Background
The order Chlamydiales contains at least four distinct family groups: Chlamydiaceae, Simkaniaceae, Waddliaceae and Parachlamydiaceae. Within the family Chlamydiaceae there are two distinct genera: Chlamydia and Chlamydophila. The Chlamydophila genus has seven recognised species, namely Chlamydophila pecorum, Chlamydophila pneumoniae, Chlamydophila psittaci, Chlamydophila abortus, Chlamydophila caviae (formerly Chlamydophila psittaci guinea pig conjunctivitis strain) and Chlamydophila felis12.
A unique developmental cycle distinguishes Chlamydophila from other intracellular bacteria 3. The infectious elementary body (EB) and the vegetative reticulate body (RB) are two major developmental forms involved in the cycle. One of the predominant proteins found on the surface of both the EB and RB forms is the major outer membrane protein (MOMP, OmpA). MOMP makes up 60% of the total outer membrane protein 4, and published data have indicated that it is critical for chlamydial infection 567.
C. psittaci has been isolated and described from various birds and mammals 8910, and has variously been subdivided into the strains/serovars/genotypes A–F, M56 and WC (referred to hereafter as genotypes). Each genotype is assumed to exhibit stringent host specificity: A in psittacine birds, B in pigeons, C in ducks and geese, D in turkeys, E in pigeons, ducks and others avian species, F in parakeets, WC in cattle and M56 in rodents 11121314. All of the C. psittaci genotypes pose a zoonotic threat and are thus of concern to human health. Psittacosis, an infection caused by C. psittaci in birds, can cause acute, protracted, chronic, or subclinical disease, and can persist in the host for months to years, often without causing obvious illness. In humans, the symptoms are mainly non-specific and influenza-like, however, severe pneumonia, endocarditis, and encephalitis are not uncommon 1516. Smith et al. 17 reported that there were a total of 935 human cases of ornithosis confirmed by the US Centers for Disease Control and Prevention (CDC) from 1988–2003. In addition, from 2005–2009, 66 human cases of ornithosis were reported to the CDC through the Nationally Notifiable Diseases Surveillance System (NNDSS). In general, these cases occurred following exposure to infected pet birds, usually cockatiels, parakeets and macaws. Vanrompay et al. 10 investigated zoonotic transmission of C. psittaci within Belgian psittacine breeding facilities. C. psittaci DNA was detected in 59 of 308 (19.2%) parrots tested, and in six of 46 (13%) bird owners. Genotypes A or E/B were detected in 14.9% of humans at these facilities. In Poland, during the reporting period of 2000–2011, only 19 human cases of ornithosis were confirmed by the National Institute of Public Health, however, no cases were diagnosed in the last three years of the reporting period (2009–2011) 18. Despite this detection in humans, little is known about the status of infection of captive parrots in Poland, which could pose a zoonotic hazard. Therefore, to obtain some baseline data for Poland, the prevalence of C. psittaci in various parrot species that did not exhibit clinical signs of infection was determined using two different polymerase chain reaction (PCR) amplification assays. We also used restriction fragment length polymorphism (RFLP) to determine the genotype of the Chlamydophila positive samples.
Methods
Sample collection
In total, 156 tracheal swab samples were collected from 34 different parrot species (Table 1). The parrots ranged in age from 8 months to 3 years old. The samples were collected from 2007–2012 from birds that were housed in private aviaries and zoological shops in Poland, and that were in contact with humans. Some of these parrots were raised in an outdoor coop; however there was no direct contact with wild birds. In some cases, the parrots were housed with pigeons. Clinical investigation and oral histories of the birds provided by the owner did not reveal any indication of diseases, including chlamydiosis. The research was conducted with the consent of the 2nd Local Ethical Committee for Animal Experiments (Wroclaw, Poland) (No. 84/2006).
<p>Table 1</p>
The incidence of
Chlamydophila psittaci
infections in various psittacine species from Poland
Genus
Species
Number of
birds tested
Number of
Ch. psittaci
positive birds
Agapornis
Agapornis roseicollis
2
0
Agapornis personata
6
1
Amazona
Amazona aestiva
6
1
Amazona barbadensis
2
0
Amazona finschi
4
2
Amazona leucocephala
2
0
Ara
Ara ararauna
12
0
Ara chloroptera
4
0
Ara severa
1
0
Aratinga jandaya
3
0
Aratinga solstitialis
2
1
Cacatua
Cacatua alba
2
0
Cacatua moluccensis
1
0
Diopsittaca
Diopsittaca nobilis
1
0
Eclectus
Eclectus roratus
8
1
Forpus
Forpus paserinus
1
0
Melopsittacus
Melopsittacus undulatus
19
2
Myiopsitta
Myiopsitta luchsi
1
0
Neophema
Neophema splendida
1
0
Nymphicus
Nymphicus hollandicus
5
0
Platycercus
Platycercus elegant
8
4
Platycercus eximius
3
0
Platycercus icterotis
1
1
Poicephalus
Poicephalus robustus
1
0
Poicephalus senegalus
1
0
Polytelis
Polytelis alexandre
1
0
Polytelis anthopeplus
1
0
Probosciger
Probosciger aterrimus
1
0
Psephotus
Psephotus haematonotus
1
1
Psittacula
Psittacula alexandri
2
0
Psittacula eupatria
5
1
Psittacula krameri
30
1
Psittacus
Psittacus erithacus
16
0
Trichoglossus
Trichoglossus haematodus
2
0
Total number (%)
156
16 (10,26)
DNA isolation
DNA was extracted from pharyngeal swab samples using a Genomic DNA Prep Plus kit (A & A Biotechnology, Gdynia, Poland) according to the manufacturer's instructions, and the extracted DNA was quantified by spectrophotometry (BioPhotometer, Eppendorf, Poland).
