Detection of Encephalitozoon Cuniculi-Infection (Strain II) by PCR in a Cat with Anterior Uveitis

Wien. Tierärztl. Mschr. - Vet. Med. Austria 97 (2010), 210 - 215

From the Institute of Parasitology1 and the Institute of Pathology and Forensic Veterinary Medicine2, Department of Patho- biology, University of Veterinary Medicine, Vienna, and the Veterinary Practice Dr. Maaß3

Detection of Encephalitozoon cuniculi-infection (strain II) by PCR in a cat with anterior uveitis

J. CSOKAI1, A. FUCHS-BAUMGARTINGER2, G. MAASS3 and A. JOACHIM1 received December 17, 2009 accepted for publication May 1, 2010

Keywords: cat, Encephalitozoon cuniculi, mouse strain, Schlüsselwörter: Katze, Encephalitozoon cuniculi, uveitis, cataract, PCR. Mäusestamm, Uveitis, Katarakt, PCR.

Summary Zusammenfassung A 4 and a half year old female neutered European Nachweis einer Encephalitozoon cuniculi-Infektion shorthair cat showed recurring bilateral anterior uveitis (Stamm II) mittels PCR bei einer Katze mit Uveitis with a cataract for 2 and a half years. Despite therapy with anterior local 1 % atropine, dexamethasone-oxytetracycline oint- Eine 4 ½ Jahre alte weibliche, kastrierte Katze der Ras- ment and systemic carprofen there was no clear improve- se Europäisch Kurzhaar zeigte seit 2 ½ Jahren beidseitige ment of the inflammation and new uveitis exacerbations rezidivierende Uveitis anterior und Katarakt. Trotz Therapie followed. Serological tests for antibodies against Toxoplas- mit einer 1 %igen Atropin-Augensalbe, einer Dexametha- ma gondii, FIP-feline corona virus, Feline leukemia virus son-Oxytetracyclin-Augensalbe und oraler Gabe von Car- and Feline immunodeficiency virus were negative. The profen gab es keine deutliche Verbesserung der Entzün- determination of antibodies against Encephalitozoon cuni- dung, und neuerliche Uveitisschübe traten immer wieder culi revealed a titre of 1:320 in the Indirect Immunofluores- auf. Serologische Tests auf Antikörper gegen Toxoplasma cence Test. Therapy with fenbendazole was initiated but gondii, FIP-Felines Coronavirus, Felines Leukämievirus finally an enucleation of the right eye had to be performed und Felines Immundefizienz-Virus verliefen negativ. Eine due to glaucoma and perforation of the cornea. In the lens, Bestimmung des Antikörpertiters gegen Encephalitozoon DNA of Encephalitozoon cuniculi (strain II) was detected by cuniculi mit einem Indirekten Immunfluoreszenz Test ergab PCR and DNA sequencing. The anatomical lens structure einen Titer von 1:320. Eine orale Therapie mit Fenbenda- was destroyed for the PCR and therefore no spores could be zol wurde gestartet. Aufgrund eines Glaukoms und einer found in pathohistological examination with special staining Hornhautperforation des rechten Auges wurde schließlich (acid-fast trichrome). Sporadic spores could be detected by eine Enukleation durchgeführt. In der Linse wurde mittels transmission electron microscopy. The presented case PCR und DNA-Sequenzierung der Mäusestamm (II) von report shows that E. cuniculi should be considered in cats Encephalitozoon cuniculi nachgewiesen. In der an- with suspected infectious uveitis and cataract. schließenden pathohistologischen Untersuchung konnten in der für die PCR aufbereiteten und damit in ihrer anato- Abbreviations: AFT = acid-fast trichrome; DNA = deoxyribonucle- mischen Struktur zerstörten Linse keine Sporen mittels ic acid; FIP = Feline infectious peritonitis; E. cuniculi = Encephali- Spezialfärbung (Acid Fast Trichrom) gefunden werden. Im tozoon cuniculi; EM = electron microscopy; FeLV = Feline Elektronenmikroskop waren vereinzelt Sporen nachweis- leukemia virus; HE = haematoxylin and eosin; ITS region = inter- bar. Der vorgestellte Fall zeigt, dass E. cuniculi bei Katzen nal transcribed spacer region; PCR = polymerase chain reaction; mit Verdacht einer infektiösen Uveitis und Katarakt in MU = million units; RNA = ribonucleic acid Betracht gezogen werden sollte.

