Bulgarian Journal of Veterinary Medicine, 2018 ONLINE FIRST ISSN 1311-1477; DOI: 10.15547/bjvm.2124

Short communication

MOLECULAR DETECTION OF PSITTACI AND CHLAMYDIA FELIS IN HUMAN KERATO- CASES

S. JAZI, A. MOKHTARI & A. EBRAHIMI KAHRIZSANGI

Department of Pathobiology, Faculty of Veterinary Medicine, University of Shahrekord, Shahrekord, Iran

Summary Jazi, S., A. Mokhtari & A. Ebrahimi Kahrizsangi, 2018. Molecular detection of and Chlamydia felis in human keratoconjunctivitis cases. Bulg. J. Vet. Med. (online first).

Given the high incidence of keratoconjunctivitis in Iran (approximately 3.6–53.9%) and low effi- ciency of clinical diagnostic measures, application of laboratory tests for detection of different kerato- conjunctivitis/conjunctivitis causes and determination of their accurate prevalence is essential. In this research, conjunctival samples were collected from 100 patients with keratoconjunctivitis signs re- ferred to an hospital of Iran. After DNA extraction, PCR was carried out for detection of Chlamy- dia psittaci and Chlamydia felis. PCR positive products were further subjected for DNA sequencing. In this study, one sample was Chlamydia psittaci positive and none was positive for Chlamydia felis. There wasn’t a statistically significant relationship between working in the field of veterinary medi- cine or keeping a pet and Chlamydia psittaci prevalence (P>0.05). This study showed a low rate of chlamydial keratoconjunctivitis and therefore further studies for detection of other causes are neces- sary. Key words: Chlamydia felis, Chlamydia psittaci, human, keratoconjunctivitis, PCR

Zoonotic potential of members of the ge- who suggested that he had acquired it nus Chlamydia is a concern, especially for from an infected . Three other cases human population in contact with animals. included a renal transplant recipient ha- Chlamydia felis is considered an impor- ving developed malaise; coughing and tant cause of infectious diseases of the abnormal liver function, a person with upper respiratory tract and in , infective endocarditis and glomeru- having zoonotic potential (Everson et al., lonephritis and a healthy man who devel- 2003). A few cases of C. felis conjunctivi- oped a febrile illness with malaise, pneu- tis have been documented in people. One monia and mental impairment. C. felis case occurred in an HIV-infected person nucleic acids have also been detected in Molecular detection of Chlamydia psittaci and Chlamydia felis in human keratoconjunctivitis cases respiratory samples collected from few psittacosis is difficult to diagnose. Harki- people, including nasal swabs from a nezhad et al. (2009) examined the occur- healthy person (Browning, 2004). rence of psittacosis in a generally healthy Avian chlamydiosis is generally called Belgian population in contact with domes- psittacosis (or ornithosis) in humans. The tic and/or companion birds using a C. psit- disease caused by C. psittaci in humans is taci-specific nested PCR and serology and called psittacosis which could be either 69 of 540 (12.7%) pharyngeal swabs were inapparent or manifested with mild influ- found to contain C. psittaci DNA. enza-like symptoms to severe and even Conjunctivitis is one of the commonest potentially fatal systemic disease with and highly contagious eye diseases in severe . Psittacosis is of con- Iran, which frequently needs in-hospital cern for public health authorities and spe- treatment. Due to the overlap of conjunc- cific control measures have been recom- tivitis symptoms it seems very difficult to mended (Aaziz et al., 2015). diagnose it precisely and definitely based Humans can be infected with any on clinical symptoms (Mokhtari et al., genotype. C. psittaci can infect people 2013). Conjunctivitis diagnosis is based asymptomatically, cause keratoconjuncti- on history taking and physical examina- vitis or result in a systemic illness. A re- tion but there are no key signs for etio- cent study from Egypt found C. psittaci logic diagnosis of conjunctivitis. Early DNA in some women presented with symptoms of the disease are not specific various gynaecological complaints (Dum- and accurate distinction between viral and ke et al., 2015). bacterial causes is almost impossible Recent studies suggest that infections (Mokhtari et al., 2014). with C. psittaci may be underdiagnosed in Better diagnostic tools are needed to some populations, such as poultry workers elucidate the role and frequency of C. (Krawiec et al., 2015). psittaci in human disease. At present, se- Since Chlamydia psittaci can infect rological tests are often used for diagnosis humans, it should be handled carefully of C. psittaci infection in birds. However, under conditions of bio-containment. The serology cannot provide a definitive diag- disease is of public health significance nosis due to the lack of specific antibody because of the popularity of birds as pets detection assays, the long persistence of and placement of birds in childcare facili- antibody titres in cured birds and the need ties, garden centres and rest homes. for convalescent sera to detect serocon- Moreover, feral pigeons in urban habitats version. There is a need to validate newly are commonly infected, presenting a developed nucleic acid amplification zoonotic risk. The disease is rarely fatal in techniques, such as polymerase chain re- properly treated patients. Therefore, action (PCR), for rapid detection of in- awareness of the danger and early diagno- fected animals and for diagnosis of infec- sis are important. tion in human patients (Damborg et al., In Belgium, seven, two and three psit- 2016). tacosis cases were reported in 2005, 2006 It is possible to identify chlamydial in- and 2007, respectively. In Australia, Ger- fection by culture, but results take longer many, Sweden and the Netherlands, 62, than the other tests. Although culture is 10, 9 and 27 cases were reported in 2007. the legal standard, it is not the gold stan- This is probably an underestimation as dard for the detection of Chlamydia. An-

