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Diagnosis of RHD

The diagnosis of RHD is usually made after death. A presumptive diagnosis of RHD can be made when more than one rabbit in the household has died suddenly, especially if they were unvaccinated adults.

The classic picture of a rabbit that has died from RHD is in opisthotonos with a haemorrhagic nasal discharge, but this is not seen in every case. Post-mortem examination is essential to make a diagnosis of RHD and to rule out other causes of death. Histopathology can confirm or refute a diagnosis RHD and gives valuable information about other causes of sudden death. PCR testing confirms the presence of RHD viral antigen and determines the serotype.

Post-mortem examination at a pathology laboratory or in-house by a veterinary practitioner?

Ideally, a comprehensive post-mortem examination, including assessment of the central nervous system, performed by a veterinary pathologist should be performed on every rabbit that dies suddenly. Facilities for further testing, such as microbiology are close, and the procedure is carried out by a trained pathologist.

However, in-house post-mortem by the veterinarian in charge of a case has many benefits:

  • The post-mortem examination can be carried out soon after death, which is particularly important in rabbits. Ideally, the examination should be carried out within 6 hours of death because autolysis occurs rapidly. Autolysis interferes with the macroscopic and microscopic appearance of the organs even if the carcase was chilled in a refrigerator.
  • Causes of sudden death with obvious gross post-mortem findings can be seen. Examples include neoplasia, gastric or intestinal rupture, peritonitis, gastric dilation and intestinal obstruction, liver lobe torsion or ureteral obstruction and hydronephropathy.
  • Fresh tissue can be collected and fixed for histopathological examination or frozen for PCR testing.
  • There may be cost benefits.

Macroscopic examination

A diagnosis of RHD cannot be made from macroscopic examination alone. Macroscopic lesions of RHD may not be present in every case and the macroscopic appearance of the internal organs may not match the histopathological changes (Harcourt-Brown et al. 2020). 

Any macroscopic lesions of RHD that are present may be subtle and variable although some findings are suggestive of RHD. These include:

  • An enlarged pale, friable liver with a distinct lobular pattern.
  • An enlarged spleen.
  • A trachea filled with foamy exudate that may or may not be blood-stained
  • Free blood in the abdomen or retroperitoneal spaces.
  • Ecchymotic haemorrhages on the serosal surfaces or in the lungs.
  • Petechiae may be seen in the muscles, including the heart.
  • Icterus may be evident on the pinnae or sclera

Microscopic changes 

Death from RHD is due to fulminant liver failure or disseminated intravascular coagulopathy (DIC) and there is microscopic evidence of this. Acute hepatocellular necrosis with a characteristic pattern is present in rabbits that have died from RHD and there may be changes in other organs. Very occasionally, death is so rapid that light microscopy cannot demonstrate these changes. There is a time lag (minimum of 4 hours) between the death of a cell and the appearance of necrosis or apoptosis. Also very occasionally some animals exhibit only very small areas where the liver changes have become visible and these are so few and far between, that they may not appear on the sections examined.

The characteristic histopathological features of RHD include:

Liver:

  • Apoptosis
  • Variable lytic and coagulative hepatocellular necrosis with a periportal to multifocal to diffuse distribution.
  • Hepatocellular necrosis may be accompanied by variable congestion, haemorrhage and sinusoidal fibrin thrombi.
  • Inflammatory infiltrates of heterophils and fewer macrophages, are generally mild but more marked in less acute cases and in cases showing larger foci of confluent necrotic hepatocytes.

Extra-hepatic:

  • Fibrin thrombi may be seen, particularly in the glomeruli of kidneys and in the lungs.
  • Haemorrhages may be seen in various organs including the heart.
  • Lymphocytolysis, deposition of fibrin and haemorrhages are sometimes evident in the spleen.

Other causes of hepatocellular necrosis

There are other conditions that can cause hepatocellular necrosis in rabbits.

Poisoning

Poisoning is a concern for many owners of rabbits that die suddenly, especially if there are multiple deaths in the same household. There are no plants that cause acute hepatic necrosis in rabbits. Aflatoxins, copper toxicity and some compounds e.g. norepinephrine can cause liver necrosis, but ingestion of these compounds is unlikely in rabbits kept as pets and death is not sudden. Histopathologically, the type of hepatic necrosis is different from RHD. In the case of aflatoxicosis, there is bile duct hyperplasia and copper toxicity is characterised by the presence of small black or greenish granules in the necrotic hepatocytes

Heatstroke

The post-mortem findings in cases of heatstroke include pulmonary haemorrhage, necrosis of the liver and signs of disseminated intravascular coagulopathy, such as fibrin thrombi in small blood vessels. The histopathological pattern of hepatocellular necrosis and the circumstances surrounding the deaths are likely to suggest heatstroke as the diagnosis.

Sepsis

Sepsis is a cause of sudden death in rabbits, but it is unlikely that multiple rabbits will be affected. Histopathologically, hepatocellular necrosis and disseminated coagulopathy may be present but the pattern of liver necrosis is different from RHD (Harcourt-Brown et al. 2020). Bacteria may be evident in the tissues.

Laboratory tests

Laboratory tests are necessary to demonstrate the presence of the virus or its antibodies and identify the viral strain. Possible tests include polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), haemagglutination (HA) tests, electron microscopy or immunohistochemistry (OIE 2019). In the UK and many other countries, PCR testing is the only laboratory test that is available to the veterinary practitioner.

PCR testing

PCR testing is available from several laboratories in the UK although samples may be sent abroad to be tested. Samples of liver, faeces, intestinal contents, blood or an oral or rectal swab may be submitted for PCR testing against RHDV1 and RHDV2 but liver samples are the most reliable. The liver is the organ with the highest quantity of viral RNA. False-negative results are more likely from other samples. False-negative results are possible, but unusual, from liver tissue collected from rabbits that have died from RHD (Harcourt-Brown et al. 2020)