Encephalitozoon cuniculi in Rabbits: The Mystery Disease AAVAC-UEP 2010 Frances Harcourt-Brown, RCVS Recognised Specialist in Rabbit Medicine and Surgery Bilton Harrogate, North Yorkshire, UK 21561176 Encephalitozoonosis is an enigmatic disease of rabbits, common but difficult to diagnose despite the vast amount of published literature. Künzel and Joachim (2010) give an excellent review of the current literature. Despite many studies and surveys, the significance of Encephalitozoon cuniculi as a pathogen of rabbits remains unclear and it is a difficult disease to diagnose with certainty, especially in the live animal. This presentation highlights the problems that E. cuniculi poses for veterinarians that treat rabbits. The Organism Encephalitozoon cuniculi is a single-cell, microsporidial organism. Microsporidia are a group of organisms that lack mitochondria. In some papers they are classified as protozoa although they have features that suggest they are related to fungi (Didier et al., 2000). Some microsporidia, including E. cuniculi, have the potential to cause disease in people (Deplazes et al., 1996). In humans, E. cuniculi can cause diarrhoea, rhinosinusitis, keratoconjunctivitis, nephritis or hepatitis, but only sporadic cases in immunocompromised people have been reported. No direct link between pet rabbits and human cases has yet been demonstrated, so the risk of humans contracting encephalitozoonosis from their pet rabbit is negligible. Owners with AIDS or who have had an organ transplant are at greater risk. To the author's knowledge, no case in a non-immunocompromised human has been reported, so although E. cuniculi might be considered a zoonosis, the risk is minimal. Strains of E. cuniculi Three strains of Encephalitozoon cuniculi have been identified (Didier et al., 1995). Strain I is associated with rabbits, Strain II with rodents and strain III with dogs. There is no morphological difference between the strains, which are differentiated by their DNA and it is not known whether the strains are species specific or if they can transfer freely between different hosts. In USA and Mexico isolates from humans have been strain III whereas strain I has been found in cases in Europe (Valencakova 2008). E. cuniculi in Other Species There are sporadic reports of Ecuniculica using disease in many species, including horses, foxes, dogs, rats, muskrats, hamsters, ground shrews, pigs, sheep, cats, mice, and a monkey (Valencakova 2008). Myocarditis, encephalitis, placentitis and abortion are diseases that have been linked with E. cuniculi infection in these species (Paterson-Kane et al., 2003; Snowden et al., 2009). In guinea pigs, encephalitis and nephritis have been reported (Illanes et al., 1993; Gannon 1980) and encephalitozoonosis has been associated with fading puppy syndrome (Snowden et al., 2009). The organism has been isolated from pigeons, cockatiels and chickens (Reetz 2003; Kasicková et al., 2007; Bart et al., 2008) and antibodies have been found in cattle and goats (Cisláková et al., 2001). The distribution of E. cuniculi appears to be worldwide with reports of the disease in USA, UK, South America, Italy, Spain, Japan, Israel, Turkey, Australia, Slovakia, Austria and Germany. Life Cycle of E. cuniculi E. cuniculi spores are oval and measure approximately 2.5 x 1.5µm. Within the spore, there is a coiled polar filament, which can be extruded and anchored to a neighbouring cell. After penetration of the cell wall, sporoplasm and the spore nucleus are transferred through the polar tube into a vacuole that forms
within the host cell. This is phase 1 of the life cycle (Schottelius et al., 2000). In phase 2, the sporoplasm divides in the host cell to form meronts that convert to sporonts, which are chain like structures. In phase 3, the sporonts subdivide into sporoblasts that transform into spores. This multiplication takes place within the vacuole until it becomes so distended that it forms a bubble-like swelling in the host cell, which eventually ruptures to release the spores. These can invade neighbouring cells. If kidney tubule cells are the host, cell rupture releases spores into the urine. Cell rupture is associated with an inflammatory response and the development of granulomatous lesions. Life Cycle of E. cuniculi.
