ORIGINAL SCIENTIFIC ARTICLE / IZVORNI ZNANSTVENI ČLANAK A preliminary study of Chlamydophila felis prevalence among domestic cats in the City of Zagreb and Zagreb County in Croatia Gordana Gregurić Gračner*, Ksenija Vlahović, Alenka Dovč, Brigita Slavec, Ljiljana Bedrica, S. Žužul and D. Gračner Introduction Feline chlamydiosis is a disease in do- Rampazzo et al. (2003) investigated mestic cats caused by Chlamydophila felis the prevalence of Cp. felis and feline (Cp. felis), which is primarily a pathogen herpesvirus in cats with conjunctivitis by of the conjunctiva and nasal mucosa using a conventional polymerase chain rather than a pulmonary pathogen. It is reaction (PCR), and discovered that 14 capable of causing acute to chronic con- out of 70 (20%) cats with conjunctivitis junctivitis, with blepharospasm, chem- were positive only on Cp. felis and mixed osis and congestion, a serous to mucop- infections with herpesvirus were present urulent ocular discharge, and rhinitis in 5 of 70 (7%) cats. (Hoover et al., 1978; Sykes, 2005). C. Helps et al. (2005) took oropharyngeal 1 psittaci infection in kittens produces fe- and conjunctival swabs from 1101 cats ver, lethargy, lameness, and reduction and by using a PCR determined Cp. felis in weight gain (Terwee at al., 1998). Ac- in 10% of the 558 swab samples of cats cording to the literature, chlamydiosis with URDT and in 3% of the 558 swab in cats can be treated successfully by samples of cats without URDT. administering potentiated amoxicillin Low et al. (2007) investigated 55 cats for 30 days, which can result in a com- with conjunctivitis, 39 healthy cats and plete clinical recovery with no evidence 32 cats with a history of conjunctivitis of a recurrence for six months (Sturgess that been resolved for at least 3 months. et al., 2001). By using conventional PCR assays to 1 Chlamydophila felis (formerly Chlamydia determine the prevalence of Cp. felis in psittaci) cats with and without conjunctivitis, they Gordana GREGURIĆ GRAČNER*, DVM, PhD, Assistant Professor (corresponding author, e-mail:ggracner@ gmail.com), Ksenija VLAHOVIĆ, DVM, PhD, Full Professor, Faculty of Veterinary Medicine, University of Zagreb, Croatia; Alenka DOVČ, DVM, PhD, Full Professor, Brigita SLAVEC, DVM, PhD, Full Professor, Faculty of Veterinary Medicine, University of Ljubljana, Slovenia; Ljiljana BEDRICA, DVM, PhD, Full Professor, Slavko ŽUŽUL, DVM, PhD, Assistant, Damjan GRAČNER, DVM, PhD, Full Professor, Faculty of Veterinary Medicine, University of Zagreb, Croatia VETERINARSKA STANICA 49 (1), 2018. 1 GORDANA GREGURIć GRAčNER, KSENIJA VLAHOVIĆ, ALENKA DOVč, BRIGITA SLAVEC, LJILJANA BEDRICA, S. ŽUŽUL and D. GRAČNER found that the overall prevalence rate of cats were both pure and mixed breeds, Cp. felis was 3.2%. different sexes, and aged from two In Sibitz et al. (2011), the presence months to 18 years old. Each cat was of Chlamydophila felis was found using clinically examined. a real-time (RT) PCR in two cats with The cats had not been vaccinated conjunctivitis out of the 49 which were against Cp. felis, and before swabs were being investigated, while Chlamydiae taken they were not treated with any related to uncultured members of medication. 48 swabs were taken from the Chlamydiales were found in three more cats’ eyes and 48 from their nostrils. Rapid cats with conjunctivitis. EIA tests were carried out immediately Of 200 cats from 19 cantons in after collecting the samples. For further Switzerland that were suspected analyses, the swabs were placed into a 2 of having calicivirus (FCV)-related mL sucrose phosphate transport medium symptoms, 8% were positive for Cp. felis, (Spencer and Johnson, 1983) and stored and this microorganism was also present at – 80 °C until analysis. Conjunctival in 1% of 100 healthy cats from the same scrapings were also taken from both eyes region. The samples were tested for for direct immunofluorescence (DIF) Cp. felis using an RT quantitative PCR tests. (qPCR) (Berger et al., 2015). Fernandez et al. (2017) discovered, by using a real- 1. Detection of chlamydial antigens time polymerase chain reaction (PCR), that 20.5% of 127 cats with URTD were Rapid (immunoenzyme assay) EIA positive for Cp. felis, as well as 19.5% of The 192 ocular and nasal swabs, were 149 with conjunctivitis and 9.1% of 154 examined using a rapid EIA (Clearview ® with gingivostomatitis (GS). Chlamydia MF, Unipath Limited , However, there could be a possible Bedford, United Kingdom) according to limitation to the molecular evidence for the manufacturer’s instructions. This test chlamydophila infection in naturally is a rapid immunoassay for the direct infected cats because of histological genus specific qualitative detection of alterations in conjunctiva caused by the Chlamydia trachomatis antigen, but chronic conjunctivitis (Kiełbowicz et al., it can also be used for Cp. felis detection 2014). (Trávnicek et al., 2002; Pavlin et al., 2005). Based on serum analyses for the presence of antibodies against Cp. felis Direct immunofluorescence (DIF) in 214 Swedish cats with no signs