Introduction described. Immunosuppressed animals as well as new- born carnivores and new-world monkeys have a higher risk Feline uveitis is a common disorder, represented in var- of an outbreak of infection. Clinical onsets of dogs occurred ious ocular signs. Uveitis in cats often has a subtle course, in South Africa and the United States of America (SHAD- leading to delayed diagnosis when alterations are already DUCK et al., 1978; BOTHA et al., 1979; SNOWDEN et al., advanced. It can have infectious, immune-mediated, trau- 2009). In Norway and Finland blue foxes also showed matic, neoplastic or idiopathic causes. Frequently no defin- symptoms (MATHIS et al., 1996; ÅKERSTEDT et al., itive cause can be found (MAGGS, 2009). 2002). Studies in Norway and Switzerland revealed no Encephalitozoon cuniculi is a microsporidian pathogen antibodies against E. cuniculi in dogs, and only one Swiss of worldwide distribution affecting primarily rabbits as cat was seropositive (DEPLAZES et al., 1996; ÅKERST- hosts. Other animals like rodents can become infected, but EDT, 2003). By contrast, in Slovakia the seroprevalence in normally do not show symptoms (CHALUPSKÝ et al., dogs were high (37.8 and 23.6 %, respectively) 1979). Clinical outbreaks in other animals are rarely (HALÁNOVÁ et al., 2003). However, the authors did not

