Prevalence of Chlamydia psittaci and Other Chlamydia Species in Wild Birds in Poland

Abstract

Avian chlamydiosis is a zoonotic disease occurring in humans, poultry, and exotic birds. It has been suggested that some wild bird species play an important role as reservoirs for Chlamydia, especially Chlamydia psittaci. Whereas C. psittaci is the predominant chlamydial agent in birds, in the present study we have determined the prevalence of different species of Chlamydia among selected wild bird species in Poland using a rapid and sensitive real-time PCR method. In total, 369 free-living birds from 35 bird species and 15 orders were examined. Samples from 27 birds (7.3%) were positive for chlamydial DNA in the PCR; 22 positive samples (81.5%) belonged to C. psittaci, three to Chlamydia trachomatis (11.1%), and two (7.4%) classified only to the genus Chlamydia. Most of C. psittaci–positive samples belonged to five orders: Anseriformes, Columbiformes, Gruiformes, Phasianiformes, and Passeriformes. All C. trachomatis samples were obtained from Eurasian coots (Gruiformes). Two Chlamydia-positive samples not classified to any Chlamydia species were obtained from a common wood pigeon (Columbiformes) and a common buzzard (Accipitriformes). Detection of C. psittaci and C. trachomatis in free-living bird populations force to think on significance of birds as reservoir of varied Chlamydia species and their epidemiological importance.

Introduction

Chlamydiosis is an important infectious disease of people and many animals, including birds. It is caused by the Gram-negative obligatory intracellular bacterium Chlamydia spp., mostly by C. psittaci in birds (Vanrompay 2013). The disease caused by this pathogen in birds is known as avian chlamydiosis and it can cause diseases in humans (Rehn et al. 2013). The groups of people in close contact with birds, like avian breeders, veterinarians, workers of poultry farms, etc., are particularly exposed to Chlamydia infections. There are few reports of cases of human-to-human psittacosis (Wallensten et al. 2014).

The Chlamydia spp. can cause an important disease of birds that is most commonly found in parrots (Psittaciformes) and pigeons (Columbiformes). The outcome of the illness is mostly chronic with conjunctivitis or is asymptomatic, but it might also appear as an acute disease. Depending on the chlamydial strain and the avian host, chlamydiae cause pericarditis, air sacculitis, pneumonia, lateral nasal adenitis, peritonitis, hepatitis, and splenitis. Generalized infections result in fever, anorexia, lethargy, diarrhea, and occasionally shock and death. Chlamydiosis is a very common chronic infection of psittacine birds. Infections cause conjunctivitis, enteritis, air sacculitis, pneumonitis, and hepatosplenomegaly (Harkinezhad et al. 2009). The asymptomatic birds can play a role in the transmition of Chlamydia in the environment, and thereby might be significant in epidemiology of chlamydiosis. C. psitaci has been isolated from more than 460 avian species in the wild (Kaleta and Taday 2003); however, little is known about the prevalence of other species of Chlamydia among wild bird population worldwide. Some reports suggest that avian infections might be caused only by C. psittaci (Harkinezhad et al. 2009), but others have confirmed infections caused by other species of Chlamydia such as C. pecorum and C. trachomatis (Sachse et al. 2012), C. abortus (Herrmann et al. 2000, Pantchev et al. 2009), and C. ibidis (Vorimore et al. 2013).

The aim of this study was to determine the prevalence of C. psittaci and other Chlamydia spp. in various species of wild birds in Poland.

Materials and Methods

The samples were collected from September, 2011, to August, 2013. This study included 369 wild birds from 15 orders and 35 different species, which are presented in Table 1. All tested birds were dead, and material, including lungs, liver, spleen as well as conjunctival swabs from both eyes, were collected during necropsy. Mallard ducks, Eurasian coots, and pheasants were obtained during the hunting season by two hunting associations in accordance with local hunting laws, special permission, and hunting programs. Samples from great cormorants were obtained during the annual population culling from the lakes of the Lower Silesia region in Poland with the consent of the Regional Directorate of Environmental Protection, Wrocław, Poland (no. WPN. 6205.67.2012.MK.1). Samples from velvet scoters were collected from birds that were found dead in fishing nets on the Baltic coast between February and April of 2013. All small garden birds were found dead and brought to our University by ornithologists, veterinarians, or private individuals. The remaining tested wild bird species were delivered after death from wildlife rescue centers, where they had died before treatment procedures were started.

