VN October 2023

Oktober 2023 35 Technical I Article The highest morbidity and mortality rates are seen in adult rabbits from naïve populations. Young rabbits less than 6–8 weeks old are less likely to become ill or die. Rabbits 4 weeks old and younger are unaffected. The age-related resistance in very young rabbits is still poorly understood. Surviving rabbits develop immunity and become fully resistant to related strains of RHDV but not to RHDV2, for which immunity is only partially protective. The disease caused by RHDV2 has typical features different from those of “classical” RHD. The mortality rate is lower but highly variable (5–70%) with an average mortality of 20% in experimentally infected rabbits; death could occur in non-vaccinated fattening rabbits and in lactating rabbits from 15 days of age onwards and the course of the diseases is usually longer (3–5 days), withmore rabbits showing subacute- chronic signs and lesions. • Inwild rabbits, outbreaks can be seasonal. In some populations, they have been associated with the breeding season. • The morbidity and mortality rates vary among populations. In Europe, RHD has caused dramatic declines in wild rabbit populations in France, Portugal and Spain, but wild rabbits in the United Kingdom and some other Northern European countries have been less severely affected. Such different evolution is most probably related to the circulation and presence in wild rabbits of non-virulent RHDV-like strains, which may induce variable levels of cross protection within each population. Clinical diagnosis While the clinical evolution of the disease can be peracute, acute, subacute or chronic, clinical manifestations have been described mainly in the acute infection, as there are usually no clinical signs of disease in the peracute form, and the subacute form is characterised by similar but milder signs. The incubation period varies between 1 and 5 days depending on the type of causative agent, and 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, nervous signs (convulsion, ataxia, paralysis, opisthotonos, paddling), groans and cries, respiratory signs (dyspnoea, frothy and bloody nasal discharge), and cyanosis of mucous membranes. During an outbreak, a certain number of rabbits (5–10% in the case of RHDV/RHDVa and significantly more if the infection is caused by RHDV2) 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 1–2 weeks later, probably due to liver dysfunction, but some rabbits survive showing very high seroconversion. Lesions Due to the rapid course of this disease, the animals are usually found in good condition after death. Gross pathological lesions are variable and may be subtle and include circulatory and degenerative disorders. Liver necrosis and splenomegaly are the primary lesions. The liver appears yellowish-brown in colour, brittle and degenerated, with a marked lobular pattern. The tracheal mucosa is hyperaemic, containing abundant frothy fluid, and the lungs are oedematous and congested. The spleen is engorged, with rounded edges and enlarged (splenomegaly). The presence of clotted blood in blood vessels is due to disseminated intravascular coagulation (DIC). Such massive coagulopathy is usually the cause of haemorrhages in a variety of organs and sudden death. In subacute and chronic disease, an icteric discoloration of the ears, conjunctiva and subcutis is clearly evident. Differential diagnosis • septicaemic pasteurellosis • poisoning • heat exhaustion • other causes of severe septicaemia with secondary DIC Laboratory diagnosis Samples • Fresh liver, spleen, and blood • Formalin-fixed samples of liver, spleen, lung, kidney and other organs The liver contains the highest viral titre (from 103 LD50 [50% lethal dose] to 106.5 LD50/ml of 10% homogenate) in acute or peracute disease and is the organ of choice for viral identification of both RHDV and EBHSV. Serum and spleen may also contain high levels of virus. In rabbits with chronic or subacute disease, RHDV may be easier to find in the spleen than the liver. RT-PCR can detect viral RNA in many organs, urine, faeces or serum. Serum should be collected for serology. Procedures Identification of the agent • Haemagglutination (HA) test: first test used for routine laboratory diagnosis of RHD, but it is less sensitive and specific than other assays and requires human typeO redblood cells – nowreplaced by virus detection enzyme-linked immunosorbent assay (ELISA). The HA test is performed on 10% tissue homogenate of liver or spleen. HAmay give false negative results: a) in the chronic form of RHD, i.e. in rabbits that die from 4 to 7 days post-infection onwards; b) with some isolates that have lost the ability to haemogglutinate. • Electron microscopy: negative-staining EM, immuno-EM, and immunogold EM. For diagnostic purposes and when other methods give doubtful results, the best EM method is an immuno-EM technique (IEM) using monoclonal antibodies (MAbs) or specific hyperimmune sera. This induces clumping of viral particles into aggregates that are quickly and easily identified by EM. • Virus detection ELISA: performed on 10% liver homogenate. An MAb-based ELISA developed at theOIE Reference Laboratory for RHD enables the subtyping of RHDV isolates. The MAb panel has been improved by including specific MAbs produced towards RHDV2. • Immunostaining: tissues fixed in 10% buffered formalin and embedded in paraffin can be immunostained using an avidin– biotin complex (ABC) peroxidise method with intense staining mainly in periportal areas of the liver, macrophages of the lungs, spleen and lymph nodes, and mesangial cells of the kidney. Tissue cryosections of liver, spleen and kidney fixed in methanol can also be directly immunostained for specific fluorescence. >>> 36