2 BJVM, ××, No × S. Jazi, A. Mokhtari & A. Ebrahimi Kahrizsangi tigen tests are no longer recommended for extracted according to phenol chloroform Chlamydia testing due to insufficient di- DNA extraction protocol. agnostic accuracy and PCR techniques are The presence of Chlamydia psittaci now the preferred option for diagnosing and Chlamydia felis was detected using Chlamydia infection. Such techniques are the primers designed by Beacon designer extremely sensitive and avoid problems software. The sequences of the forward with poor viability of the organisms and reverse primers were 5’-GCT (Browning, 2004) ACACGCATAAGTTTACC -3’ and 5’- In previous studies reported from Iran, TGGAGAGAGACCTCTAGG -3’ for the prevalence of the various bacterial and C. felis and 5’- GCATTACTCGCTATA viral conjunctivitis and keratoconjunctivi- TTCC-3’ and 5’-TGTGGAAGGAGA tis varies from 3.6% to 53.9%. Data about GGGAAC -3’ for C. psittaci respectively. the possible involvement of Chlamydia in Each PCR reaction was performed in a these complications from Iran are not final volume of 25 µL containing 11 µL of available (Sohrabi et al., 2011; 2014; deionized sterile water, 10 µL of Taq Mokhtari et al., 2013; 2014). DNA Polymerase 2× Mix Red-MgCl2 2 Considering the increasing prevalence mM (GeneAll, Cat. no. A180301), 1 pmol of Chlamydia in abortion cases of Isfahan of each primer and 2 µL of DNA tem- and Chaharmahal and Bakhtiari (neigh- plate. bouring province) and the increasing in- The thermal cycling conditions for the tensity of contacts between humans and amplification were 1 cycle for 4 min at animals (Sohrabi et al., 2011; Damborg et 94 ºC, 30 cycles of 30 s at 94 ºC, 30 s at al., 2016), we have designed this study as 53 ºC for C. psittaci and 50 ºC for C. felis a cross-sectional survey on a target group and 25 s at 72 ºC, with a final extension comprising all patients referred to an ocu- step of 3 min at 72 ºC. Positive and nega- lar disease hospital with general kerato- tive controls (from Iranian Biological Re- conjunctivitis symptoms. One hundred source Center) were included in each conjunctival swab samples were obtained analysis. Six µL of the amplified products during 2013–2014 and were analysed to were loaded on a 1.3% agarose gel, and detect vaccinia . All the procedures visualised by staining with ethidium bro- followed in this study were in accordance mide and compared to DNA markers (50 with the ethical standards and with the ladder, Fermentas). Helsinki Declaration of 1975, as revised PCR positive sample in a volume of in 2000. A complete history of work ex- 50 mL was sent to Bioneer Company for perience in the lab or health centres was sequencing. Sequencing was performed collected. The description of samples such on the ABI 3730XL DNA Analyzer, with as sex and age groups, association with a high quality of sequence analysis data veterinary activities or pet keeping is (Phred Score (QV): ≥ 20, Guaranteed read available in Table 1. The patients’ eyes lengths: ≥ 600 bp). After obtaining all were washed with sterile saline and a ste- results, the data was analysed using IBM rile cotton swab was rubbed onto the con- SPSS 22 software. junctiva. Then it was immersed in Eagle’s Out of the 100 conjunctival swab sam- minimal essential medium supplemented ples, 1 sample (1%) was positive for C. with penicillin, amikacin, amphotericin B psittaci fragment. Thirty-two and 1% foetal bovine serum. DNA was of the cases had a history of working in

