high after an outbreak, due to the possible persistence of the virus in the environment. • The level of cross protection induced by vaccination with RHDV/RHDVa vaccines against RHDV2 is poor and does not prevent infection and losses due to clinical disease. Therefore combined vaccination with both antigenic types or the use of a vaccine homologous to the RHDV strain identified during the epizootics or the outbreak are highly advisable. Medical prophylaxis • Immunity is solid followingnatural infection. However, because the virus is hardy in the environment and the disease becomes endemic in populations, it is probable that recovered animals are repeatedly exposed to the virus to re-boost immunity. • Inendemic areaswherecontrol isdesirable, avaccineconsisting of clarified liver suspension that has been inactivated and adjuvanted is used. This inactivated vaccine is administered initially twice at a 2-week interval, and then annually. In many countries, different types of vaccines, including either RHDV or RHDVa, are commercially available and commonly used. Vaccines specific for the “new” RHDV2 have been registered in Technical I Article