Richter, M; Matheis, F; Gönczi, E; Aeby, S; Spiess, B M; Greub, G (2010). Parachlamydia acanthamoebae in domestic cats with and without corneal disease. Veterinary Ophthalmology, 13(4):235-237. Postprint available at: http://www.zora.uzh.ch University of Zurich Posted at the Zurich Open Repository and Archive, University of Zurich. Zurich Open Repository and Archive http://www.zora.uzh.ch Originally published at: Veterinary Ophthalmology 2010, 13(4):235-237.

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Year: 2010

Parachlamydia acanthamoebae in domestic cats with and without corneal disease

Richter, M; Matheis, F; Gönczi, E; Aeby, S; Spiess, B M; Greub, G

Richter, M; Matheis, F; Gönczi, E; Aeby, S; Spiess, B M; Greub, G (2010). Parachlamydia acanthamoebae in domestic cats with and without corneal disease. Veterinary Ophthalmology, 13(4):235-237. Postprint available at: http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch

Originally published at: Veterinary Ophthalmology 2010, 13(4):235-237. Parachlamydia acanthamoebae in domestic cats with and without corneal disease

Marianne Richter1, Franziska Matheis1, Enikö Gönczi2, Sébastien Aeby3, Bernhard Spiess1, Gilbert Greub3,*

1 Section of Ophthalmology, Equine Department, Vetsuisse Faculty, University of Zurich, Switzerland 2 Clinical Laboratory, Department of Farm Animals, Vetsuisse Faculty, University of Zurich, Switzerland 3 Centre of Research on Intracellular (CRIB), Institute of Microbiology, University Hospital Centre and University of Lausanne, Switzerland [email protected] * Corresponding author:

Gilbert Greub Centre for Research on Intracellular Bacteria Institute of Microbiology University of Lausanne Bugnon 48 1011 Lausanne SWITZERLAND Phone: (00) 41 21 314 49 79 Fax: (00) 41 21 314 40 60 Email: [email protected]

Keywords : Ocular infection, , Intracellular bacteria, cats, keratitis, emerging pathogens

Word count = 1137

ABSTRACT Corneal samples of cats with and without corneal diseases were screened with a pan- PCR and specific PCRs for Parachlamydia, Protochlamydia, felis, and feline herpesviruses (FHV-1). Several corneal samples tested positive for Parachlamydia and related Chlamydiales, indicating cat exposure to these intracellular bacteria.

Introduction. Chlamydophila felis ( family) is the only member of the Chlamydiales order, whose pathogenic role in cats has been established. Novel members of the Chlamydiales order have been recently detected in samples of humans and a number of animal species with respiratory, urogenital, granulomatous and ocular diseases (1). These new members of the Chlamydiales order have been assigned to different families such as the , the , and the Waddliaceae families (2). All these new bacterial species exhibit the same developmental stage than the members of the Chlamydiaceae family and share 80 to 90% 16S rRNA sequence similarity with the Chlamydiaceae (2). Parachlamydiaceae, such as Parachlamydia acanthamoebae and Protochlamydia neagleriophila have been associated with pneumonia in humans (2, 3). Parachlamydia acanthamoebae and other members of the Parachlamydiaceae family have also been detected in ocular samples from humans (4, 5) and animals (6-8) with conjunctivitis and/or keratitis. The presumed pathogenic potential of Parachlamydia acanthamoebae may be mediated by its ability to infect and multiply within human macrophages (9). Acanthamoeba are natural protozoan hosts for Parachlamydiaceae (10) and for other prokaryotes (11), and are causative organisms of granulomatous amoebic encephalitis and amoebic keratitis in humans (11). Although feline herpesvirus type 1 (FHV-1) is considered the main etiologic agent for corneal diseases in cats (12), an etiologic diagnosis is infrequently made in chronic corneal diseases (13), suggesting that other pathogens may be involved. No data is are currently available about the route of transmission, infectivity and pathogenicity of Parachlamydiaceae in humans and animals. The role of animals and especially of pets as source of infection for humans is still unknown. The aim of this study was thus to investigate the presence of Parachlamydia acanthamoebae, Protochlamydia neagleriophila and Protochlamydia amoebophila in eyes of domestic cats with and without corneal disease by using PCRs specific for the Parachlamydia acanthamoebae species and for the Protochlamydia genus, respectively. These samples have also been tested for the presence of Chlamydophila felis by using a species specific PCR and for the presence of any other Chlamydiales using a pan-Chlamydiales PCR, i.e a PCR amplifying any members of the Chlamydiales order, including Chlamydiaceae (Chlamydophila felis) and Parachlamydiaceae (Parachlamydia acanthamoebae, Protochlamydia neagleriophila). Moreover, these samples have been screened for FHV- 1 and for the Acanthamoeba protist by using specific PCRs.