BJVM, ××, No × 3 Molecular detection of Chlamydia psittaci and Chlamydia felis in human keratoconjunctivitis cases the veterinary fields or keeping a pet. One signed for detection of Chlamydia psittaci out of these 32 cases were C. psittaci was confirmed. positive. Chlamydia prevalence results Psittacosis can cause severe serious according to factors such as age, sex, ex- health problems among humans. All geno- posure and Chlamydia positivity status are types of Chlamydia psittaci can be trans- listed in Table 1. In this study there wasn’t mitted to humans and cause psittacosis or a statistically significant association be- parrot fever. From 1988 through 2003, a tween Chlamydia and working in the vet- total of 935 human cases of psittacosis erinary fields or keeping a pet (Chi square were reported to the US Centers for Dis- test; P>0.05). ease Control and Prevention; most were Chlamydia psittaci was detected using related to contact with Psittaciformes. The PCR test specific for the 16s rRNA nu- incidence of psittacosis in men seems to be cleotide fragment. The Chlamydia psit- increasing in industrialised countries and is taci-specific band with the size of 122 bp related to importation of exotic birds. Other was detected in Chlamydia psittaci posi- cases may not be correctly diagnosed or tive control. The positive PCR product reported (Vanrompay et al., 2007). was of the same size as those from the During the 1980s, approximately 70% positive control. There was no Chlamydia of the psittacosis cases with a known felis positive among samples while band source of infection resulted from human with the size of 159 bp was detected in exposure to caged pet birds; of these per- Chlamydia felis positive control (Fig. 1). sons, the largest group affected (43%) After sequencing, alignment of ex- included bird fanciers and owners of pet pected amplicon with the read sequences birds (Anonymous, 1996). Similarly, in confirmed the presence of the 16s rRNA the present study, one person who had nucleotide fragment in the positive sam- positive result for Chlamydia psittaci PCR ple. There was 98% identity between PCR test was owner of pet birds. Human cases product sequences and expected ampli- of psittacosis has been reported from con. Therefore, the result of PCR test de- many countries such as Austria, Argen-

Table 1. The prevalence of C. psittaci among the studied keratoconjunctivitis patient cohort

C. psittaci positive C. psittaci negative Total Sex: female 13 37 50 male 15 35 50 Age groups (years): <15 0 25 25 15–30 1 24 25 30-45 0 25 25 >45 0 25 25 Working at veterinary medicine field/keeping pet: yes 1 31 32 no 0 68 68 Exposure: previous 0 10 10 current 1 20 21