Table 1.

Orders and Species of Birds Examined in the Present Study

Order of birds	Birds species (no.)	Total of samples
Accipitriformes	Common Buzzard (5), Sparrow Hawk (1)	6
Anatiformes	Mallard Duck (120), Velvet Scoter (30), Mute Swan (5), Long-Tailed Duck (1)	156
Apodiformes	Common Swift (9)	9
Charadiformes	Razorbill (1), Guillemot (1), Woodcock (1)	3
Ciconiiformes	White Stork (3)	3
Columbiformes	Common Wood Pigeon (2), Rock Pigeon (2)	4
Falconiformes	Kestrel (8)	8
Gaviiformes	Red-Throated Loon (1)	1
Gruiformes	Eurasian Coot (7)	7
Passeriformes	Eurasian Siskin (7), Eurasian Coot (7), Eurasian Tree Sparrow (5), Hooded Crow (2), Great Tit (6), Bochemian Waxwing (3), Rook (6), Greenfinch (4), Black Red Start (3), Blue Tit (2), House Sparrow (1), Blackbird (1), Barn Swallow (1), Magpie (1)	49
Pelecaniformes	Gray Heron (3)	3
Phasianiformes	Common Pheasants (7)	7
Podicipediformes	Great Crested Grebe (1)	1
Strigiformes	Tawny Owl (1), Little Owl (1)	2
Suliformes	Great Cormorants (110)	110
Total		369

DNA was extracted and purified with a Syngen Tissue DNA Mini Kit, according to the manufacturer's manual (Syngen Biotech, Poland). The DNAs of reference strains were included as positive controls and were as follows: C. suis VR-1474 (American Type Culture Collection [ATCC], USA), C. trachomatis and C. abortus (NRVI, Puławy, Poland), Chlamydophila felis strain 905 (Merial, France), and Chlamydophila psittaci (field isolate obtained from birds in our laboratory described previously by Piasecki et al. (2012). Mycoplasma spp. ATCC 2391 (LGC Standards, Poland) and Staphylococcus epidermidis ATCC 35984 (PAN, Wroclaw, Poland) were included as negative controls.

All DNA samples were tested by using the quantitative PCR (qPCR) procedure described by Everett et al. (1999). Products obtained with the PCR were sequenced (Genomed, Poland) and identified using BLAST (blast.ncbi.nlm.nih.gov). This test targeted the 23S ribosomal RNA gene. The oligonucleotides were synthesized by Sigma-Aldrich (Germany). Primer and probe sequences used during the study are provided in Table 2. Each 20-μL reaction mixture for real-time PCR with the TQF/TQR primers and probe, as described by Everett et al. (1999), contained 10 μL of KAPA PROBE FAST Bio-Rad iCycler 2x qPCR Master Mix (KapaBiosystems, USA), 10 μM of each primer, 10 μM of the probe, 4.2 μL of Milli-Q water, and 5 μL of genomic DNA. The cycling conditions were 40 cycles of 0:15 min at 95°C and 1:00 min at 60.5°C, with an initial denaturation for 3:00 min at 94°C. Real-time PCR was performed using Bio-Rad iQ5 (Bio-Rad, Poland).

Table 2.