Material and methods and results. Among 63 cats (63 eyes) included in this study, 44 cats had outdoor access, 14 cats were strictly kept indoors, and the habitat of 5 cats was not recorded. Ocular examination prior to sampling was performed for 40 eyes of 40 cats that presented to the Ophthalmology unit of our veterinary hospital with keratitis and/or other corneal lesions. As controls, we studied 23 eyes from 23 cats without any ocular disease and euthanized for unrelated reasons in our veterinary hospital. Ocular signs were evaluated using a portable hand-held binocular biomicroscope (KOWA SL-14, Kowa, Tokyo, Japan). Fluorescein (Fluoreszein, Haag Streit, Koniz, Switzerland) staining was performed to visualize corneal erosions. Among the 40 eyes with keratitis and/or other corneal lesions, 7 eyes presented with active keratitis (vascularisation, cellular infiltrates) but intact epithelium, 9 eyes with corneal necrosis, 9 eyes with chronic corneal erosion, 11 eyes with mixed signs (erosion+necrosis, erosion+active keratitis, active keratitis+necrosis), and 4 eyes with eosinophilic keratitis. To confirm eosinophilic keratitis, cytologic examination was done from corneal samples of cats with clinical evidence of eosinophilic keratitis, i.e. corneal vascularization and superficial multifocal whitish cellular infiltrates. Cytology smears were air-dried, stained with Wright's stain and evaluated by certified pathologists. Corneal samples for PCRs were obtained by scraping of the corneal surface using a cytobrush (Cytobrush® plus, Medscand Medical®, Malmoe, Sweden) in 22/40 eyes with keratitis and in 22/23 normal eyes, by keratectomy in 8/40 eyes with keratitis and in 1/23 normal eyes or by both methods in 10/40 eyes with keratitis and/or other corneal lesions. Samples were put into a sterile tube and either processed immediately or stored at -20C until DNA extraction was performed. Nucleic acids were extracted from corneal swab samples or keratectomy specimens using the DNeasy Tissue Kit (Qiagen, Hombrechtikon, Switzerland). Each sample was mixed with 20l proteinase and 200l ATL-buffer and processed according to manufacturer’s instructions. One negative extraction control was tested in each extraction run. Contamination of reactions by PCR products was avoided by strict separation of working areas and the use of filter-plugged pipette tips. Distilled water negative controls and DNA positive control samples were included in each PCR. A Chlamydiales specific PCR was performed on all samples to screen for the presence of any member of the Chlamydiales order. Practically, the complete 16SrRNA encoding gene (about 1500 bp) was amplified using primers 16SIGF and rP2Chlam as described (14). DNA of Chlamydiales was detected in 3/50 corneal samples from 3/40 cats with keratitis and/or other corneal lesions (40 eyes) (Table 1), and in 5/23 samples from 5/23 cats with normal eyes (23 eyes). To screen for infections with FHV-1 and Chlamydophila felis, well known and common ocular pathogens in cats, specific real-time TaqMan PCRs were performed according to Vogtlin et al. (15) and Helps et al. (16), respectively, using the ABI PRISM 7700 (Applied Biosystems, Rotkreuz, Switzerland). Feline herpesvirus DNA was detected in 13 of 50 samples from 12/40 cats with keratitis and/or other corneal lesions (Table 1), and in 11/20 samples from 11/23 cats with normal eyes. Chlamydophila felis DNA was detected in 2/50 samples from 2/40 cats with keratitis and/or other corneal lesions, and was not detected in any sample from 23 cats with normal eyes. Detection of P. acanthamoebae and Protochlamydia spp. DNA were performed by real-time TaqMan PCRs as described elsewhere (3, 17) using the ABI PRISM 7000 (Applied Biosystems, Rotkreuz, Switzerland). Each sample was amplified in duplicate. Parachlamydia acanthamoebae DNA was detected in 5/50 corneal samples from 5/40 cats with keratitis and/or other corneal lesions (Table 1), and in 6/23 samples from 6/23 cats with normal eyes. Protochlamydia DNA was not detected in any sample from any cat. Three samples were positive both with the broad-range Chlamydiales PCR and the Parachlamydia PCR, hence excluding false positivity by PCR contamination since both PCRs amplify different DNA segments. Samples positive for 16S rDNA, but negative for Chlamydophila felis, Parachlamydia acanthamoebae and any member of the Protochlamydia genus were submitted for sequencing. Sequence analyses did not reveal any meaningful results, perhaps due to low number of copies and/or due to use of primers amplifying large sequences (about 1400 base-pairs), at the expense of sensitivity. Statistical analysis (Chi-Square test, Fisher’s exact test) revealed no significant difference in the rate of Parachlamydia acanthamoebae and Chlamydiales order specific PCRs positivity between cats with and without corneal disease. However, among 8 diseased eyes positive for Parachlamydia acanthamoebae DNA and Chlamydiales order specific 16S rDNA, 7 suffered from chronic corneal erosions and/or corneal necrosis with damaged epithelium, and the remaining eye exhibited an active keratitis with intact epithelium (Table 1). The habitat of the cat (indoors versus outdoors) did not correlate with presence of Parachlamydia acanthamoebae DNA and Chlamydiales order specific DNA. All samples were also screened with an Acanthamoeba specific 18SrDNA gene PCR for the presence of Acanthamoeba species, which are natural hosts for Parachlamydia acanthamoebae. The Acanthamoeba specific primer pair JDP1 and JDP2 were used as described by Schroeder et al. (18), with slight modifications of the forward primer JDP1-mod (5′ GGCCCAGATCGTTTACCGTG-3′) and the reverse primer JDP2- mod (5′-CACAAGCTGCTAGGGGAGTC-3′) (6). Acanthamoeba DNA was not detected in any sample of any cat.