4 BJVM, ××, No × S. Jazi, A. Mokhtari & A. Ebrahimi Kahrizsangi tina, Australia, Belgium, Bosnia and Her- search was higher than the present study zegovina, Chile, Croatia, Czech Republic, which is probably due to this fact that Denmark, Finland, Germany, Hungary, their sampling performed from the popu- Japan, Poland, Slovakia, Spain, Sweden, lation at the risk of transmission, but in The Netherlands, UK/Great Britain, the present study, random sampling was UK/N. Ireland, Ukraine, USA from 1996 used. to 2009, In Iran there are reports of Chla- A study in China reported that severe mydia psittaci but not from keratocon- pneumonia in peacocks and peacock junctivitis cases (Browning, 2004). farmers was caused by C. psittaci (Brown- ing, 2004). The rate of infection in the study was similar to our study; however the sampling site was different. Harkinezhad et al. (2009) reported that the prevalence of Chlamydia psittaci infections in a human population was 12.7% using PCR and that 40.6% sera of PCR-positive individuals reacted posi- tively in the rMOMP ELISA. Among 28 C. psittaci positive people, 24 and 4 had daily or weekly contact with domestic or companion birds, respectively (Browning, 2004).. They concluded that since previ- ous studies were based on serological tests; psittacosis occurs more often in Belgium than reported and incidence of psittacosis is most likely also underesti- mated in other European countries So, in our study we used PCR test for detection of Chlamydia to have more accurate re- sults. Fig. 1. C. felis and C. psittaci PCR amplifica- Beeckman et al. (2009) showed that tion products following electrophoresis. Lane human psittacosis is a zoonotic infectious 1: C. felis positive control, lane 2: C. felis disease and due to a low awareness of the negative control, lane 3: C. psittaci positive disease and a variable clinical presenta- sample, lane 4: C. psittaci positive control, tion psittacosis is often not recognised by lane 5: C. psittaci negative control, lane 6: 50 bp DNA ladder. general practitioners. Overall, because human infection can result from brief, Vanrompay et al. (2007) studied passing exposure to infected birds or their zoonotic transmission of contaminated droppings, persons with no psittaci in 39 breeding facilities for Psit- identified leisure-time or occupational risk taciformes that frequently used antimicro- can become infected (Johnston et al., bial drugs. Chlamydia psittaci was de- 2000). tected in 14.9% of humans at these facili- Another Chlamydia we tried to ties (Beeckman et al., 2009). The preva- detect was Chlamydia felis. It more com- lence of Chlamydia psittaci in their re- monly causes conjunctivitis in cats (Hala-

BJVM, ××, No × 5 Molecular detection of Chlamydia psittaci and Chlamydia felis in human keratoconjunctivitis cases nova et al., 2011). The majority of cases gen or not. He concluded that based on are frequently associated with chronic seven case reports C. felis may occasion- conjunctivitis (Wills et al., 1988) and ally cause keratoconjunctivitis in humans more severe ocular disease (Fernandez et and although at a lower rate, it can cause al., 2016). serious systemic disease or pneumonia. C. felis was suspected of causing hu- Based on the previous studies, the man disease, particularly follicular con- likelihood of Chlamydia felis isolation junctivitis (Ostler et al., 1969; Schachter from humans is low and therefore it may et al., 1969) or atypical pneumonitis (Cot- be considered as one of the reasons for ton & Partridge, 1998). Endocarditis with not achieving a positive Chlamydia felis secondary glomerulonephritis (Regan et sample or a low prevalence of C. psittaci al., 1979) and severe liver breakdown in in this study. an immunocompromised patient (Griffiths Since the organism is intracellular, it is et al., 1978) were reported. Although C. necessary to obtain good quality swabs felis is a zoonotic , there is few that include cells (Segarra et al., 2011). epidemiological evidence for a significant We used PCR test for identifying two zoonotic risk. Conjunctivitis caused by C. Chlamydia strains. PCR is a good test for felis has been reported in an HIV-infected detection of Chlamydia species. Other patient (Hartley et al., 2001). techniques for demonstrating the organism Since pet cats and dogs are considered are less sensitive and less reliable than to be faithful friends and companions of PCR (Streeten & Streeten, 1985). humans, they play an important role in Harkinezhad et al. (2009) concluded human life. Unfortunately, cats and dogs clinicians should be strongly encouraged may be important sources of C. felis infec- to use nucleic acid amplification tests for tion in humans (Aaziz et al., 2015). C. diagnosing Chlamydia rather than sero- felis can induce conjunctivitis in people logy because molecular tests are ex- who are in close contact with infected tremely sensitive and specific and present cats, but the risk is extremely low, as indi- direct evidence of a Chlamydia infection. cated by the comparison of the seropreva- Although C. felis has a zoonotic poten- lence in cats and their owners (Yan et al., tial, we couldn’t detect it in this study. 2000). This result is in concordance with previ- Travnicek et al. (2002) documented ous studies that showed that the preva- that Chlamydia felis has a zoonotic poten- lence of C. felis is very low. However, the tial. They showed presence of C. felis in failure in detection of it may be due to 13 and 10 investigated cats and human in sampling condition, because we used only nasal mucosa smears and . We ocular swab samples. Overall, the present couldn’t found any C. felis positive sam- study showed a low rate of C. psittaci ple. Given the fact that the number of infection among human keratoconjunctivi- samples taken from the pet owners or tis cases in Iran and no statistically sig- animal-related occupations was low in our nificant relationship obtained between study, and the global prevalence of C. felis keeping pet animals and Chlamydia in humans is low, this result was likely. prevalence (P>0.05). Since previous stud- Browning (2004) reviewed all the re- ies emphasise that chlamydial zoonotic ports and texts to find whether Chlamydo- infections are rarely identified, some pre- phila felis is a significant zoonotic patho- caution is warranted when handling pet