of DIF was performed on thirty ocular infectious disease, Ström Holst et al. swabs with a commercially FITC-labelled (2006) found that 11% of the cat serums monoclonal antibody specific for the were positive but had low antibody major outer membrane protein (MOMP) titres. The aim of this preliminary study of Chlamydia trachomatis was to investigate the prevalence of (CHLAMYDIA DIRECT IF IDENTIFI- Chlamydophila felis (Cp. felis) among CATION, Biomerieux, France) according domestic cats in City of Zagreb and to the manufacturer’s instructions. It can Zagreb County, Croatia. also be used for Cp. felis detection. DNA extraction, conventional Materials and methods polymerase chain reaction (PCR) and 48 cats were investigated. All of them sequencing came as patients to clinics of the Faculty PCR was performed on six samples of Veterinary Medicine in Zagreb. The (three nostril samples, three eye samples). 2 VETERINARSKA STANICA 49 (1), 1-7, 2018. A preliminary study of Chlamydophila felis prevalence among domestic cats in the City of Zagreb and Zagreb County in Croatia Preliminarno istraživanje učestalosti pojave bakterije Chlamydophila felis u domaće mačke u Gradu Zagrebu i na području Zagrebačke županije Before DNA extraction ocular and Wizard PCR Preps DNA Purification nasal swabs were placed in 2 mL of System (Promega Corp., Madison Chlamydia transport medium and WI, USA) following manufacturer’s homogenised by vortexing. Total instructions and sent for sequencing to DNA was extracted from 200 µL of Macrogen laboratory (Macrogen Inc, the sample using QIAamp®DNA Seoul, Korea). Nucleotide sequence Mini Kit (Qiagen, Hilden, Germany) data were analysed by BLAST (Altschul according to manufacturer`s protocols et al., 1990) for finding similarity for Blood and body fluid spin protocol. with sequences from NCBI sequence The presence of Chlamydiaceae was database. confirmed using specific primers 16SF2 (5` CCGCCCGTCACATCATGG 3`) and 23R (5` TACTAAGATGTTTCAGTTC Results 3`) for amplifying approximately 600 Most of the examined cats were in bp long segment containing partial 16S good health. Their coats were shiny, and RNA gene, intergenic spacer region and not brittle or coarse. Based on the cats’ partial 23S RNA gene (Everett et al., owners’ statements, all of the cats had 1999). The PCR was preformed using a good appetite and normal behaviour. HotStartTaq®Plus PCR (Qiagen, Hilden, Their body temperatures were normal, as Germany) in 25 µL reaction according were their pulses and breathing. However, to manufacturer`s instruction. The PCR examination of their conjunctival mucosa program consisted 5 minute at 95 °C showed that 54% had unilateral or for PCR initial activation step followed bilateral mild conjunctivitis. with 33 cycles of 30 second at 94 °C, 1 One cat, whose biological samples minute at 50 °C and 1 minute at 72 °C, were PCR positive, was underfed and with a final extension of 7 minute at run-down, and its fur was unkempt and 72 °C. Amplicons were visualized by in an unhealthy condition. This cat also gel electrophoresis on a 1,8% bromide showed bilateral conjunctivitis with stained agarose gel. PCR products were marked mucopurulent discharge from excised from the gel, purified with the left eye and nasal discharge from both Fig. 1. The results (%) of rapid immunoenzymme assay (EIA) performed on conjunctival, nostril and rectal swab samples VETERINARSKA STANICA 49 (1), 1-7, 2018. 3 GORDANA GREGURIć GRAčNER, KSENIJA VLAHOVIĆ, ALENKA DOVč, BRIGITA SLAVEC, LJILJANA BEDRICA, S. ŽUŽUL and D. GRAČNER Table 1. Results of direct immunofluorescence performed on conjunctival smears from cats Left eye Right eye DIF number (%) number (%) Positive result 3 (20.0) 1 (6.7) Negative result 11 (73.3) 13 (86.6) Inconclusive result 1 (6.7) 1 (6.7) Total 15 (100) 15 (100) nostrils. According to the cat’s owner, The eye samples which were positive the mucopurulent ocular discharge when using the EIA were examined using had appeared the day before she came DIF, and the results are shown in Table 1. to the clinic and the cat had not been Of six clinical samples (three nostril treated with any medication. A thoracic samples and three eye samples), only radiograph was performed on this cat two were found to be positive for Cp. because of a suspicion of pneumonia and felis using a PCR. More precisely, the it was examined for FeLV and FIV using positive samples were taken from the left FASTest FeLV and FASTest FIV tests nostril and left eye of the same cat. The (Mega Cor Diagnostic). positive and control samples produced Using a rapid immunoenzyme an approximately 600 bp long product assay (EIA) produced 5-35% positive in the first round of PCR (Fig. 2). For results, depending on which mucosa confirmation of the positive results and were examined. The results are shown determination of Chlamydia, species in Fig. 1. sequencing was performed. Fig. 2. Agarose gel electrophoresis of PCR products extracted from clinical samples (left nostril and left eye of one cat): Column M = 100 bp long DNA standard: Column CP = positive control Cp.