Determination of the Prevalence of Chlamydia psittaci by PCR in Iranian Pigeons

Many areas in Iran such as parks and gardens can be highly contaminated with pigeon feces. Chlamydia psittaci is a lethal bacterial that causes endemic avian chlamydiosis, epizootic outbreaks in mammals, and respiratory psittacosis in humans. Chlamydia psittaci strains in birds infect mucosal epithelial cells and macrophages of the respiratory tract. The aim of this study was to determination of prevalence of Chlamydia psittaci in feces of pigeons in Iran using PCR assay. DNA was extracted from 445 fecal samples of pigeons. The prevalence of this pathogen was 14.3% in region of this study. These results indicate that pigeon feces are a source of several zoonotic agents for humans, bird and animals. We suggested that continuous surveys can estimate, and thus help to minimize the risk of humans contracting diseases from pigeons.


Introduction
Chlamydophila psittaci (C.psittaci), an obligate intracellular, gram negative bacterium, has 7 known genotypes (A-F and E/B) (Geens et al., 2005).All genotypes can be transmitted to humans and cause psittacosis or parrot fever.Genotypes are distinguished by sequencing of the outer membrane protein A (ompA) gene (Vanrompay et al., 1997).C. psittaci can infect 465 avian species in 30 avian orders, with at least 153 species in the order Psittaciformes (Vanrompay et al., 2007).It is known that pigeons, like many other bird species, can harbor C. psittaci (Heddema et al., 2006).C. psittaci is a bacterium that can be transmitted from pet birds to humans (Johnston et al., 2000).The genus Chlamydia consists of three species: C. psittaci, C. trachomatis, and C. pneumoniae.Recently, a fourth species, C. pecorum, has been proposed (Fukushi & Hirai, 1992).C. trachomatis is primarily a human pathogen and contains three biovars and 15 serovars.C. pneumoniae is also a human pathogen (Vanrompay et al., 1993).These bacteria are obligate intracellular organisms that are transmitted as metabolically inactive particles called elementary bodies (EBs) (Binet & Maurelli, 2007).C. psittaci has the ability to remain infectious in the environment for months, presenting a variety of public health issues, including economically devastating outbreaks in poultry farms and occasionally severe pneumonia in humans (Kaltenboeck et al., 1991).Transmission of this atypical respiratory pathogen can occur through direct contact with infected birds, bird feces, nasal discharges, and aerosols, causing respiratory disease in both mammals and birds (Smith et al., 2005).In birds that have avian chlamydiosis, cloudy air sacs and an enlarged liver and spleen usually are observed, but no specific gross lesion is pathognomonic.The chromatic or immunologic staining of tissue-impression smears can be used to identify organisms (Johnston et al., 2000).Persons at risk include those exposed to pet birds, pigeons, and poultry and in specific occupations such as laboratory and wildlife workers.Human infection can result from even brief exposure to the contaminated excretions or secretions of infected birds (Smith et al., 2005).In contrast to veterinary medicine, psittacosis in humans is not a notifiable disease anymore as the regulation was changed some years ago (Zweifel et al., 2009).From 1941 to 2003, 78 cases in humans were reported (Haag-Wackernagel & Moch, 2004), due to contact with feral pigeons.Transmission is mainly by inhalation of nasal and fecal secretions.The aim of this study was to determination of the prevalence of C. psittaci in pigeon population in Iran by PCR method.

Sampling and DNA extraction
In present study, 445 samples of fresh feces pigeons without contamination in different areas of Iran were isolates during three months and analyzed by PCR for detection of ompA gene of C. psittaci.Genomic DNA was extracted from 100 ng fecal samples with DNA extraction kit (Qiagen, Germany), according to the manufacturer's instructions.The quality and quantity of extracted DNA was measured at 260 nm optical density according to the method described by Sambrook and Russell (Sambrook and Russell, 2001).The extracted DNA of each sample was kept frozen at -20°C until used.C. psittaci strain ATCC VR-125 (Genekam Biotechnology AG, Germany) was used as positive control and a negative-DNA control was performed by adding 1 μl of sterile ultrapure deionized water.