210 Wien. Tierärztl. Mschr. - Vet. Med. Austria 97 (2010) describe the isolation of the parasite to confirm the results; per os once daily for 3 weeks, a knife point of L-Lysin pow- therefore these results should be interpreted with care. So der (L-Lysin®, Hecht-Pharma GmbH, Stinstedt, German) far, only 2 studies of naturally infected cats are published twice a day into the food, 9 mg/kg famciclovir (Famvir®, (RENSBURG and DU PLESSIS, 1971; BUYUKMIHCI et Novartis Pharma GmbH, Vienna, Austria) once a day per al., 1977), and pathogen determination in those cases was os and topical recombinant interferon omega (Virbagen carried out only to the genus level (Encephalitozoon spp.). Omega 10 MU®, Virbac S.A., Carros, France) 3 times a day Our case report describes a clinical manifestation of E. because of the corneal erosion which could be caused by cuniculi (strain II) in a cat. felines herpesvirus. Despite intensive therapy no significant improvement of Case report the uveitis was noticed and medication was finally discon- tinued after 5 months. Approximately 1 and a half years later a bilateral exacerbation of the anterior uveitis re- History and clinical appearance occurred, which was again treated with atropine ointment, A 4 and a half year old female neutered European dexamthasone-oxytetracycline ointment, carprofen and shorthair cat showed recurring bilateral anterior uveitis doxycycline. A little improvement was notable. with a cataract, which had been present for 2 and a half After 9 months the cat was presented to a veterinarian years. The cat was born on a farm and housed indoors since the age of 2 months. It was kept with a litter mate and in a private practice with bilateral total mature cataract an older cat. In the past there were 2 rabbits in the house- beside the anterior uveitis. In addition, vascularisation hold as well, which had no contact with the cats. One of between the iris and the anterior lens capsule was present these rabbits developed a head tilt. A determination of anti- in the region of the posterior synechiae in both eyes (Fig. bodies against E. cuniculi was not performed despite a 1a,b). The suspicion of an infection with E. cuniculi arose suspicion of encephalitozoonosis. The rabbit was finally because of the negative results in serological tests for oth- euthanized. er infectious diseases as well as the similarity with anoth- At the age of 2 years the cat was presented the first time er case where a cat, seropositive for E. cuniculi, showed an with eye problems at the Veterinary University in Vienna. anterior uveitis with keratitic precipitates. In both cats a The left eye showed a mildly swollen conjunctiva. The pupil vascularisation between the iris and the lens was remark- was miotic and the pupillary light reflex was complete. A able, where the lens was adhered with the iris. This vascu- swollen and reddened iris was presented. Keratitic precip- larisation was reminiscent of the beginning of a phaco- itates were visible on the ventral half of the corneal clastic uveitis like those found in rabbits due to E. cuniculi. endothelium and a fibrin clot was located in the anterior The cataract of the first cat appeared conspicuous and chamber. A total mature cataract was present. Therefore a unusual, a filiform opacity, which terminated in an enlarge- fundus examination was not possible. The intraocular pres- ment in the area of the lens capsule (Fig. 1c,d). In the pre- sure was 3 mmHg. The ophthalmic examination of the right sented cat the determination of antibodies against E. cuni- eye offered a mildly dilated pupil and a normal pupillary culi was also positive with a titer of 1:320. The litter mate light reflex. The menace response and the dazzle reflex showed seroconversion as well, while the older cat in the were normal. The lens showed an immature cortical and household was seronegative. The cat was treated with 20 ® nuclear cataract. At the retina areas of chorioretinitis were mg/kg fenbendazole (Panacur , Intervet, Vienna, Austria) found. The intraocular pressure measured 11 mmHg. For once daily per os in intervals of 2x10 days application, clarification of the cause serological test for Toxoplasma interrupted by a 10 day break, topical gentamicin ointment gondii, FIP-feline corona virus, Feline leukemia virus (Gentax®, Agepha, Vienna, Austria) twice daily, topical vita- (FeLV) and Feline immunodeficiency virus were per- min A-containing ointment (Oleovit, Fresenius Kabi Austria formed. All tests were negative. GmbH, Graz, Austria) once daily and 1.5 mg/kg pred- The cat was treated with topical atropine ointment nisolone (Aprednislon®, Merck, Vienna, Austria) once a day (Atropin POS 1 %®; Ursapharm Arzneimittel GmbH, Saar- per os. After 1 week prednisolone therapy was gradually brücken, Germany) once daily, topical dexamethasone- reduced and finally phased out. A corneal erosion on the oxytetracycline ointment (Corti Biciron®, S&K Pharma right eye of 6 mm diameter developed 12 days after the Schumann und Kohl GmbH, Perl, Germany) 3 times daily start of the fenbendazole treatment. Topical eye medication and 3 mg/kg carprofen (Rimadyl®, Pfizer Corporation Aus- (Solcoseryl, Solco Pharma Austria, Salzburg, Austria) was tria, Vienna, Austria) per os once daily. given to stimulate the corneal regeneration. 3 days later Endothelial precipitates and fibrin receded. Carprofen the intraocular pressure was increased to 30-40 mmHg. treatment was reduced to every second day and finally dis- Despite brinzolamid eye drops (AZOPT®, Alcon Pharma continued, while the application of dexamethasone- GmbH, Freiburg im Breisgau, Germany) 3 times daily, oxytetracycline ointment was continued. 3 weeks later the there was no improvement and finally a corneal perforation cat suffered a relapse with bilateral, anterior uveitis and developed. An enucleation of the right eye was performed bilateral endothelial precipitates in the cornea. The therapy and the eye was submitted to the Institute of Parasitology was restarted with carprofen. 2 weeks later a corneal and Zoology for PCR examination. oedema developed in the left eye and finally corneal erosion was present. The cornea was dabbed with iodine, and Further analyses a debridement was performed. The therapy included topi- Molecular methods cal oxytetracycline ointment (Oxytetracyclin®, Jenapharm The right eye-ball was slivered with a scalpel and lens GmbH & Co. KG, Jena, Germany) twice a day. In addition was removed and minced with a scalpel blade. The bulbus the cat was medicated with 7 mg/kg doxycycline and the remnants of the lens were stored at -20 °C. DNA (Vibramycin®, Pfizer Corporation Austria, Vienna, Austria) isolation was performed with a High Pure PCR Template