CpsiA/B PCR assay
PCR amplification of the pmp gene was performed using the primers CpsiA, 5′-ATG AAA CAT CCA GTC TAC TGG-3′, and CpsiB, 5′-TTG TGT AGT AAT ATT ATC AAA-3′19, and the following cycling conditions: initial denaturation at 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 50°C for 30 s and 65°C for 2 min, and a final extension at 65°C for 10 min. The PCR reaction contained 50 ng of template DNA, 1 U Green Taq polymerase (Fermentas, Vilnius, Lithuania), 10× PCR buffer (Fermentas), 3 mM MgCl2, 200 μM dNTPs (Fermentas, Vilnius, Lithuania) and 20 pmol of each primer (Genomed, Warszawa, Poland). The expected product size amplified by these primers was ~300 bp 20.
MOMP gene PCR assay
DNA was extracted as described above and then amplified by PCR using the primers CTU, 5′-ATG AAA AAA CTC TTG AAA TCG G-3′, and CTL, 5′-CAA GAT TTT CTA GAY TTC ATY TTG TT-3′21, which target an ~1.1-kb fragment of the highly conserved region of the outer membrane protein A gene, ompA, the structural gene for MOMP 22. PCR reactions contained 50 ng template DNA, 1 U Green Taq polymerase, 10× PCR buffer, 1.5 mM MgCl2, 200 μM dNTPs and 20 pmol of each primer in a final volume of 25 μl. Reaction conditions were as follows: 30 cycles of 1 min at 95°C, 1 min at 51°C and 2 min at 72°C, followed by a final extension at 72°C for 7 min.
The amplification products were resolved on a 1.5% agarose gel, stained with ethidium bromide and visualised using a Gel-Doc UV transilluminator system (BioRad, Warszawa, Poland) and Quantity-One software (BioRad).
PCR-RFLP genotyping
Samples that were positive for C. psittaci were then examined by restriction enzyme digestion. ompA PCR products from the positive samples were digested with 2 U of AluI for 5 h at 37°C to determine their ompA genotype. The restriction products were examined by electrophoresis using a 6% agarose gel (Resolva, GQT, PRONA, ABO, Gdańsk, Poland) stained with ethidium bromide. Gels were viewed under UV illumination and analysed using Quantity-One software. Following agarose gel electrophoresis, cleavage patterns were compared with the sizes and patterns of the DNA bands from of each of the previously described C. psittaci genotypes, as described by Sayada et al. 23. Additionally, RFLP-PCR profiles were analyzed by composing a data matrix built on the presence (1) or absence (0) of any fragment appearing in each strain. Cluster analysis of the pairwise similarity values was performed using the UPGMA (unweighted pair group method using averages) algorithm 24 and Dice similarity coefficients.
Results
Sixteen of 156 the birds tested were positive for C. psittaci using the PCR MOMP gene assay, corresponding to 10.3% of the examined population (Table 1). The CpsiA/B PCR assay showed 15 positive samples (Table 2), all of which corresponded to positive samples from the MOMP assay. RFLP analysis of the sixteen ompA amplicons revealed that most of the samples displayed a genotype-A pattern (13 samples), with only two samples showing a genotype-B pattern (Figure 1). We were unable to assign a genotype to one sample because it did not match any of the previously published patterns. Interestingly, no product was amplified from this sample in the CpsiA/B PCR assay.
<p>Table 2</p>
The list ofCh.psittaci-possitive parrots
Species
Laboratory
No.
PCR
RFLP-PCR
Genotype
OMP
gene
assay
CpsiA/B
The results of genotyping. Legend: *the birds which were kept with pigeons, NN-unidentified.
