A review of Encephalitozoon cuniculi infection in the domestic rabbit

Emma Keeble BVSc Cert ZooMed MRCVS

Royal (Dick) School of Veterinary Studies, University of Edinburgh, Hospital for Small Animals, Easter Bush Veterinary Centre, Roslin, Midlothian, EH25 9RG

Encephalitozoon cuniculi is an emerging disease in pet rabbits, although the infection has been recognised for some time in farm and laboratory rabbits. More recently human infections with this parasite have been diagnosed. Whilst much is still unknown about the disease in pet rabbits, this article will attempt to clarify what we do know about the clinical signs, diagnosis, treatment and control of this parasitic infection and also summarise current research in this area.

What is it?

E.cuniculi is a tiny single celled organism called a protozoon, which has to live inside a host cell to survive (an obligate intracellular parasite). It belongs to the phylum Microspora. These are spore-forming parasites with a wide host distribution. Recent research has indicated that they may be more closely related to fungi than to protozoa (Wasson and Peper, 2000), which means that novel antifungal drugs may be of use in treating this disease. Microsporidia contain a unique internal structure called the polar filament, which plays an important role in infection of the host cell.

E.cuniculi primarily infects rabbits and is a significant cause of disease. It is also important to rabbit owners since it is potentially zoonotic (can spread to humans) and has been associated with infections in immunocompromised people. Infection has been diagnosed in rabbits in Europe, Africa, America and Australia. In the United Kingdom this parasite is frequently encountered in laboratory rabbits (Gannon, 1980), but is thought to be rare in the wild rabbit population (Cox and Ross, 1980). It has been diagnosed in pet rabbits (Harcourt-Brown and Holloway, 2003) and a recent study has shown a 52% incidence of exposure in normal healthy domestic rabbits (Keeble and Shaw, in press).

Background

The first mammalian infections were described in rabbits in the 1920's (Wright and Craighead, 1922) and the infection was recognised in humans in the 1950's, with increasing reports during the 1980's related to the AIDS pandemic. Microsporidia are now also thought to be a cause of diarrhoea in children and travellers (Hollister and others, 1991). E.cuniculi may also be found in other mammal species, including rodents, guinea pigs, foxes, monkeys, cats, dogs, sheep, goats, pigs, llamas and snakes (Shadduck and others, 1979, Pang and Shadduck, 1985, Illanes and others, 1993, Lobo and others, 2003).

Three strains of E.cuniculi have been identified (Didier and others, 1995). Strain I is found in rabbits ('rabbit strain') and has been isolated from humans in Switzerland (Deplazes and others, 1996). Strain II is found in rodents ('rodent strain') and has so far not been seen in people. Strain III occurs in dogs ('dog strain') and has been seen in immunocompromised humans in the Unites States (Didier and others, 2000) and one case in the United Kingdom (Hollister and others, 1996). In the human infections in Switzerland there was no history of recent contact with rabbits. E.cuniculi strains from humans can infect rabbits (Mathis and others, 1997) and have been shown to be the same as those found in rabbits. It is likely that E. cuniculi infections in rabbits pose a zoonotic risk to immunocompromised humans. Close contact between owners and susceptible pet species could lead to an increase in human exposure to the parasite. Infection in humans is thought to be from the environment following contamination with spores from other infected humans or animals.

Lifecycle and Transmission

Once a rabbit has the disease it passes infectious spores in its urine. Transmission to another rabbit occurs by eating these spores in urine contaminated food and water. The unborn kits may also be infected across the placenta during pregnancy. Inhalation of spores has been shown to cause infection, although these last two routes are thought to occur less frequently (Cox and others, 1979). Once the parasite has entered the rabbit's body it is carried in the blood circulation to target organs such as the liver, kidney and central nervous system (brain and spinal cord). It then infects the cells of these organs by ejecting the polar filament, which injects the spore into the cell. Once inside the host cell the parasite multiples causing the cell to enlarge and eventually rupture, which releases spores. These in turn infect nearby cells and pass into the blood to other organs. The life cycle lasts three to five weeks. Rupture of the host cell causes inflammation, primarily in the liver, kidney, brain and spinal cord and results in clinical signs in the rabbit.