211 Wien. Tierärztl. Mschr. - Vet. Med. Austria 97 (2010)

Fig. 1: Alterations in the eyes of cats with encephalitozoonosis; (a, b): presented case; (c, d): previous case with anti-E. cuniculi serum antibodies which was not confirmed by PCR; (a) total mature cataract with posterior synechiae (arrow) of the right eye; (b) vascularisation between the iris and the anterior lens capsule (left eye); (c) filiform opacity of the lens, which terminated in an enlargement in the area of the lens capsule; (d) swollen and reddened iris; pupil margin is adhered with the lens.

Preparation Kit (Roche Diagnostics, Vienna, Austria) according to instructions of the kit manufacturer. A PCR of the 16S small subunit ribosomal RNA gene was performed to identify E. cuniculi. PCR (primers Ence_549_forward and Ence_549_reverse) was done according to ROSSI et al. (1998) with minor changes (CSOKAI et al., 2009). The PCR product was controlled by sequencing. A second PCR of the internal transcribed spacer (ITS) region was performed with the primer MSP3 and MSP4A (KATZWINKEL-WLADARSCH et al., 1996) for the determination of the genotype. The protocol was performed according to GUSCETTI et al. (2003) and the PCR product was sequenced afterwards for discovering the strain. For sequencing PCR-amplicons were obtained from the agarose gel using a QIAquick® Gel Extraction Kit (Qiagen, Vienna, Austria). The sequencing of the 2 products was Fig. 2: Transmission electron micrograph of lens tissue performed by AGOWA (Berlin, Germany). with an electron dense microsporidial spore; bar = 0.6 μm

