Detection of Chlamydia psittaci Genotypes Among Birds in Northeast Iran.
Key words: Chlamydia psittaci, chlamydiosis, avian, Psittaciformes, Columbiformes, ompA gene, nested-PCR
Chlamydiosis, or psittacosis/ornithosis in people, is caused by infection with Chlamydia psittaci, an obligate intracellular gram-negative bacterium. (1) According to the most recent classification, the Chlamydiaceae family with a single genus Chlamydia consists of 12 species including C pneumoniae, C abortus, C caviae, C felis, C psittaci, C muridarum, C pecorum, C suis, C trachomatis, C avium, C gallinacea, and C ibidis. (2,3) Recently, two new Chlamydia species (C avium in pigeons and C gallinacea in poultry) have been detected. C psittaci is considered to be the main agent of chlamydiosis in birds. (4)
C psittaci is transmitted primarily from infected birds to other susceptible birds in close proximity, and it has been detected in at least 467 species of birds belonging to 30 various bird orders. (5) The order Psittaciformes contains 153 Chlamydia-positive bird species. (6) Parrots (Psittaciformes) and pigeons (Columbiformes) have the highest infection rates. (6) Clinical disease depends on the chlamydial strain, avian host, age of the bird, degree of exposure, and stress and environmental factors. (7,8) Clinical signs and lesions can include pericarditis, hepatitis, splenitis, peritonitis, conjunctivitis, air sacculitis, rhinitis, sinusitis, anorexia, weight loss, and urate discoloration. (9,10) The main reservoirs of C psittaci are captive and wild birds. (11) All C psittaci genotypes are transmissible to people. (1) Infection is transmitted through inhaling the urine, dried feces, or respiratory secretions of an infected bird; a bite from or mouth-to-beak contact with an infected bird; or handling the plumage and tissues of an infected bird. (10,12) The symptoms of the disease in people are mainly nonspecific, similar to influenza. Severe pneumonia, endocarditis, and encephalitis are uncommon symptoms of ornithosis in humans, which can progress and sometimes cause death. (13) Unfortunately, a licensed vaccine against chlamydial infection in humans has not yet been promoted. (14)
Different diagnostic methods are used to detect C psittaci infection. (15) Polymerase chain reaction (PCR) is one specific and sensitive method. (16) Nested-PCR, developed by Kaltenbock et al., has higher sensitivity compared to other diagnostic protocols to detect C psittaci infection. (17,18) Previous studies on bird populations in Iran have shown C psittaci infection prevalence of 23.5% in pet birds, (9) 14.3% in pigeons, (19) 12.6% in pet and wild birds, (12) and 0% in turkey flocks. (20)
Our study was performed in Khorasan from September 2015 to June 2016. To our knowledge, no previous studies have reported the prevalence and genotyping of C psittaci infection in symptomatic and asymptomatic birds in northeastern provinces (formerly Great Khorasan) of Iran.
Materials and Methods
A total of 200 (70 symptomatic and 130 asymptomatic birds) samples from 2 avian orders and 12 avian species were collected between 2015 and 2016 (Table 1) in the northeast of Iran and evaluated for the presence of C psittaci. The collected samples were choanal cleft and/or cloacal swab samples or freshly voided feces in live birds and/or samples of visceral organs including liver, lungs, and spleen from dead birds. Symptoms related to chlamydiosis (anorexia, weight loss, ocular or nasal secretions, and diarrhea with yellow-green urates) were used to select symptomatic birds. Afterwards, swab samples were placed into 1.5-mL (sucrose phosphate glutamate [SPG]) (21) dilution.
