Prevalence of Chlamydophila psittaci in Fecal Droppings from Feral Pigeons in Amsterdam, The Netherlands

In many cities, the feral rock dove is an abundant bird speciesthat can harbor Chlamydophila psittaci. We determined the prevalenceand genotype of C. psittaci in fresh fecal samples from feralpigeons in Amsterdam, The Netherlands. The prevalence was 7.9%overall (26/331; 95% confidence interval, 5 to 11). Ten genotypedPCR-positive samples were all genotype B.

INTRODUCTION

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Introduction

In many European cities, the feral rock dove (Columbia livia)is an abundant bird species that often lives in close contactwith humans. It is known that pigeons, like many other birdspecies, can harbor Chlamydophila psittaci. This bacterium isa pathogen of birds but can cause zoonotic disease (5). Birdscan shed this bacterium in the environment when they are eitherovertly ill or without any symptoms. In birds, the bacteriumcan be isolated from feces, the cloacae, and respiratory andconjunctiva secretions. For this study, we determined the prevalenceof C. psittaci shedding in feces from feral pigeons in Amsterdam,The Netherlands, and the genotype of the PCR-positive samples.C. psittaci in these specimens was detected with a recentlydeveloped real-time PCR (6).

Setting and sampling.

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Setting and Sampling.

The city of Amsterdam consists of 14 town councils. Pigeon sampleswere obtained from nine locations in eight town councils. Theselocations were geographically widely distributed in Amsterdam,and all were situated in public areas and chosen based on previousresearch of assembling locations for feral pigeons in Amsterdam(3). At these locations, pigeons were attracted with food, andtheir fresh fecal droppings were sampled with sterile cottonswabs (MW&E, United Kingdom). Since shedding occurs intermittentlyand can be activated by stress factors, such as breeding, sampleswere taken on 3 February and 8 March 2005, when breeding activitywas low (low-breeding period), and on 2 May 2005, when breedingwas frequent (12).

DNA extraction and PCR.

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Dna extraction and pcr.

The cotton swabs were placed in 1.5-ml tubes in 300 µlBaker water (Boom B.V. Meppel, The Netherlands) and vortexedthoroughly. Fifty microliters of each fecal suspension was usedas input for the DNA extraction procedure (1). C. psittaci PCRwas performed as previously described (6). Briefly, this real-timePCR targets an 82-bp fragment of the ompA gene of C. psittacias well as an internal control plasmid, using primers CpsittF(5'-CGCTCTCTCCTTACAAGCC-3') and CPsittR (5'-AGCACCTTCCCA CATAGTG-3').The internal control, which was added to each sample, has thesame primer sites, length, and nucleotide content as the C.psittaci amplicon but has a shuffled probe binding region. Toprevent false-positive reactions due to amplicon carryover,we used the uracil N-glycosylase system and a unidirectionalworkflow combined with separation of PCR mix preparation andDNA extraction from all (post)amplification activities.

Genotyping.

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Genotyping.

PCR-positive samples were genotyped by ompA sequence analysis.The gene was amplified with the primers CPsittGenoFor (5'-GCTACGGGTTCCGCTCT-3';nucleotides 400 to 416) and CPsittGenoRev (5'-TTTGTTGATYTGAATCGAAGC-3';nucleotides 1420 to 1441) (nucleotide positions are accordingto the C. psittaci 6BC ompA gene [GenBank accession no. X56980]),resulting in a 1,041-bp amplicon. These primers target the conservedregions of the ompA gene enclosing the four variable domains.Genotype PCR was performed in a LightCycler 2.0 instrument (RocheDiagnostics, Germany). The final reaction mixture (20 µl)included 8 µl eluate and was essentially the same as thatdescribed previously (6). The real-time PCR steps were as follows:50°C for 10 min; 95°C for 10 min; and 45 cycles of 95°Cfor 10 s, 62°C for 5 s, 72°C for 50 s, and 30°Cfor 30 s. Ten microliters of the PCR product was analyzed by1% agarose gel electrophoresis. The expected amplicon was excisedfrom the gel, purified by a simplified guanidinium thiocyanateextraction procedure (2.5 µl silica; wash cycles withL2, ethanol, and acetone), and eluted in 15 µl TE buffer(10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) (2). To obtain sufficientproduct for sequence analysis, reamplification for only 20 cycleswas performed in a GeneAmp 9700 thermocycler (Perkin-Elmer).The reaction mixture for reamplification (50 µl) included2 µl of eluate, 5 µl (10x) PCR II buffer, 5 µgbovine serum albumin, 0.25 U AmpliTaq Gold, a 0.16 µMconcentration of each primer, and 4.5 mM MgCl₂. The PCR stepswere as follows: 95°C for 10 min; 20 cycles of 95°Cfor 1 min, 55°C for 1 min, and 72°C for 2 min; and 72°Cfor 10 min. When a single band of approximately 1,041 bp wasobtained by agarose gel electrophoresis, the PCR product wassubjected to sequence analysis (BigDye Terminator sequencingkit; Applied Biosystems). Overlapping sequences were obtainedwith four sequencing primers, including the above-mentionedgenotype primers and two inner primers, CPsittFinner (5'-CGCTCTCTCCTTACAAGCC-3')and CPsittRinner (5'-GATCTGAATCGAAGCAATTTG-3'). We used theC. psittaci ORNI (genotype A) strain and a C. abortus strainas positive controls. To prevent amplicon carryover, the samemeasures as those described for real-time PCR were taken. Theresulting sequences were aligned, and a similarity index basedon the resulting amino acids was calculated for an 894-bp fragmentof the ompA gene. Similarity (1 – distance) was calculatedby using the pairwise distance method generated by MEGA3 (8).Reference ompA genotype sequences A to F (available in the GenBankdatabase under accession numbers AY762608 to -12 and AF269261)were included in this analysis (4).