Primer and Probe Sequences Used During the Study by Everett et al. 1999

Test	Primer	Sequences of oligonucleotides
Real- time PCR	TQF	5′-GAAAAGAACCCTTGTTAAGGGAG-3′
	TQR	5′-CTTAACTCCCTGGCTCATCATG-3′
	Probe	FAM-CAAAAGGCACGCCGTCAAC-TAMRA

Results

Chlamydia-positive results were obtained from 27 out of 369 birds, comprising 7.3% of all of examined samples. Chlamydia-positive birds belonged to 13 species of birds (from eight orders) including: Hooded Crow, Common Wood Pigeon and Rock Pigeon, Eurasian Coot, Pheasant, Waxwing, Eurasian Tree Sparrow, Common Buzzard, Great Tit, Eurasian Siskin, Common Swift, Great Cormorant, and Mallard Ducks (Table 3).

Table 3.

Chlamydial DNA Detected in Free-Living Birds

			No. (%) of Chlamydia spp.–positive birds
Order of birds	Bird species/total no. of samples	No. (%) of positive samples	C. psittaci	C. trachomatis	None classified Chlamydia
Accipitriformes	Buteo buteo	1 (20.0)	0 (0.0)	0 (0.0)	1 (100.0)
	Common Buzzard
	n = 5
Anseriformes	Anas platyrhynchos	5 (4.16)	5 (100.0)	0 (0.0)	0 (0.0)
	Mallard Duck
	n = 120
Apodiformes	Apus apus	1 (11.11)	1 (100.0)	0 (0.0)	0 (0.0)
	Common Swift
	n = 9
Columbiformes	Columba palumbus	1 (50.0)	0 (0.0)	0 (0.0)	1 (100.0)
	Common Wood Pigeon
	n = 2
Columbiformes	Columba livia	1 (50.0)	1 (100.0)	0 (0.0)	0 (0.0)
	Rock Pigeon
	n = 2
Gruiformes	Fulica atra	3 (42.86)	0 (0.0)	3 (100.0)	0 (0.0)
	Eurasian Coot
	n = 7
Passeriformes	Spinus spinus	1 (14.28)	1 (100.0)	0 (0.0)	0 (0.0)
	Eurasian Siskin
	n = 7
Passeriformes	Corvus cornix	1 (100.0)	1 (100.0)	0 (0.0)	0 (0.0)
	Hooded Crow
	n = 1
Passeriformes	Parus major	1 (16.67)	1 (100.0)	0 (0.0)	0 (0.0)
	Great Tit
	n = 6
Passeriformes	Bombycilla cedrorum	1 (33.33)	1 (100.0)	0 (0.0)	0 (0.0)
	Waxwing
	n = 3
Passeriformes	Passer montanus	1 (25.0)	1 (100.0)	0 (0.0)	0 (0.0)
	Eurasian Tree Sparrow
	n = 5
Phasianiformes	Phasianus colchicus	3 (42.86)	3 (100.0)	0 (0.0)	0 (0.0)
	Pheasant
	n = 7
Suliformes	Phalacrocorax carbo	7 (6.36)	7 (100.0)	0 (0.0)	0 (0.0)
	Great Cormorant
	n = 110
Others	Others (22 species)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
	n = 85
Total	n = 369	27 (7.30)	22 (81. 48)a	3 (11.10)a	2 (7.40)a

^aThe rate of respective Chlamydia species number calculated from the number of all Chlamydia-positive (27) birds.

Twenty-two positive samples (81.5%) belonged to C. psittaci, three (11.1%) to C. trachomatis, and two (7.4%) were identified just as Chlamydiacae but were not classified to any Chlamydia species. All positive results came from DNA obtained from tissues (lungs, liver, spleen). All spleens and livers of Chlamydia-positive birds showed a positive result in the real-time PCR, but only nine lungs were Chlamydia-positive in the same test. No positive results were seen in the swabs.

The PCR products were sequenced. Sequencing confirmed 99–100% genetic similarity to the C. psittaci (gi|485099180|ref| NR 102574.1|:508-637 C. psittaci 6BC strain 6BC 23S ribosomal RNA complete sequence [2]) and C. trachomatis (gi|527130011|gb|CP002677.1|:882052-882179 C. trachomatis RC-F(s)/342 complete genome) gene fragment sequences obtained from GenBank, respectively. Two products were identified only as Chlamydia spp., but sequencing did not confirm a high degree of similarity of these two products to any Chlamydia species.