Histological section of a brain from a rabbit which was infected with E.cuniculi (stained with H and E).

If the kits are infected in-utero, spores are able to cross into the lens of the eye. Later on in the rabbit's life the spores multiply and erupt causing cataracts and lens rupture resulting in inflammation within the eye (uveitis). This is a serious condition and is painful to the rabbit.

Spores can survive in the environment in extreme cold or heat (Kucerova-Pospisilova and others, 1999); but at average temperatures and in dry conditions their survival time is four weeks. They are easily killed by routine disinfectants (Waller, 1979).

Clinical signs

Recent research has indicated that over 50% of normal healthy pet rabbits have already been exposed to this parasite and are positive on blood sampling to look at antibody levels (Keeble and Shaw, in press). This implies that infection in rabbits is often not associated with any clinical signs. Instead the infection remains hidden and the rabbit can carry the parasite with the possibility of passing it on to in-contact rabbits. This makes control of the disease in a multi-rabbit household more difficult.

The rabbit-parasite relationship has yet to be fully understood. Establishment of infection and development of clinical signs are thought to be dependent on several factors; the parasite species and strain, the route of infection, the age of the rabbit at the time of the infection and the rabbit's immune status (Weber and others, 1994). If there is a balance between the rate of infection and the rabbit's immunity then clinical signs may not develop, as is commonly seen. If for some reason the rabbit becomes immunosuppressed following exposure to this parasite, clinical disease is likely to develop. In immunodeficient rabbits, or young and newborn animals with immature immune systems, disease is more likely. In rabbits with a healthy immune system clinically silent chronic infection develops (Didier and others, 2000). It is possible in these cases that clinical signs could develop at a later date associated with a concurrent infection, stress or immunosuppression. This is thought to be the case in the pet rabbit situation, where normal healthy seropositive animals can go on at a later date to develop clinical signs associated with infection.

A proportion of rabbits develop clinical signs associated with E.cuniculi infection. These include neurological disease (head tilt, unsteadiness, weakness of the hind legs, neck spasm and urinary incontinence), kidney disease and eye disease (Stiles and others, 1997). Other causes of head tilt and neurological disease in rabbits should be ruled out, such as spinal trauma, inherited congenital abnormalities ('splay leg'), abscesses, middle ear infections, listeria infection, toxoplasma infection and lead toxicity.

Rabbit with severe head tilt associated with E.cuniculi infection.

Diagnosis

A major problem with this disease is the difficulty diagnosing an active infection in live rabbits. Post mortem is often the only definite way of testing for this parasite, with typical lesions seen in stained slide sections of the kidney and/or brain on microscopic examination. Some texts state that the organism may not be found, even on post mortem examination, despite classic clinical signs and changes on histopathology (Percy and Barthold, 1993). This makes absolute diagnosis difficult and questions whether this parasite is truly involved in these cases.

More recently polymerase chain reaction (PCR) has been developed in humans for identification of E.cuniculi antigen in patient's urine or sputum (De Groote and others, 1995) and is in the process of being developed for commercial use on urine samples from pet rabbits (J.MacDonald - personal communication). This should greatly aid the diagnosis of this disease in pet rabbits, identifying infections at the acute phase. Biopsy of the kidney has been used to diagnose infection in humans (Aarons and others, 1994) and could be used in rabbits using endoscopy or laparotomy.

ELISA tests are used at present in the UK to record the presence of antibody (IgG) to E.cuniculi in the blood (Boot and others, 2000). Measurement of actual antibody levels is now possible, with rising levels of antibodies indicating an active infection.

Once the rabbit is infected antibody levels start to rise at about two weeks later and peak at 10 weeks. Spores may be present in the urine after 3-4 weeks (Cox and others, 1979). A wide variation in antibody response has been reported in rabbits, with some animals having persistently high levels of IgG for a long time and others becoming negative after eight weeks. The severity of the clinical signs does not appear to be related to how high antibody levels are in the blood (Kunstyr and others, 1986).