6 BJVM, ××, No × S. Jazi, A. Mokhtari & A. Ebrahimi Kahrizsangi

animals. tions. Journal of Comparative Pathology, Further studies for determination of 155, S27–S40. the prevalence of Chlamydia species in Fernandez, M., E. G. Manzanilla, A. Lloret, keratoconjunctivitis cases in a larger scale M. León & J. C. Thibault, 2016. Preva- and consequently, preventive methods are lence of feline herpesvirus-1, feline necessary in Iran. calicivirus, Chlamydophila felis and My- coplasma felis DNA and associated risk factors in cats in Spain with upper respira- ACKNOWLEDGEMENTS tory tract disease, conjunctivitis and/or gingivostomatitis. Journal of Feline Medi- The authors are grateful to microbiology labo- cine and Surgery, 19, 461–469. ratory personnel of Faculty of Veterinary Gaydos, C. A., L. Palmer, T. C. Quinn, S. Medicine, University of Shahrekord for their Falkow & J. J. Eiden, 1993. Phylogenetic collaboration. relationship of to Chlamydia psittaci and Chlamydia tra- chomatis as determined by analysis of 16S REFERENCES ribosomal DNA sequences. International Ahmadi, M. H., A., Mirsalehian & A. Baha- journal of Systematic Bacteriology, 43, dor, 2015. Prevalence of genital Chlamy- 610–612. dia trachomatis in Iran: A systematic re- Halanova, M., Z. Sulinova, L. Cislakova, A. view and meta-analysis. and Trbolová, L. Páleník, T. Weissová, M. Global Health, 109, 290–299. Halán, Z. Kalinová & M. Holičková, 2011. Aaziz, R., P., Gourlay, F. Vorimore, K. Chlamydophila felis in cats – are the stray Sachse, V. I. Siarkou & K. Laroucau, cats dangerous source of infection? Zoo- 2015. in North Atlantic noses and Public Health, 58, 519–522. seabirds admitted to a wildlife rescue cen- Harkinezhad, T., K. Verminnen, M. De Buyz- ter in Western France. Applied and Envi- ere, E. Rietzschel, S. Bekaert & D. Van- ronmental Microbiology, 81, 4581–4590. rompay, 2009. Prevalence of Chlamydo- Anonymous, 1996. Compendium of measures phila psittaci infections in a human popu- to control Chlamydia psittaci infection lation in contact with domestic and com- among humans (psittacosis) and pet birds panion birds. Journal of Medical Microbi- (avian chlamydiosis), 1998. Morbidity and ology, 58, 1207–1212. Mortality Weekly Report, 47 (RR10), 1–9. Hartley, J. C., S. Stevenson, A. J. Robinson, J. Beeckman, D. S. & D. C. Vanrompay, 2009. D. Littlewood, C. Carder, J. Cartledge, C. Zoonotic Chlamydophila psittaci infec- Clark & G. L. Ridgway, 2001. Conjuncti- tions from a clinical perspective. Clinical vitis due to Chlamydia felis (Chlamydia psittaci feline pneumonitis agent) acquired Microbiology and Infection, 15, 7–11. from a cat: Case report with molecular Browning, G. F., 2004. Is Chlamydophila felis characterization of isolates from the pa- a significant zoonotic pathogen? Austra- tient and cat. Journal of Infection, 43, 7– lian Veterinary Journal, 82, 695–596. 11. Damborg, P., E. M. Broens, B. B. Chomel, S. Hashemi, F. B., B. Pourakbari & J. Zaeimi Guenther, F. Pasmans, J. A. Wagenaar, J. Yazdi, 2007. Frequency of Chlamydia tra- S. Weese, L. H. Wieler, U. Windahl, D. chomatis in Women with Cervicitis in Te- Vanrompay & L. Guardabassi, 2016. Bac- hran, Iran, Infectious Diseases in Obstet- terial zoonoses transmitted by household rics and Gynecology, 2007, 67014–67018. pets: State-of-the-art and future perspec- tives for targeted research and policy ac- Johnston, W. B., M. Eidson & M. G. Stobier- ski, 2000. Compendium of measures to