For DNA extraction, we used the High Pure PCR preparation kit (Denazist Asia, Mashhad, Iran) according to the manufacturer's instructions. Based on the primers described by Sachse and Hotzel (15) and under similar conditions, C psittaci was detected by nested PCR using 191CHOMP (5'-GCI YTI TGG GAR TGY GGI TGY GCI AC-3')-CHOMP371 (5'-TTA GAA IC [GT] GAA TTG IGC [AG] [TC] IA GTG IGC IGC TT-3') primers. These produced an amplicon of 576 to 597 base pairs (bp; genus specific) in the first round, which can detect all chlamydial organisms. Then 218PSITT(5'-GTA ATT TCI AGC CCA GCA CAA TTY GTG-3')-CHOMP336 (5'-CCR CAA GMT TTT CTR GAY TTC AWY TTG TTR AT -3') primers (species-specific) were used, which produced 389 to 404 bp amplicon in the second round with some modifications that can only detect C psittaci, C abortus, C felis, and C caviae. (15) The temperature-time profile steps were based on the study by Sachse and Hotzel. (15)
CTU/CTL PCR for sequencing
Positive samples in nested-PCR were used for outer membrane protein A (ompA) gene sequencing with a 1050 bp DNA PCR product using oligonucleotide CTU(5'-ATG AAA AAA CTC TTG AAA TCG G-3')/CTL(5'-CAA GAT TTT CTA GAY TTC ATY TTG TT-3') primers. (22) The DNA extract of IR110SR isolate, which has been described previously, (23) was used as positive control, and sterile water was used as negative control for all PCR reactions. Amplification steps were based on the study by Sayada et al. (22)
Sequencing and genotyping
Manual editing of the sequenced samples was performed by ChromasPro (version 2.1.4; Technelysium Pty Ltd, South Brisbane, Australia) software, partial sequences were aligned with CLC software (CLC Main Workbench version 7; Qaigen, Aarhus, Denmark), and the phylogenetic tree was constructed using different genotype sequences deposited in Gen Bank. Reference sequences of C psittaci and C caviae ompA genes are available in GenBank. The accession numbers of the reference genotype sequences used for multiple alignment analysis are shown in Table 2. To test the association between the presence of clinical signs and the occurrence of C psittaci infection, a [chi square] test was used (IBM SPSS Statistics 21.0; available in the public domain at https://www.ibm.com/ products/spss-statistics). Results were considered significant at P [less than or equal to] .05.
In this study, we detected 18.5% (37/200) of C. psittaci by nested PCR among asymptomatic and symptomatic birds (25.7% [18/70] and 14.6% [19/ 130], respectively). Only 14 PCR products of the 37 positive samples (8 symptomatic and 6 asymptomatic birds) were CTU/CTL PCR-positive and also were subjected to sequencing (Table 3). Positive samples in symptomatic birds included 9 cloacal, 7 choanal cleft, and 2 double swab samples (cloaca and choanal cleft swabs). All positive samples of asymptomatic birds were cloacal swab samples, except for 2 choanal swab samples. All asymptomatic bird samples were collected from Psittaciformes, whereas symptomatic bird specimens were collected from Psittaciformes and Columbiformes. In the Psittaciformes order, 26.2% and 14.6% were C psittaci-positive in symptomatic and asymptomatic birds, respectively. Moreover, 20% of symptomatic Columbiformes were positive for C psittaci. Most swab samples were collected from cockatiels (N hollandicus) with a high detection rate of chlamydiosis. In symptomatic birds, the highest prevalence rate of chlamydial infection was diagnosed in Alexandrine parakeets (P eupatria; 40%) in asymptomatic birds, highest prevalence rates were in ring-necked parakeets (P kramerr, 45.5%) and also in orange-winged Amazon parrots (Amazona amazonica; 100%, 1 positive of 1 sample collection).
Of 14 positive samples by CTU/CTL PCR assay genotyped by ompA gene sequencing, 10 had 100% sequence identity with genotype A, comprising 5 cockatiels, 2 ring-necked parakeets (P krameri), and 3 Congo African grey parrots (P erithacus). Genotype B was detected in 2 ring-necked parakeets and 1 rock dove (C livia). Provisional genotype I was detected in 1 cockatiel. All positive samples of asymptomatic birds belonged to genotype A, whereas positive samples of symptomatic birds showed various genotypes, including A, B, and provisional genotype I. Accordingly, 100% of asymptomatic and 50% of symptomatic positive samples showed genotype A, whereas 37.5% and 12.5% of symptomatic positive samples showed genotype B and provisional genotype I, respectively (Table 3, Fig. 1). No significant association was observed between presence of clinical signs and occurrence of C psittaci infection in birds in this study.