Discussion. The detection of Chlamydiales order specific DNA and Parachlamydia acanthamoebae DNA in corneal samples from cats with and without corneal disease indicates exposure of cats, or strictly speaking, of the feline eye to these obligate intracellular bacteria. Eyes of cats living strictly indoors and eyes of cats having outdoor access were equally exposed to Parachlamydia acanthamoebae and other Chlamydiales. Interestingly, Acanthamoeba DNA was not detected in any cat. Therefore, transmission of Parachlamydia acanthamoebae and other Chlamydiales to the feline eye remains unknown. The absence of Acanthamoeba DNA in samples positive for Parachlamydia acanthamoebae is unlikely to be due to false negative Acanthamoeba PCR, but rather suggest that epithelial cells of cat’s eyes might allow persistence of Parachlamydia acanthamoebae in the absence of an amoebal host, as suggested by the persistence of this strict intracellular bacteria in different mammalian cell lines (19-21). Since cats frequently live in close contact with humans, cats might be a possible source of infection for humans and the associated zoonotic risk merits further investigation.

Acknowledgments

We thank Dr. Felix Grimm from the Institute of Parasitology, Vetsuisse Faculty, University of Zurich for providing us with a cloned control target as positive control for the Acanthamoeba specific PCR. We also thank Philip Tarr (Basel, Switzerland) for reviewing the manuscript. Gilbert Greub is supported by the Leenards Foundation through a career award entitled “Bourse Leenards pour la relève académique en médecine clinique à Lausanne”. This study was partly funded by a competitive research grant from the Vetsuisse Faculty of the University of Zürich received by Marianne Richter. References 1. Corsaro D, Greub G. Pathogenic potential of novel and diagnostic approaches to infections due to these obligate intracellular bacteria. Clin Microbiol Rev 2006;19:283-97. 2. Greub, G. Parachlamydia acanthamoebae, an emerging agent of pneumonia. Clin Microbiol Infect 2009; 15:18-28. 3. Casson N, Michel R, Muller KD, Aubert JD, Greub G. Protochlamydia naegleriophila as etiologic agent of pneumonia. Emerg Infect Dis 2008;14:168- 72. 4. Fritsche TR, Gautom RK, Seyedirashti S, Bergeron DL, Lindquist TD. Occurrence of bacterial endosymbionts in Acanthamoeba spp. isolated from corneal and environmental specimens and contact lenses. J Clin Microbiol 1993;31:1122-6. 5. Fritsche TR, Horn M, Wagner M, Herwig RP, Schleifer KH, Gautom RK. Phylogenetic diversity among geographically dispersed Chlamydiales endosymbionts recovered from clinical and environmental isolates of Acanthamoeba spp. Appl Environ Microbiol 2000;66:2613-9. 6. Lutz-Wohlgroth L, Becker A, Brugnera E, et al. Chlamydiales in guinea-pigs and their zoonotic potential. J Vet Med A Physiol Pathol Clin Med 2006;53:185- 93. 7. von Bomhard W, Polkinghorne A, Lu ZH, et al. Detection of novel chlamydiae in cats with ocular disease. Am J Vet Res 2003;64:1421-8. 