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Histological examination Feline immunodeficiency virus (MAGGS, 2009). Frequent- For the histopathologic examination the bulbus and the ly no cause can be found. 2 studies evaluated cases of lens were thawed, fixed in 7 % buffered formaldehyd and feline uveitis and could determine the cause of disease in embedded in paraffin wax. 3 to 4 micrometer sections were 30 % or 67 %, respectively (DAVIDSON et al., 1991; PEIF- stained with haematoxylin and eosin (HE) and acid-fast FER and WILCOCK, 1991). The most common diagnoses trichrome (AFT). were Feline infectious peritonitis (FIP), FeLV-associated lymphosarcoma and traumata (DAVIDSON et al., 1991; Transmission electron microscopy PEIFFER and WILCOCK, 1991). Cataracts, glaucoma and For electron microscopy (EM) areas of the lens slides lens luxation are the most common sequelae (DAVIDSON were dissected out, rehydrated, fixed with osmium tetro- et al., 1991; ROZE, 1998). xide and embedded in Epon. Ultrathin sections were The presented cat as well as the first cat with the con- stained with uranyl acetate and lead citrate and examined spicuous and unusual cataract (a filiform opacity, which ter- with a Zeiss EN 900 transmission electron microscope. minated in an enlargement in the area of the lens capsule) had both a vascularisation between the iris and the lens Results which was reminiscent of the beginning of a phacoclastic uveitis like those found in rabbits due to E. cuniculi. Rabbits infected with E. cuniculi can also show a polar anterior The first PCR (primers Ence_549_forward and cataract or focal linear opacities on the anterior lens cap- Ence_549_reverse) yielded a DNA-fragment of the expect- sule at the beginning of a phacoclastic uveitis (ASHTON et ed size (549 bp). The sequenced amplicon was compared al., 1976; GIORDANO et al., 2005; WALDE et al., 2008). with other sequences (BLAST search in GenBank Vascularisation develops in cases with posterior synechiae. [www.ncbi.nlm.nih.gov/blast/Blast.cgi]) and showed a 99 % The occurrence of a white intraocular mass due to a lens homology with E. cuniculi small subunit ribosomal RNA rupture is remarkable in rabbits with phacoclastic uveitis gene (accession number: X98467). (STILES et al., 1997; FELCHLE and SIGLER, 2002; GIOR- The second sequence of the PCR with primers MSP3 DANO et al., 2005). This sign was not macroscopically and MSP4A showed a two 5`-GTTT-3` repeat in the inter- present in the cat. nal transcribed spacer region. Compared with the strains Histologically the presented cat showed a cataract and described by DIDIER et al. (1995) the mouse genotype (II) a non-suppurative uveitis anterior. Interestingly, the cat did could be confirmed. The obtained sequence was submitted not develop a lens capsule rupture leading to a pyogranu- to GenBank (accession number: GU213880). lomatous phacoclastic uveitis, which is the usual sequela Histological examination of the HE-stained eye was dif- to ocular E. cuniculi infection in rabbits (ASHTON et al., ficult because of artefacts due to freezing and destruction 1976; WOLFER et al., 1993). of tissue for PCR sampling. The oedematous cornea Also in humans microsporidia can have an ophthalmic showed a central ulcus and breaks in the Descemet’s manifestation. E. cuniculi infects the conjunctival and membrane demarcated by neutrophils and fibrin. Nearby corneal epithelium and occurs in immunocompromised the limbus endothelial aggregates of lymphocytes and patients (FRIEDBERG et al., 1990; CALI et al., 1991; MET- plasma cells were visible. Lymphocytes and plasma cells CALFE et al., 1992; KODJIKIAN et al., 2005). Stromal dominated the anterior uveitis. Remnants of the anterior keratitis caused by e.g. Vittaforma corneae (formerly Nose- surface of the lens capsule had remained in the zonula ma corneae), Trachipleistophora hominis, Microsporidium area after removal of the lens for PCR. These remnants spp. and Nosema spp. (not identical with E. cuniculi, for- showed plaques of fibrous tissue on the external surface, merly named Nosema cuniculi ) rarely occur in immuno- but lens epithelium and fibers were not evident. Separate competent patients (FONT et al., 2003; RAUZ et al., 2004). examination of the lens remnants showed signs of cataract These pathogens can lead to corneal ulcers and finally by focal fragmentation of lens fibers. Neither with HE- nor also to perforation (PINNOLIS et al., 1981). A perforating with AFT-stain spores consistent with microsporidia could ulcer was also found in the presented case. As in the be detected in lens or bulbus. cornea no microsporidial spores could be found it is more With the EM few mature spores (about 2.0 μm x 1.0 μm) likely that the corneal ulcer was a sequelae of the glauco- with a thick electron-lucid wall including an outer and inner ma and not a corneal manifestation of E. cuniculi. electron-dense membrane could be seen in the lens. Oth- Cats can be infected experimentally with E. cuniculi er developmental stages could not be detected. It was not (PANG and SHADDUCK, 1985), whereas natural infec- possible to further identify the internal structures. A more tions with Encephalitozoon are described rarely in the lit- exact identification of the microsporidia was therefore not erature. In only 2 cases the pathogen was determined by feasible (Fig. 2). light microscopy (haematoxylin and eosin, Giemsa and Gram stain) and electron microscopy and could only be Discussion identified to the genus Encephalitozoon (RENSBURG and DU PLESSIS, 1971; BUYUKMIHCI et al., 1977). In one In this work the case of a cat with an E. cuniculi (strain case report a 3 and a half year old cat showed keratocon- II) infection showing recurrent anterior uveitis and cataract junctivitis and uveitis and Encephalitozoon was found in is presented. Feline uveitis can have infectious, immune- the stromal cornea (BUYUKMIHCI et al., 1977). The sec- mediated, traumatic, neoplastic and idiopathic causes ond publication describes 3 littermate kittens with severe (MAGGS, 2009). Systemic causes often lead to bilateral spasms, twitching of muscles and depression. One of uveitis. Systemic infectious reasons include infections with these cats was necropsied and showed nonpurulent T. gondii, FIP-feline corona virus, Feline leukemia virus or meningoencephalitis and interstitial nephritis. Spores could