Chlamydia psittaci can cause symptomatic and asymptomatic infections in avian species with intermittent shedding of the organism from infected birds. We examined the prevalence of C psittaci in 200 samples from birds comprising 12 avian species using a sensitive, nested-PCR detection assay. Although various methods are available to identify C psittaci infection, such as cell culture, serology, embryonated chicken egg inoculation, and so forth, PCR is a more sensitive technique. (24,25) Based on results reported by Sachse and Hotzel, (15) sensitivity was higher with nested-PCR compared to cell culture. Conversely, there is risk of contamination and false-positive results with nested PCR. However, PCR and cell culture were nearly equivalent in specificity. (15) Historically, cell culture has been accepted as the gold standard for diagnosis; however, molecular techniques currently are much preferred. (25) Isolation by cell culture can only be performed in specialized biosafety level 3 (BSL3) laboratories (Iranian Universities have no access to such laboratories). Therefore, cell culture requires experienced laboratory personnel, is time consuming, some chlamydial strains simply do not grow in cell cultures or embryonated eggs, and finally, isolation depends on the presence of a sufficient number of viable infectious chlamydial elementary bodies. (8,15)
We examined the prevalence of C psittaci infection in 11 species of psittacine and one species of nonpsittacine birds (pigeons). Choanal and cloacal samples were taken from asymptomatic (n = 130) and symptomatic (n = 70) birds. Of 200 double samples, C psittaci was detected in 37 birds; 25.7% and 14.6% in symptomatic and asymptomatic birds, respectively. Few studies regarding chlamydial prevalence among avian species in Iran, especially in asymptomatic birds, have been published. In 2013, Madani and Peighambari (12) reported that in the Tehran province, 12.6% of 253 samples from 27 avian species belonging to 7 orders were positive; however, they did not report the number of samples obtained from symptomatic or asymptomatic birds. Cell culture isolation of 4 of these samples was reported. (9)
Detecting Chlamydia species from >45% of parrot species sampled confirmed that psittacine birds could be considered as the predominant reservoir of this bacterium among birds. (26) We only tested 5 samples taken from different pigeon squabs with 1 (20%) positive sample; however, some studies have suggested pigeons as the natural reservoir of C psittaci, with a high prevalence. (26-29) Madani and Peighambari (12) also reported that 5 of 28 (17.8%) rock doves tested in Tehran province were positive, which is in agreement with our findings (20%), although we had fewer samples. Similarly, Sariya et al (1) found that 10.8% of the asymptomatic feral pigeons in central Thailand were positive.
An important finding of this study is that the samples from asymptomatic household parrots had the same positive rate (51%; 19/37) as symptomatic parrots (49%; 18/37). This shows almost equal frequency of C psittaci infection among symptomatic and asymptomatic pet birds and could be regarded as a potential infection risk for owners and veterinary staff.
Only 14 of 37 positive samples were CTU/CTL PCR-positive, and this could be due to a lower sensitivity of CTU/CTL PCR compared to the diagnostic nested PCR, as noted by other researchers especially in clinical samples. (12,30)
Phylogenetic tree construction based on neucleotide sequences from this study and published reference sequences deposited in the GenBank revealed that our sequenced strains fall into 3 distinguished clusters mainly with 100% identity with genotype A and in second place with reference genotype B (Fig 1).
Genotype A is endemic among Psittaciformes (African grey parrots, cockatoos, lories, and parakeets) and is considered a major source of human infection. This genotype also has been identified in chickens, turkeys, and wild birds. (30) Genotype B is endemic among pigeons and it also has been reported in psittacine birds, turkeys, and chickens, (31,32) while provisional genotype I was detected in 6 African grey parrots. (12) Madani and Peighambari (12) reported that the provisional genotype I is related to African grey parrots; however, our study has suggested a wider range of hosts for this genotype, because we have detected provisional genotype I in a cockatiel, which is the first report of this genotype in this species.
To our knowledge, this is the first report demonstrating the presence of C psittaci in a superb parrot (Polytelis swainsonii), a green-cheeked parakeet (Pyrrhura molinae), and an orange-winged Amazon parrot in Iran.
All C psittaci genotypes are transmissible to people, and all genotypes are considered a serious threat to those who are in close contact with infected birds. (5) The high virulence of genotype A in people could possibly be related to: (1) high virulence of this genotype for psittacine species, which excrete the bacterium in large amounts for long periods of time, and (2) intensive exposure to Psittaciformes, as the main bird source of these genotypes, which are kept frequently as pets inside the home. (6)
The high infection rate found in our study is a potential threat to public health, especially in Mashhad as the second biggest pilgrimage city in the Middle East and as one of the trade centers for psittacine and passerine birds. Further studies are required to determine the prevalence of C psittaci in pigeons, captive passerine birds, and specific species of popular parrots in Iran and also to establish the genotypes circulating in these birds.
In conclusion, we recommend that health administrators and local veterinary organizations implement control programs, such as regular sampling from all imported birds to prevent smuggling and regular monitoring, international quarantine protocols, and educational programs for owners and domestic breeders to prevent possible transimission of C psittaci.
Acknowledgments: Supported by Grants 40447 and 41920 from the Research Council of the University of Mashhad. We thank Ali Kargar for his assistance in laboratory work.
Abbasi Mina, DVM, Akbarzadeh Fatemeh, DVM, and Razmyar Jamshid, DVM, DVSc
From the Department of Clinical Sciences, Faculty of Veterinary Medicine. Ferdowsi University of Mashhad. Mashhad. Iran (Mina. Fatemeh. Jamshid); the Department of Avian Diseases (Jamshid), Faculty of Veterinary Medicine. University of Tehran. Tehran. Iran (Jamshid).