Gene amplification
The ompA region was amplified by PCR using primers CPsitt-F (5΄-GCTACGGGTTCCGCTCT-3΄) and CPsitt-R (5΄-TTTGTTGATYTGAATCGAAGC-3΄) with accession number AB512087.1 as described by Heddema for ompA region (Heddema et al., 2006).Primers performed at the NCBI using the experimental GENINFO BLAST Network Service to assess degree of homology between these primers and other reported sequences.The samples were placed in a thermal cycler (Mastercycler gradient, Eppendrof, Germany) with an initial denaturation step for 5 min at 95°C, then amplified for 30 cycles of denaturation for 1 min at 94°C, alignment for 1 min at 57°C, extension for 1 min at 72°C and, final extension step for 7 min at 72°C.PCR products were separated by 2% agarose gel electrophoresis and visualised by ethidium bromide staining.The DNA molecular weight marker was used as a size marker.

Results
Out of 445 feces samples 64 (14.3%) were positive for C. psittaci infection using PCR.Analysis of PCR products for presence of ompA gene of C. psittaci on agarose gel revealed a 1041 bp fragment (Figure 1).
The positive control showed the excepted amplification product specific for C. psittaci (1041 bp).To investigate the source of infection, PCR on feces revealed positive results for C. psittaci although these pigeons did not show any symptoms of chlamydial infections.In our study the pigeons were asymptomatically carriers for C. psittaci in their body excretions as feces or respiratory discharges.These results suggest that C. psittaci infection of pigeons is rare or arises only as a result of very close contact, for example, in nests where the risk of infection with various fecal pathogens increases.

Discussion
Chlamydia psittaci is a causative agent of psittacosis, systemic diseases inpsittacine birds which can be of acute, protracted, chronic, or subclinical manifestation and which represents the most important animal chlamydiosis of zoonotic character (Goellner et al., 2006).The Chlamydiaceae are etiological agents of many important human and animal diseases.Formerly called Chlamydia, with only two recognized species 25 years ago (C.trachomatis and C. psittaci), the family now contains nine species divided into two genera, Chlamydia and Chlamydophila (Bush & Everett, 2001).From 2005 to 2009, 66 human cases of psittacosis were reported to the Centers for Disease Control and Prevention (CDC) (Smith et al., 2005).C. psittaci is currently grouped into seven avian genotypes (A through F and a newly identified genotype, E/B) and two non avian genotypes (M56 and WC).Recent reclassification of C. psittaci has resulted in the separation of C. abortus and C. caviae into distinct species, although these species are genetically closely related (Van Loock et al., 2003).
The presence of C. psittaci in current study was 14.3% (64 of 445).Tanaka et al. found C. psittaci in 106 of 463 fecal samples obtained from feral pigeons (Tanaka et al., 2005).Salinas et al. reported the prevalence of C. psittaci in feral pigeons was found by culture in 18% of fecal samples (Heddema et al., 2006).Heddema et al. determined the prevalence and genotype of C. psittaci in fresh fecal samples from feral pigeons in Amsterdam, the Netherlands.The prevalence was 7.9% overall (26/331).Ten genotyped PCR-positive samples were all genotype B (Heddema et al., 2006).
In conclusion, feces of pigeons can spread several zoonotic diseases such as chlamydiosis in Iran.According to these findings it's suggested a vaccine and information on sensible use of antimicrobial drugs in Psittaciformes for prevent to psittacosis in humans and development of drug-resistant bacterial strains.

Figure 1 .
Figure 1.An agarose gel stained with ethidium bromide, for detection of ompA gene in Chlamydia psittaci (Line M: 1 kb DNA ladder (Fermentas, Germany), lines 1 and 2: negative and positive controls, respectively, lines 3, 4 and 7: positive tests and line 5 and 6: negative tests)