Spread of Infection in Rabbits E. cuniculi infection in rabbits may be verticalor horizontal. Vertical transmission has been confirmed by demonstrating E.cuniculi DNA in placentas and foetuses of seropositive dams (Baneux and Pognan 2003). Horizontal spread of infection can be caused by ingestion of food contaminated with infected urine. The spores can survive for long periods in the environment and places that house many rabbits, such as rescue centres and pet shops, could easily harbour spores, although common disinfectants are effective in killing them. After ingestion, the spores invade the intestinal epithelium and gut-associated lymphoid tissue before spreading through the blood stream, either as free spores or in infected monocytes, to other organs including the kidney, brain, liver and heart. The most common sites are the brain and kidney and in infected animals spores may be seen in these tissues, usually in association with granulomatous inflammation. Lesions in the brain are most commonly seen in the cerebrum (Csokai et al., 2009). Clinical Diseases Associated with Encephalitozoon cuniculi Neurological Disease Neurological disorders are common in pet rabbits, especially vestibular disease and ataxia. More severe problems, such as seizures or even sudden death have been attributed to E. cuniculi, while, at the other end of the scale, encephalitozoonosis may be blamed for vague neurological signs, such as unresponsiveness, swaying or nodding. Neurological examination in rabbits is not easy and definitive diagnosis of E. cuniculi as a cause of neurological problems is impossible to prove, even after death. In a recent study of the histopathological changes in the brains of 118 rabbits that had exhibited neurological signs, although E. cuniculi was present in many of the cases, many rabbits had concurrent problems that could have contributed to neurological signs, such as lymphoma, otitis media, aspergillosis toxicity, hepatic encephalopathy, hypocalcaemia, hypoglycaemia, endotoxaemia, traumatic haemorrhage, HSV (herpes simplex virus) infection, cardiac disease and other structural brain problems (Gruber et al., 2009). It is interesting to note that in this study, none of the rabbits had toxoplasmosis lesions and middle ear infection was only confirmed in only four cases. Two of these had concurrent E. cuniculi infection. Vestibular disease is the neurological sign that is most commonly associated with E. cuniculi and is often attributed to acute cell rupture and inflammation (Feaga 1997). Vestibular disease is usually sudden in onset and often follows a stressful event in the rabbit's life. Cerebral disease. Even though vestibular disease appears to be the most common clinical manifestation of encephalitozoonosis, a comprehensive post-mortem study of 71 pet rabbits has shown that spores and inflammatory lesions are more commonly found in the cerebrum than the vestibular nuclei (Csokai et al., 2009). In this study, lesions were found in cerebrum of 98%ofthe rabbits with confirmed E. cuniculi infection, many of which were asymptomatic. The severity of the brain lesions did not correlate with the severity of the clinical signs. Although this finding could cast doubt on the significance of E. cuniculi as a pathogen, it does not rule it out as a cause of neurological disease. Altered mental status is associated with cerebral disease and is difficult to assess in rabbits. Altered mental status has been classified as: Bright and alert (normal) Obtunded: Conscious but inactive, unresponsive to the environment, tends to sleep when undisturbed
Stuporous: Sleeps when undisturbed, will not arouse to innocuous stimuli such as noise but will awaken with an apparently painful stimulus Comatose (Vernau et al., 2007) Many pet rabbits could fall into the 'obtunded' category and be considered to be very tame and well behaved by their owners. Ataxia, with or without bladder atony and urinary incontinence, is another clinical sign that appears to be linked with E. cuniculi infection. Anecdotally, treatment for encephalitozoonosis often brings about a temporary, or occasionally a permanent, improvement in these cases. However, histopathological studies rarely confirm this link. Trauma and degenerative disc disease are major differential diagnoses for the ataxia and the list is even longer for urinary incontinence. Renal Disease Renal disease is a feature of E. cuniculi infection and results in characteristic scarring of the kidneys. This is a common post mortem finding, and is an easy and cheap way to confirm infection with E. cuniculi, although the rabbits have shown no obvious signs of encephalitozoonosis or renal disease during life. Acute renal failure due to encephalitozoonosis appears to be rare. No reported cases could be found in the literature although the author has encountered a few cases. Chronic renal failure is a common and easily overlooked disease of older rabbits. Affected animals cannot vomit like dogs and cats with advanced renal disease do. Rabbits with chronic renal failure also often continue to eat well, in contrast with dogs and cats. Weight loss is the main clinical feature. Polydypsia/ polyuria may be evident in some cases. Marked elevations in urea and creatinine only occur late in the course of the disease. Hypercalcaemia is often present in rabbits with kidney disease as calcium