Discussion

The detection rate (7.3%) of Chlamydiacae in all birds examined in the present study is in compliance with the rates reported by other authors, which showed high variability, ranging from 1% to 83% (Blomquist et al. 2012, Madani and Peighambari 2013, Ribeiro et al. 2013). The Chlamydia-positive detection rate in our study was also variable among different orders, as in results obtained by Kaleta and Taday (2003), who reported that many positive wild birds came from orders such as Lariformes (28%), Alciformes (26%), Sphenisciformes (25%), and Anseriformes (21%). Although all birds seem to be a host of C. psittaci, there are some groups and orders that are supposed to be more naturally infected than others. Waterfowl seem to have a high carrier state. In present study, the water and wetland birds (especially Anseriformes, n = 120, and Suliformes, n = 110) were the biggest group of all examined birds. However, the percentage of Chlamydia-positive birds for the Suliformes and Anseriformes orders were 6.36% and 4.16%, respectively. The result obtained in the order Anseriformes was lower than described by Kaleta and Taday (2003).

On the basis of the available data, C. psittaci has been traditionally assumed to be the only relevant chlamydial agent in birds. Results obtained in our study show that more than only the classical C. psittaci, such as some atypical avian strains of Chlamydiacae, might be detected in free-living birds. C. trachomatis, a typical human Chlamydia species was detected in three Eurasian coots. C. trachomatis was previously described in other species of birds by other authors. Sachse et al. (2012) provided evidence of the occurrence of C. trachomatis in urban pigeons. Some authors reported the prevalence of other chlamydial species than C. psittaci, such as C. pecorum (Sachse et al. 2012) and C. abortus (Herrmann et al. 2000, Pantchev et al. 2009), as well as C. ibidis (Vorimore et al. 2013) in birds.

Further reports on wild birds or feral pigeons as a source of C. psittaci for humans and other animals exist (Travnicek et al. 2002, Harkinezhad et al. 2009, Zweifel et al. 2009, Dickx et al. 2010). The detection of C. trachomatis in the three Eurasian coots might suggest a crossover of chlamydial infections between humans and free-living birds. The three positive Eurasian coots were caught at one pond, a location at which people and whole families spend their free time.

Very often birds might be carriers of Chlamydia asymptomatically, but sometimes avian chlamydiosis manifests as an initial upper respiratory tract infection or progresses to a severe pneumonia, polyserositis, and/or hepatitis (Harkinezhad et al. 2009). Most of the birds examined by us did not show any pathological changes during the necropsy related to a chlamydial infection. Only one Eurasian Siskin had nodular lesions in the liver and spleen induced by a concurrent salmonellosis. This Salmonella infection case has been described by Krawiec et al. (2014). These findings confirm the report of Harkinezhad et al. (2009) regarding the severity of clinical signs, which depend on the host species immunity and which may be triggered by a stress factor, such as another accompanying infection.

Conclusions

The present study shows the prevalence of Chlamydia spp. in a population of wild birds. Mainly C. psittaci but also C. trachomatis were detected in various bird orders. The Chlamydia-positive birds belonged to Accipitriformes, Anatiformes, Apodiformes, Columbiformes, Gruiformes, Passeriformes, Phasianiformes, and Suliformes. Different infection rates of Chlamydia in wild birds as well as the detection of other species than C. psittaci confirm that free-living birds might be a reservoir of this zoonosis. It also shows that our knowledge on chlamydial organisms in birds is still fragmentary.

Acknowledgments

This research project was financed by the National Centre for Research and Development, project number no. 12 0126 10. The authors are grateful to Paweł Lis, PhD, for help with the biotechnological analyses and to Mieczysław Łyskawa (M.Sc., Eng.) and Piotr Zajiczek for their help with collecting the samples.

Author Disclosure Statement

No competing financial interests exist.