Results.

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Results.

In total, 331 fecal samples were obtained, with 160 samplesobtained before and 171 samples obtained during the breedingperiod (Table 1). At least 15 samples were collected at eachlocation. During the low-breeding period, 5% (8/160; 95% confidenceinterval [CI], 2% to 10%) of all samples were PCR positive.Among samples obtained during the breeding period, 10% (18/171;95% CI, 6% to 16%) were positive, and hence the prevalence ofpositive samples during the breeding period was twice that inthe low-breeding period (P = 0.07 by Fisher's exact test; GraphPadSoftware, San Diego, CA). The overall prevalence was 7.9% (26/331;95% CI, 5% to 11%). All negative samples were truly negativesince all internal controls were amplified correctly, thus excludingthe occurrence of PCR inhibition. It was possible to genotype10 of the 26 PCR-positive samples. The obtained sequences wereall 100% similar to reference genotype B. Similarities basedon amino acid sequences were 98% (genotype A), 56% (C), 43%(D), 99% (E), and 51% (F). The positive controls (C. psittaciORNI and C. abortus) were amplified as expected and could subsequentlybe sequenced.

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TABLE 1. Number of C. psittaci PCR-positive fecal samples in feral pigeons by sampling location in Amsterdam, The Netherlands

Discussion.

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Discussion.

This study shows that between 5 and 10% of our sample of urbanferal pigeons in Amsterdam shed C. psittaci in their feces.Only genotype B was found in these isolates. We were unableto genotype all PCR-positive samples. For genotyping, a 1,041-bpfragment had to be amplified; the genotyping PCR was less sensitivethan the optimized diagnostic real-time PCR, which amplifiesa fragment of only 82 bp. Therefore, samples with relativelylow C. psittaci loads could not be amplified by genotyping PCR.The major advantage of this study was the use of an internallycontrolled real-time PCR assay. PCR is a sensitive and specifictest compared to enzyme-linked immunosorbent assays and tissueculture assays available for C. psittaci detection in birds(7, 9). Salinas et al. reported one of the largest series onthe prevalence of C. psittaci in feral pigeons (10). In theirstudy, C. psittaci was found by culture in 18% (7/39; 95% CI,9% to 33%) of fecal samples, a prevalence that is similar toour results obtained by PCR. Recently, Tanaka et al. found C.psittaci in 106 of 463 (22.9%; 95% CI, 19% to 27%) fecal samplesobtained from feral pigeons. However, they did not use exclusivelyfresh fecal samples and applied a nested PCR protocol, whichis known to be particularly prone to contamination (13).

A previous study indicated that in 2001, the pigeon populationsize in Amsterdam averaged approximately 30,000 (3). Combinedwith our results, the number of feral pigeons shedding C. psittaciin their feces would be, on average, about 2,400 (95% CI, 1,500to 3,300). Our isolates were all identical to genotype B. Currently,at least nine genotypes are known. Each genotype is more orless associated with a specific group of birds from which itis most commonly isolated. Geens et al. and Vanrompay et al.also found genotype B to be particularly associated with thepigeon host (4, 14). However, this genotype has been recoveredfrom many bird species, including turkeys, parakeets, and ducks(11, 15). Whether shedding of C. psittaci by feral pigeons inAmsterdam poses a substantial zoonotic risk for humans has tobe determined. Besides the zoonotic potential, there is alsothe risk of infection of domesticated birds, such as pet birdsand poultry, which live in closer contact with human beings.Diagnosing C. psittaci infections has been hampered by a lackof sensitive and specific methods. Culture is only performedin some select laboratories, serologic tests do not fully differentiateinfections with the various Chlamydia spp., and PCR is not routinelyperformed. However, PCR can provide a definitive diagnosis ofpsittacosis. We recommend that psittacosis in humans be diagnosedby detection of the agent by PCR, with or without serologictesting, instead of by serologic testing alone. Subsequent ompAgene sequence analysis can identify the responsible genotype.This approach could lead to a better understanding of the epidemiologyof the different genotypes of C. psittaci in infected bird populationsand human psittacosis cases and determine the relationship betweenthe two.

Nucleotide sequence accession numbers.

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Nucleotide sequence accession...

The ompA sequences of the positive control strains and the genotypeB sequence obtained from the fecal pigeon samples were submittedto GenBank under accession no. DQ267973, DQ435299, and DQ435300.

ACKNOWLEDGMENTS

Y. Pannekoek, University of Amsterdam, Amsterdam, The Netherlands,provided the C. psittaci ORNI strain. D. Vanrompay, Ghent University,Ghent, Belgium, provided the C. abortus strain.

FOOTNOTES

* Corresponding author. Mailing address: Department of Medical Microbiology, Room L1-245, P.O. Box 22660, 1100 DD Amsterdam, The Netherlands. Phone: 31 20 5665731. Fax: 31 20 5669745. E-mail: e.r.heddema{at}amc.uva.nl.

REFERENCES

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