• A single positive antibody test in a healthy rabbit:

A single positive antibody result on its own in a healthy animal indicates that the rabbit has been exposed to the parasite, but does not indicate what will happen. There could be any one of three scenarios in this case; the rabbit could just have become infected (early infection), prior to the development of clinical signs, could be chronically infected, with no clinical signs or could have been previously infected and then recovered.

• A single positive antibody test in a rabbit showing signs associated with E.cuniculi infection:

If a single positive titre is recorded in a rabbit with neurological or kidney disease this suggests an active infection may be present, although other differential diagnoses would need to be ruled out (see earlier).

To differentiate between all the above scenarios and diagnose an active infection, two separate antibody tests should be carried out on the blood, at least 4 weeks apart and a rising antibody titre would need to be shown. It is important to note, however that antibody levels can persist for years in rabbits without any clinical signs (Scharmann and others, 1986) and as previously stated there is considerable individual variation in immune response in each case.

• A single negative antibody response in a healthy animal

This does not rule out early infection with the parasite since antibody levels rise two weeks after initial infection. This result in a healthy rabbit should be followed by a repeat test four weeks later to rule out the possibility of early infection prior to seroconversion, especially if there is a history of the rabbit already being in contact with another positive animal.

• A single negative antibody test in a rabbit showing signs associated with E.cuniculi infection:

In cases associated with clinical signs however, a single negative result rules out E.cuniculi as a cause of clinical disease.

Treatment and Prognosis

In affected rabbits the inflammation and release of spores results in clinical signs, particularly affecting the target organs (brain, spinal cord and kidney). Treatment is aimed at reducing inflammation, using anti-inflammatory drugs such as steroids and stopping spore formation. Most studies have been carried out in vitro, with little research in the live animal. Treatment has been described in rabbits using benzimidazole drugs (Franssen and others, 1995, Katiyar and Edlind, 1997). These are microtubule inhibitors and prevent extrusion of the polar filament, so stopping the host cell from becoming infected by the parasite. Albendazole is successfully used to treat infections in humans (Kotler and Orenstein, 1999). Adverse reactions have been reported in rabbits, possibly related to albendazole toxicity resulting in bone marrow disease, fever and liver failure (Mortiz, 2004). There is also evidence that the drug causes foetal abnormalities in rabbits (Kotler and Orenstein, 1999). The possible long term side effects of albendazole are not known. A study in laboratory rabbits used fenbendazole (another benzimidazole drug) which was safe and effective in reducing clinical signs in less advanced cases and importantly also prevented infection in exposed in-contact rabbits (Suter and others, 2001).

More recent in vitro studies have looked at drugs affecting chitin synthetase enzymes, since chitin is a basic component of the E.cuniculi spore. Polyoxin D and nikkomycin Z are known competitive inhibitors of these enzymes and have shown good in vitro activity against a human-derived strain of E.cuniculi (Sobottka and others, 2002). They could be useful in the future to treat cases in rabbits.

The response to treatment varies a lot and depends on how advanced the disease is. Chronic cases usually have neurological signs associated with severe cell damage and successful treatment may not be possible (Suter and others 2001). In acute cases urine dribbling may resolve with fenbendazole. There is a good prognosis with eye disease. These may be initially treated with steroid eye drops and benzimidazole drugs by mouth (Stiles and others 1997). In severe cases however the lens or eye itself may need to be removed.

Eye disease is a common problem associated with E.cuniculi infection of the lens in utero. Successful treatment is possible in these cases.

In suspect clinical cases the author typically prescribes a 28 day course of oral fenbendazole once daily, with covering broad spectrum antibiotics. If a rabbit presents with nervous system signs early on in the disease then steroids are used by subcutaneous injection every other day for 3 doses. Glucocorticoids, such as prednisolone should be used with care in rabbits, since they may also lower the white cell count and put the rabbit at risk of other infections. In one study 50% recovery rates were seen in rabbits with neurological signs treated with glucocorticoids (Ewringmann and Gobel, 1999). Severe neurological signs may require sedation of the rabbit using diazepam by injection.