213 Wien. Tierärztl. Mschr. - Vet. Med. Austria 97 (2010) be found in several organs (RENSBURG and DU increasingly electron dense (WASSON and PEPER, PLESSIS, 1971). 2000). Therefore it was not possible to further identify the In other carnivores (dogs and blue foxes), especially internal structures of these spores, but these ultrastructur- puppies and young animals, E. cuniculi infection results in al features which were present were consistent with spores anorexia, failure to gain body mass, tremors, ataxia, pare- of microsporidia (WASSON and PEPER, 2000). sis, circling, convulsions, nephritis and death (SHADDUCK Further investigations will be required to determine the et al., 1978; BOTHA et al., 1979; ÅKERSTEDT et al., 2002; relevance of E. cuniculi-infections in cats and their clinical SNOWDEN et al., 2009). Minks can develop cataractous appearance. It is unclear how far an systemic anti- lenticular lesions and blindness. Neurological signs are microsporidial therapy can influence an ocular infection. rarely noted, perhaps due to the mild lesions in the nerv- Phacoemulsification is often performed in rabbits with pha- ous tissue (BJERKÅS, 1990). Maybe cats are also less coclastic uveitis (KÜNZEL et al., 2008) and can be utilised prone to a fatal course of the infection, in contrast to dogs in cats with recurring anterior uveitis as well. Moreover, E. and blue foxes. cuniculi is a zoonotic pathogen which can affect immuno- Due to a possible lack of awareness for an ocular mani- deficient humans (MATHIS et al., 2005). It should be point- festation of an infection with E. cuniculi this disease is ed out that cats could be a potential source for human probably underdiagnosed in cats. infections if they harbour infections of other organs includ- In rabbits with phacoclastic uveitis an infection can be ing the kidneys and excrete spores with the urine. detected by serological testing, histological examination of the eye (after enucleation) and by PCR (after enucleation Acknowledgements or phacoemulsification). Many rabbits are infected subclini- The authors want to thank Walpurga Wille-Piazzai and cally (KÜNZEL et al., 2008) and serological tests therefore Ildikó Barki from the Institute of Parasitology for their tech- cannot exclude other diseases. By contrast, seropreva- nical support and Christine Glatzer of the Institute of lences in cats appear to be generally low (DEPLAZES et Pathology for her excellent technical assistance. We would al., 1996), and together with a corresponding clinical pre- also like to thank Dr. Petra Benz from the Ophthalmology sentation encephalitozoonosis should be considered. How- Unit of the Veterinary University of Vienna for critical review ever more detailed seroprevalence studies are necessary. of the clinical descriptions in this manuscript. In addition to specific serum antibodies E. cuniculi DNA could be detected by PCR in affected tissue. For E. cuniculi References 3 strains (strain I = rabbit strain, strain II = mouse strain, strain III = dog strain) have been described, which can be ÅKERSTEDT, J. (2003): Serological investigation of canine encepha- differentiated by the number of 5`-GTTT-3` repeats in the litozoonosis in Norway. Parasitology Research 89, 49-52. internal transcribed spacer region of the ribosomal RNA ÅKERSTEDT, J., NORDSTOGA, K., MATHIS, A., SMEDS, E., gene (DIDIER et al., 1995). To our knowledge, this is the DEPLAZES, P. (2002): Fox encephalitozoonosis: isolation of first time that