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Caption: Figure 1. The phylogram was constructed from nucleotide sequences of the ompA gene. The phylogenetic tree was rooted by the ompA gene sequence of Chlamydia caviae. The name of the isolate and genotyping were used to represent our isolates and related references.
Table 1. The incidence of Chlamydia psittaci infection in various symptomatic and asymptomatic bird species that were sampled clinically and diagnosed by nested PCR targeting of the ompA gene. Samples (n) Order Species Scientific Symptomatic name birds Psittaciformes Congo Psiltacus 9 African erithacus grey parrot Timneh Psittacus 4 African timneh grey parrot Ring-necked Psittaca 9 parakeet humeri Alexandrine Psittacii 5 parakeet eupatria Cockatiel Nymphicus 28 hollandicus Lovebird Agapornis 2 roseicollis Budgerigar Melopsittacus 4 undulatus Orange- Amazona 1 winged amazonica Amazon Superb Polytelis -- parrot Swainsonii Barred Bolhorhynclius 3 parakeet lineola Green- Pvrrhura -- cheeked molinae parakeet Columbiformes Rock dove Columha livia 5 Samples (n) Number (%) positive Order Species Asymptomatic Symptomatic birds samples Psittaciformes Congo 12 1 (11.1%) African grey parrot Timneh -- 0 African grey parrot Ring-necked 11 3 (33.3%) parakeet Alexandrine 4 2 (40%) parakeet Cockatiel 62 10 (35.7%) Lovebird 20 0 Budgerigar -- 1 (25%) Orange- 1 0 winged Amazon Superb 4 -- parrot Barred 6 0 parakeet Green- 10 -- cheeked parakeet Columbiformes Rock dove -- 1 (20%) Number (%) positive Order Species Asymptomatic Total samples positive (%) Psittaciformes Congo 4 (33.3%) 23.8% African grey parrot Timneh -- 0% African grey parrot Ring-necked 5 (45.5%) 40% parakeet Alexandrine 0 22.2% parakeet Cockatiel 5 (8.1%) 16.7% Lovebird 1 (5%) 4.5% Budgerigar -- 25% Orange- 1 (100%) 50% winged Amazon Superb 1 (25%) 25% parrot Barred 0 0% parakeet Green- 2 (20%) 20% cheeked parakeet Columbiformes Rock dove -- 20% Table 2. Accession numbers of ompA gene sequences of Chlamydia psittaci and Chlamydia caviae used in this study to detect Chlamydia species in samples collected from 200 Psittaformes and Columbiformes birds. Psittaciformes Genotype Acccssion No. C psittaci A AY762608 C psittaci B AY762609 C psittaci C L25436 C psittaci D AF269266 C psittaci E X12647.1 C psittaci F AF269259 C psittaci E/B AY762613 C psittaci M56 AF269268 C psittaci WC AF269269 C psittaci Provisional I HQ845540 C psittaci Provisional J HQ845545 C caviae -- AF269282 Table 3. Genotyping using CTU and CTL primers (ompA partial DNA sequencing) of Cpsittaci-positive samples from 14 Psittaciformes and Columbiformes birds. Psittaciformes Host Asymptomatic/ symptomatic MFR100IR Ring-necked parakeet S (a) MFR201IR Cockatiel S MFR202IR Cockatiel S MFR005IR Cockatiel S MFR006IR Rock dove s MFR4731R Cockatiel s MFR4381R Ring-necked parakeet s MFR459IR Cockatiel s MFR001IR Ring-necked parakeet A (b) MFR002IR Ring-necked parakeet A MFR008IR Cockatiel A MFR012IR Congo African grey parrot A MFR424IR Congo African grey parrot A MFR325IR Congo African grey parrot A Psittaciformes Genotype GenBank accession number MFR100IR B KY924646 MFR201IR A KY924636 MFR202IR A KY924637 MFR005IR A KY914493 MFR006IR B KY924638 MFR4731R Provisional I KY924639 MFR4381R B KY924641 MFR459IR A KY924640 MFR001IR A KY924647 MFR002IR A KY924644 MFR008IR A KY924645 MFR012IR A KY914492 MFR424IR A KY924642 MFR325IR A KY924643 (a) Symptomatic. (b) Asymptomatic.
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|Author:||Mina, Abbasi; Fatemeh, Akbarzadeh; Jamshid, Razmyar|
|Publication:||Journal of Avian Medicine and Surgery|
|Date:||Mar 1, 2019|
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