Control and Prevention

Research has shown that this parasite is common in pet rabbits (Keeble and Shaw, in press) and so it may not be possible to prevent this infection in your pet. It has been shown that an E.cuniculi free breeding colony can be formed, but this process requires time and costs money. To achieve this healthy E.cuniculi negative young rabbits should be housed in complete isolation from other rabbits and in separate cages. Serum antibody levels are then tested every two weeks for two months and all positive animals, however weak the titre, are removed. Testing continues until all animals are negative for a month. These animals are then used to set up the breeding colony, but testing should continue on a monthly basis to confirm the disease free status (Cox and others, 1977).

If a single positive blood test occurs in an otherwise healthy rabbit, treatment may not be necessary. Antibody titres should be taken again 4 weeks later to see if there is an active infection. If this is diagnosed or clinical signs develop then treatment with fenbendazole should be started.

Ideally in a group situation all rabbits should be blood tested and any positive rabbits isolated and monitored. Antibody titres should be taken again 4 weeks later to see if there is an active infection. Antibody levels to the parasite rise before infective spores are shed in the urine, so in-contact rabbits can be screened to identify infected animals before the parasite is excreted, in an outbreak. These animals may be isolated and treatment with fenbendazole commenced.

If this is not practically feasible measures which may reduce the likelihood of infection are prophylactic fenbendazole in feed, regular disinfection of the rabbit's environment, including the food bowls and water containers, lifting the food bowls off the floor to reduce the risk of urine contamination, using water bottles rather than bowls again to reduce the risk of urine splash and housing animals in separate hutches, rather than tiered systems where urine may drop onto the lower cages. Wild rabbits and rodents could also act as a potential source of infection to pet rabbits and contact should be avoided.

If contact between rabbits not normally housed together is necessary, for example at rabbit shows and studs then stringent hygiene practices should be followed. Urine contact between rabbits at shows should be avoided.

Recent Research

A study at Edinburgh University in 2000 found that screening for this parasite was not routine in veterinary practice, due to the cost involved. Thirty percent of vets questioned at this time were not aware of this disease in pet rabbits (Beazley and Goodman 2001, unpublished data).

A further study, funded by the Rabbit Welfare Fund from 2001-2003, was carried out at the University of Edinburgh to determine the level of exposure in healthy domestic rabbits to E.cuniculi in the UK. This found that 52% of rabbits had already been exposed to the parasite. Veterinary surgeons were asked to fill in a questionnaire giving information about the rabbit's husbandry, diet, vaccination status, health status and any preventative medicine routines. None of these factors were found to make an animal more likely to be positive for E.cuniculi. Blood samples were also screened to see if there was any evidence of kidney disease or other changes in positive healthy animals. No significant difference was found in these values between positive and negative animals.

Areas for future research

• At present only IgG antibody is measured. If measurement of IgM antibody was also available it would be possible to differentiate between an active infection and a chronic infection. If both IgM and IgG antibodies are raised, this indicates very recent infection. Mildly raised IgG levels alone could indicate a chronic infection course.

• Hyperimmune responses to infection with E.cuniculi could be one of the causes of kidney disease in chronic cases (Didier and others, 2000). Clinically this may be associated with hypergammaglobinaemia on serum protein electrophoresis and could be used diagnostically in the future in suspect clinical cases. A study is at present underway to look into this.

• Detection of E.cuniculi by PCR analysis of rabbit urine would greatly aid diagnosis of this disease and may become available in the future.

• More research is needed to monitor healthy E.cuniculi positive rabbits and document how infection progresses in these cases.

There are an estimated 1.3-3 million pet rabbits in the UK: Over fifty percent of these are likely to have been exposed to E.cuniculi. Increased awareness of the parasite in rabbit keepers is important so that clinical cases may be identified and treated at an early stage. This is an important emerging disease in pet rabbits and should be considered wherever there are suspicious clinical signs.

Acknowledgements:

The author would like to thank the Rabbit Welfare Fund for funding the Edinburgh University study, Medlab MacDonald Laboratories Limited for processing of samples for the study, and all those owners and veterinary surgeons who supported the study by sending in samples.

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