Agapornis personata
987
+
+
A
Amazona aestiva
1235
+
+
A
Amazona finschi
542
+
-
NN
Amazona finschi
541
+
+
A
Aratinga solstitialis
1214
+
+
A
Eclectus roratus*
624
+
+
B
Melopsittacus undulatus*
1240
+
+
B
Melopsittacus undulatus
1306
+
+
A
Platycercus elegans
902
+
+
A
Platycercus elegans
903
+
+
A
Platycercus elegans
1229
+
+
A
Platycercus elegans
1023
+
+
A
Platycercus icterotis
1026
+
+
A
Psephotus haematonotus
831
+
+
A
Psittacula eupatria
1028
+
+
A
Psittacula krameri
1220
+
+
A
<p>Figure 1</p>Ch.psittaci RFLP profiles generated folowing RE (restriction endonuclease) digestion of OMP PCR gene products from 11 strains
Ch.psittaciRFLP profiles generated folowing RE (restriction endonuclease) digestion ofOMPPCR gene products from 11 strains. M-100bp marker, Lane 2 (No. 542)- unclassified genotype pattern, Lane 3 (No. 624) and lane 11 (No. 1240) – genotype-B pattern, residual- genotype-A pattern.
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Dendrogram of OMP- RFLP patterns of AluI digests was shown on Figure 2. A cluster analysis grouped all 16 strains into one cluster (A), which was divided into two smaller clusters (A1 and A2) with a 78% similarity coefficient. The two samples showing a genotype-B pattern belonged to the A1 cluster and showed 88% similarity in their restriction profiles. The remaining strains, all with a genotype-A pattern, belonged to the second, smaller cluster (A2), and had a high level of similarity. The remaining untypeable strain showed 82% similarity to the genotype-A pattern in its restriction profile.
<p>Figure 2</p>Dendrogram of OMP- RFLP patterns of AluI digests
Dendrogram of OMP- RFLP patterns ofAluI digests.
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Discussion
C. psittaci is generally an avian pathogen, however it can cause zoonotic disease in humans (ornithosis). Birds can shed this bacterium into the environment both when overtly ill, and when asymptomatic. The purpose of this investigation was to survey and collect baseline data, for the first time in Poland, on the prevalence of C. psittaci in parrots, and to determine the genotypes of any positive isolates. All birds examined in this investigation were clinically healthy at the time of sampling; however it is possible that the birds had asymptomatic infections. Asymptomatic animals pose a threat to individuals working with these birds in various facilities and capacities.
We determined a prevalence of C. psittaci of 10.3% amongst captive parrots in Poland. According to official statements, C. psittaci was not reported in humans in Poland between 2009 and 2011. It is possible that many more cases do occur but are not correctly diagnosed or reported. Diagnosis of ornithosis could be hampered by the lack of sensitive detection methods. Culturing-based identification is only performed in select laboratories, and serologic tests do not fully differentiate the various chlamydial microbes.
We noted that C. psittaci genotype-A is the most prevalent amongst parrots in Poland, which is consistent with the global predominance of genotype-A C. psittaci in parrots 14. Interestingly, C. psittaci genotype-B is commonly found in pigeons 2526. The two samples showing a genotype-B pattern in the current study were isolated from parrots that had been raised in pigeon-shared aviaries, thereby indicating cross-species transmission, and the susceptibility of parrots to this genotype. The ungenotypeable strain may belong to another member of the Chlamydophila genus, as this sample was not amplified by the CpsiA/B PCR assay, which Larocau et al. 20 showed only amplifies C. abortus, C. psittaci and C. caviae strains.
This study showed that the two PCR-based methods yielded similar results, both with a high degree of sensitivity. However, the advantage of the MOMP PCR assay is the downstream genotyping by RFLP. This is especially important, as each genotype is assumed to exhibit stringent host specificity. Thus, RFLP-ompA genotyping could be a sensitive method for identifying the susceptibility of animals to different genotypes or cross-species transmission. This assay may also be helpful for better understanding the epidemiology of C. psittaci genotypes. Nevertheless, Larocau et al. 20 showed that a CpsiA/B RFLP assay distinguished between the different serovars of avian chlamydia strains, other than between serovars B and E.
Conclusions
We determined that the prevalence of C. psittaci in asymptomatic captive parrots in Poland was 10.3%. However, no ornithosis was reported in humans during the corresponding sampling period. Nonetheless, there may have been unreported or misdiagnosed cases amongst people in contact with these birds. We determined that the PCR-based typing techniques were rapid and sensitive for identifying C. psittaci genotypes in the majority of cases, particularly when using subsequent RFLP of the MOMP gene amplification products. Large scale analysis of parrots and their handlers using the ompA PCR-based assays could shed light on the incidence of chlamydiosis and ornithosis, and the epidemiology of C. psittaci in Poland.
Competing interest
The authors declare that we have no competing interest.
Authors’ contributions
TP conceived and designed the study, performed laboratory assays and revised the manuscript. KC participated in design the study, laboratory assays, drafted and revised the manuscript. AW participated in coordination the study and helped to draft the manuscript. All authors read and approved the final manuscript.