8. Bodetti TJ, Viggers K, Warren K, et al. Wide range of Chlamydiales types detected in native Australian mammals. Vet Microbiol 2003;96:177-87. 9. Greub G, Mege JL, Raoult D. Parachlamydia acanthamoebae enters and multiplies within human macrophages and induces their apoptosis [corrected]. Infect Immun 2003;71:5979-85. 10. Amann R, Springer N, Schonhuber W, et al. Obligate intracellular bacterial parasites of acanthamoebae related to Chlamydia spp. Appl Environ Microbiol 1997;63:115-21. 11. Marciano-Cabral F, Cabral G. Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev 2003;16:273-307. 12. Stiles J. Feline herpesvirus. Vet Clin North Am Small Anim Pract 2000;30:1001-14. 13. Stiles J, McDermott M, Bigsby D, et al. Use of nested polymerase chain reaction to identify feline herpesvirus in ocular tissue from clinically normal cats and cats with corneal sequestra or conjunctivitis. Am J Vet Res 1997;58:338-42. 14. Thomas V, Casson N, Greub G. Criblamydia sequanensis, a new intracellular Chlamydiales isolated from Seine river water using amoebal co-culture. Environ Microbiol 2006;8:2125-35. 15. Vogtlin A, Fraefel C, Albini S, et al. Quantification of feline herpesvirus 1 DNA in ocular fluid samples of clinically diseased cats by real-time TaqMan PCR. Journal of Clinical Microbiology 2002;40:519-23. 16. Helps C, Reeves N, Egan K, Howard P, Harbour D. Detection of Chlamydophila felis and feline herpesvirus by multiplex real-time PCR analysis. J Clin Microbiol 2003;41:2734-6. 17. Casson N, Posfay-Barbe KM, Gervaix A, Greub G. New diagnostic real-time PCR for specific detection of Parachlamydia acanthamoebae DNA in clinical samples. J Clin Microbiol 2008;46:1491-3. 18. Schroeder JM, Booton GC, Hay J, et al. Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge. J Clin Microbiol 2001;39:1903-11. 19. Casson, N., N. Medico, J. Bille, and G. Greub. 2006. Parachlamydia acanthamoebae enters and multiplies within pneumocytes and lung fibroblasts. Microbes Infect 8:1294-300. 20. Greub, G., J. L. Mege, J. P. Gorvel, D. Raoult, and S. Meresse. Intracellular trafficking of Parachlamydia acanthamoebae. Cell Microbiol 2005;7:581-9. 21. Greub, G., J. L. Mege, and D. Raoult. Parachlamydia acanthamoebae enters and multiplies within human macrophages and induces their apoptosis Infect Immun 2003; 71:5979-85.

Table 1: PCR results of 50 samples taken from 40 cats (40 eyes) with keratitis, conjunctivitis and/or other corneal lesions. Please note that some cat’s eyes presented corneal necrosis and corneal erosion or active keratitis with or without corneal necrosis, explaining that the total of the denominators provided in a column is higher than 50 and that the total of the numerators is higher than the actual number of eyses (cats) positive for a given pathogen.

Chlamydiales ° Parachlamydia C. felis FHV-1 active keratitis 2/19* 1/19 0/19 6/19 corneal necrosis 2/20 3/20 1/20 4/20 corneal erosion 1/20 3/20 0/20 4/20 eosinophilic 0/4 0/4 ¼ ¼ keratitis conjunctivitis 0/4 0/4 ¼ ¾ ° Unidentified Chlamydiales other than Parachlamydia acanthamoebae and Chlamydophila felis