excretion is impaired. It may be associated with soft tissue calcification and osteosclerosis, especially in blood phosphorus levels are also elevated. Nephrolithiasis is common in rabbits. Affected rabbits are almost invariably seropositive for E. cuniculi (Harcourt-Brown 2007) although spores are rarely found, perhaps because the clinical signs are due to longstanding tissue damage rather than acute inflammation. In the study by Csokai et al. (2009) 46/48 rabbits with spores in the brain also had interstitial nephritis and the rabbits with severe brain lesions also had severe kidney lesions. Myocardial lesions occur in pet rabbits (Csokai et al., 2009) and are often inflammatory and focal. Myocardial fibrosis is a frequent post-mortem finding (Harcourt-Brown, unpublished data) in both rabbits with no sign of heart failure and in those with cardiac signs or that have unexpectedly died. It is easy to speculate that E. cuniculi might be the cause of these lesions. Cataracts, hypopyonor uveitis can be caused by E.cuniculi and rabbits with these signs are invariably seropositive (Ewringmann, Göbel 1999; Felche, Sigler 2002; Donnelly 2003; Giordano 2005). Ocular lesions are reputedly vertically transmitted when the lens is predeliction site for the parasite in the foetus. After birth, the lens ruptures, which initiates an inflammatory response. At this point, E. cuniculi organisms or their DNA are invariably found in the lens material, which confirms the diagnosis. This contrasts with renal disease, presumably because the clinical signs are associated with an acute inflammatory response before the parasite has been eliminated.
Diagnosis of E. cuniculi as a Cause of Disease A definitive diagnosis of encephalitozoonosis is difficult. Blood samples, radiology, MRI and CT scans can be useful to confirm or exclude other diseases and show exposure to E. cuniculi but never give an absolute diagnosis of encephalitozoonosis, which is only definitive if the organism can be shown to be present and causing disease. In the live rabbit, organisms have been detected in urine, tears or lens tissue harvested during phacoclastic emulsification (Giordano 2005) but this is only applicable to a few cases. Even after death, the diagnosis of encephalitozoonosis is often presumptive and based on presence of characteristic inflammatory lesions in tissues rather than the presence of the organism, although the absence of organisms on histopathological examination does not mean they are not there. It could mean that the histopathologist has looked in the wrong place. Brain histopathology is a timeconsuming process as several sections need to be examined and the tissue needs to be collected and properly fixed. In recent years, many surveys of encephalitozoonosis in pet rabbits have been published (Ewringmann, Göbel 1999; Harcourt-Brown, Holloway 2003; Igarashi et al., 2008; Künzel et al., 2008) and the usefulness of diagnostic tests such as PCR testing, examination of CSF and urine, serological titres, serum electrophoresis and special staining of post-mortem material have been evaluated (Jordan et al., 2006; Reusch et al., 2007; Jass et al., 2008; Csokai et al., 2009; Cray et al., 2009; Jeklova et al., 2010). The most significant findings of these studies, and the author's clinical experiences are summarised below: Although antibody titre levels might be useful, they are not reliable indicators of the presence of severity of the disease. In a comprehensive post-mortem study conducted by Csokai et al. (2009), there was a high correlation between seropositivity and presence of lesions in the body, but the actual titre levels did not correspond with severity of lesions or severity (or even presence) of clinical signs of encephalitozoonosis. The course of infection under natural conditions does not seem to follow a predictable pattern with regard to antibody production and inflammatory changes so laboratory studies into humoral response only give guidance to interpret serology results. Laboratory rabbits are usually exposed to a single dose of antigen rather than repeated doses as in pet rabbits. They are not kept for as many years as pet rabbits. In pet rabbits titre levels appear to fluctuate with time. A negative result is the most significant result of serological testing as it usually rules out encephalitozoonosis as a diagnosis but it is only significant in long standing, rather than acute, cases. A post-mortem study by Csokai et al. (2009) found spores and histopathological lesions in some seronegative rabbits in contact with a positive rabbit. The authors postulated that the rabbits developed lesions before the antibody response. Although, testing for both IgG and IgM antibodies gives more information about whether infection is active or not, it does not give a definitive diagnosis. Young rabbits with clinical signs are often seronegative but seroconvert within weeks, presumably due to interference by maternal immunity. Old rabbits that were seropositive in their youth can become seronegative with time (Harcourt-Brown, personal experience). Toxoplasma gondii infection should be considered as a differential diagnosis for neurological and ocular disorders in rabbits (Jeklova et al., 2010). Special stains are required to identify spores; ZN and AFT. These stains differentiate E. cuniculi spores from those of T. gondii.