the strain II was identified in a cat. In other the agent from an outbreak in farmed blue foxes (Alopex lago- carnivores, strain III was found in dogs and strain II in blue pus) in Finland and some hitherto unreported pathologic foxes (SHADDUCK et al., 1978; ÅKERSTEDT et al., 2002). lesions. Journal of Veterinary Medicine Ser. B 49, 400-405. ASHTON, N., COOK, C., CLEGG, F. (1976): Encephalitozoonosis Strain II was isolated from mice and a rat as well (DIDIER (nosematosis) causing bilateral cataract in a rabbit. British et al., 1995; MÜLLER-DOBLIES et al., 2002). Therefore Journal of Ophthalmology 60, 618-631. rodents may be a reservoir for wild foxes (HERSTEINS- BJERKÅS, I. (1990): Brain and spinal cord lesions in encephalito- SON et al., 1993) and perhaps for cats as well. In rabbits zoonosis in mink. Acta Veterinaria Scandinavica 31, 423-432. with phacoclastic uveitis an intrauterine infection is BOTHA, W.S., DELLEN, A.F.van, STEWART, C.G. (1979): Canine assumed to have taken place because of the very thin or encephalitozoonosis in South Africa. Journal of South African absent lens capsule at this stage (ASHTON et al., 1976; Veterinary Medical Association 50, 135-144. WOLFER et al., 1993) and cats may also contract E. cuni- BUYUKMIHCI, N., BELLHORN, R.W., HUNZIKER, J., CLINTON, J. (1977): Encephalitozoon (Nosema) infection of the cornea in culi in utero. In the presented case the ocular manifestation a cat. Journal of the American Veterinary Medical Association and the seropositivity of the litter mate might indicate 171, 355-357. intrauterine infection, while a transmission from the rabbit CALI, A., MEISLER, D.M., LOWDER, C.Y., LEMBACH, R., to the cat is unlikely because usually strain I (“rabbit”) is AYERS, L., TAKVORIAN, P.M., RUTHERFORD, I., LONG- found in this host. WORTH, D.L., MCMAHON, J., BRYAN, R.T. (1991): Corneal In rabbits organisms can be detected histologically in microsporidioses: characterization and identification. Journal of the lens within the liquefied cortex in close approximation Protozoology 38, 215S-217S. CHALUPSKÝ, J., VÁVRA, J., BEDRNÍK, P. (1979): Encephalito- to the lens epithelium (WOLFER et al., 1993) or the lens zoonosis in laboratory animals-a serological survey. Folia Para- epithelial cells (GIORDANO et al., 2005). Due to the sitologica (Praha) 26, 1-8. destruction of the lens for PCR in the sample for histologi- CSOKAI, J., JOACHIM, A., GRUBER, A., TICHY, A., PAKOZDY, cal examination lens epithelium and presumably adjacent A., KÜNZEL, F. (2009): Diagnostic markers for encephalito- lens fibers were not present, and it can be assumed that zoonosis in pet rabbits. Veterinary Parasitology 163, 18-26. this is the reason why no spores could be found by special DAVIDSON, M.G., NASISSE, M.P., ENGLISH, R.V., WILCOCK, staining. B.P., JAMIESON, V.E. (1991): Feline anterior uveitis: a study of Damage of the tissue due to refreezing, inadequate fix- 53 cases. Journal of the American Animal Hospital Assiciation 27, 77-83. ation and handling of the wax embedded and stained tis- DEPLAZES, P., MATHIS, A., MÜLLER, C., WEBER, R. (1996): sue for electron microscopic examination yielded poor Molecular epidemiology of Encephalitozoon cuniculi and first electron microscopic results. Moreover, as the proliferative detection of Enterocytozoon bieneusi in faecal samples of pigs. forms differentiate to spores the cytoplasma becomes Journal of Eukaryotic Microbiology 43, 935.

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