BJVM, ××, No × 7 Molecular detection of Chlamydia psittaci and Chlamydia felis in human keratoconjunctivitis cases

control Chlamydia psittaci infection Streeten, B. W. & E. A. Streeten, 1985. “Blue- among humans (psittacosis) and pet birds body” epithelial cell inclusions in conjunc- (avian chlamydiosis). Morbidity and Mor- tivitis. Ophthalmology, 92, 575–579. tality Weekly Report, 49, 1–17. Trávnicek, M., S. Mardzinová, L. Cisláková, I. Meyer, T., 2016. Diagnostic procedures to de- Valocký & T. Weissová, 2002. Chlamydial tect infections. infection of cats and human health. Folia Microorganisms, 4, 25. Microbiologica, 47, 441–444. Mokhtari, M., A. R. Laripour Tehranfar, S. Vanrompay, D., T. Harkinezhad, M. van de Moghim, A. R. Dehghani, Z. Farajzadegan Walle, D. Beeckman, D. van Droogen- & N. Hosseini, 2013. Determination of broeck, K. Verminnen, R. Leten, A. Martel prevalence, sensitivity and specificity of & K. Cauwerts, 2007. Chlamydophila psit- clinical signs and symptoms of adenoviral taci transmission from pet birds to hu- keratoconjunctivitis. Journal of Isfahan mans. Emerging Infectious Diseases, 13, Medical School, 30, 2029–2038. 1108–1110. Mokhtari, M., K. Vosta, T. Narimani, N. Wills, J. M., P. E. Howard, T. J. Gruffydd- Hosseini & Sh. Moghim, 2014. Prevalence Jones & C. M. Wathes, 1988. Prevalence of herpetic keratitis in patients referred to of Chlamydia psittaci in different cat Feiz Ophthalmology Center, Isfahan, Iran, populations in Britain. Journal of Small using polymerase chain reaction method. Animal Practice, 29, 337–339. Journal of Isfahan Medical School, 32, 321–329.

Segarra, S., K. Papasouliotis & C. Helps, 2011. The in vitro effects of proxymeta- caine, fluorescein, and fusidic acid on real- time PCR assays used for the diagnosis of Paper received 15.12.2017; accepted for Feline herpesvirus 1 and Chlamydophila felis infections. Veterinary Ophthalmo- publication 16.03.2018 logy, 14, 5–8. Sohrabi, M. & Z. Goodarzi, 2014. The preva- lence of viral conjunctivitis in patients Correspondence: who referred to eye specialist hospitals in Tehran, Iran. Iranian Journal of Ophthal- A. Mokhtari mology, 26, 29–32. Department of Pathobiology, Sohrabi, N., M. Tebianian & H. Moeini, 2011. Faculty of Veterinary Medicine, Analyzing bacterial agents of keratocon- University of Shahrekord, junctivitis in patients referred to Ophthal- P.O Box 115, Shahrekord, Iran, mology Ward of Feiz Hospital in Isfahan. Tel: +983832324427, Journal of Fasa University of Medical Fax: +9832324427, Sciences, 2, 95–100. email: [email protected]

8 BJVM, ××, No ×