Even in cases with severe histopathological lesions, only a few spores may be detected. Inflammatory lesions are often present without spores, especially in the kidney. Sometimes spores are found without inflammatory lesions. Standard PCR is not as reliable as nested PCR for detecting DNA. Brain tissue (and lens tissue in cases of phacoclastic uveitis) contain the highest concentration of spores and are the best organs for detecting organisms, especially by nested PCR. Examination of urine, either by PCR or direct examination for spores is unreliable because of intermittent excretion. Spores may be detected in the urine of symptomatic as well as asymptomatic rabbits. Any CSF changes (increased WBC count, increased protein) are non specific. Antibodies and spores have not been found, even in rabbits with lesions and organisms in other parts of the body. Blood parameters, such as SPE, may be indicative (but not diagnostic) of encephalitozoonosis. Encephalitozoonosis may flare up because of immunosuppressive effects of other diseases so concurrent disease is often present. Treatment of Encephalitozoonosis Several medications have been used to treat E. cuniculi although it is difficult to prove their efficacy. The subjective opinion of the owner of whether their rabbit 'seems better' may be the only indicator that is available to assess the success of treatment. Priorto 2001, albendazole was the treatment that was recommended because of its success in human patients. Now, in the UK, fenbendazole is used more than albendazole since the publication of a small study that described the eradication of E. cuniculi organisms in rabbits treated with fenbendazole for 28 days at a dose rate of 20 mg/kg/day (Suter et al., 2001). A shorter course of 9 days prior to infection appeared to be protective and this is now recommended by the manufacturers as a prophylactic measure. Time may tell if this is successful. There are anecdotal reports of bone marrow suppression in a small number of rabbits treated with benzidamazoles, but these claims of toxicity have not been substantiated by post mortem examination or publication. Toxicity studies suggest that benzidamazoles are very safe. The LD 50 is very high (>5000 mg/kg for fenbendazole) although some teratogenic effects have been recorded for albendazole, there are no published reports of bone marrow suppression. This does not mean that an idiosyncratic reaction could occur in some individuals. Other products that are used to treat E. cuniculi in rabbits include oxytetracycline and pyrimethamine, even though in vitro studies suggest that pyrimethamine is ineffective against E. cuniculi (Beauvais et al., 1994). Similar studies have found that toltrazuril is also ineffective (Franssen et al., 1995), which questions the anecdotal use of ponazuril as a remedy in USA. Some veterinarians recommend no treatment at all for E. cuniculi, especially for vestibular disease, because they believe the syndrome is idiopathic and self-limiting, as it is in elderly dogs. This could be the case. It is true that many cases of vestibular disease resolve with or without treatment and a few cases continue to deteriorate despite treatment. Without a definitive diagnosis and more therapeutic studies, it is difficult to make any real sense of the various treatment protocols. Corticosteroids are often used for acute neurological signs associated with E. cuniculi. They appear to be effective and are indicated to suppress the inflammatory response associated with cell rupture. The author uses a single dose of dexamethasone (0.5 2 mg/kg) for rabbits that have recently developed acute neurological signs. If further treatment is required, antiinflammatory, rather than immunosuppressive doses of 0.2 mg/kg dexamethasone are used and only for
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