目的2015年3-5月调查吉林省长春市和吉林市肉鸽与信鸽中鹦鹉热衣原体的流行情况及基因型分布。方法本研究共采集鸽子粪便样本399份,其中长春市样本282份,吉林市样本117份。利用PCR技术进行鹦鹉热衣原体 ompA基因扩增、测序以及基因型分析。结果本实验结果显示:鹦鹉热衣原体的感染率为5.01%(21/399),其中吉林市鹦鹉热衣原体的感染率(9.40%)明显高于长春市的感染率(3.19%)。此外,品种也是与衣原体感染相关的主要风险因素, 肉鸽的感染率为7.49%,而信鸽的感染率为0。 ompA基因的序列分析显示,这些鹦鹉热衣原体都属于B型。结论综上所述,我国吉林省肉鸽具有较高的B型鹦鹉热衣原体流行,给人类的健康带来了潜在的威胁。
Chlamydia psittaci is a causative agent of psittacosis, which can infect a wide range of hosts including birds and humans. However, information regarding C. psittaci infection in pigeons is scarce. In the present study, a total of 399 fecal samples from pigeons were collected from Jilin Province, northeastern China, between March and May 2015, and examined by nested PCR amplification of outer membrane protein A ( ompA) gene. The overall Chlamydiosis prevalence was 5.01% (21/399), with 3.19% in Changchun City and 9.40% in Jilin City. Furthermore, breed was the major risk factor associated with Chlamydia infection in pigeon, boiler pigeons had a prevalence of 7.49%, whereas no C. psittaci was detected in racing pigeons. Sequence analysis of the ompA gene revealed that all the identified isolates represented C. psittaci genotype B. Our results firstly indicated the presence of zoonotic C. psittaci in boiler pigeons in Jilin Province, northeastern China, and effective measures should be implemented to reduce the risk of C. psittaci transmission from pigeons to humans.
Chlamydia is a kind of parasitic gram-negative bacteria that can infect a range of hosts worldwide. Chlamydiosis (also called psittacosis) caused by C. psittaci has been reported in humans, birds and mammals[1, 2]. More than 400 avian species have been identified as reservoir hosts for C. psittaci[3]. Hosts acquire C. psittaci infection mainly through inhalation of infected excretions and discharges[4, 5], showing the symptoms of clinical (gastrointestinal disease and encephalitis) and/or subclinical, and even death[4].
C. psittaci is classified into nine genotypes, namely A to F, E/B, M56 and WC, based on sequences of the outer membrane protein A (ompA) gene[6]. Interestingly, they seem to be animal-specific[7]. For example, genotype A was commonly found in Psittaciformefas (cockatoos, parakeets, lories), C in Anseriformes (mainly ducks and geese), E/B in ducks, D in turkeys and B in Columbiformes (doves and pigeons)[8]. However, occasionally, A, C, D, E, E/B have also been identified in pigeons[9, 10, 11].
In 1940, pigeon was firstly identified as the reservoir host of C. psittaci[12]. Thereafter, a large number of studies concerning C. psittaci infection in pigeons have been recorded[9, 10, 11, 13], but limited information about the C. psittaci genotypes in pigeons in China is available[14, 15]. The aims of the present study were to estimate the C. psittaci prevalence in pigeons in Jilin Province and characterize their genotypes.
The investigation was carried out in two cities in Jilin Province, northeastern China. Changchun (n=282, 43° 05'-45° 15'N, 124° 18'-127° 05'E) is the capital of Jilin Province, and it is one of the central cities in Northeast China. Jilin City (n=117, 42° 31'-44° 40'N, 125° 40'-127° 56'E) is also located in Jilin Province. The climate of Jilin Province is northerly continental monsoon type, and the average annual temperature is -10 ℃ to -23 ℃, moreover, the annual precipitation is 400-600 mm.
A total of 399 fecal samples were collected from 282 pigeons in Changchun City, and from 117 pigeons in Jilin City, northeastern China, between March and May 2015. Each of these samples were collected into a separate sterile glove after the fecal defecation onto the ground, immediately, and then were sent to the laboratory, and stored at 4 ℃ until further analysis. Information about breed, age and geographic origin of pigeons were acquired, and listed in Tables 1 and 2.
The Stool DNA kit (Omega, USA) was used to extract the genomic DNA from feces. All the operations were performed according to the manufacturer̓s recommendations. All the obtained DNA samples were stored at -20 ℃ until PCR analysis. The ompA gene was used to determine the C. psittaci species/genotypes by semi-nested PCR[16, 17]. The PCR reaction was performed under the conditions of 5 min at 95 ℃ for initial denature, followed by 40 cycles of 20 sec at 95 ℃, 1 min at 55 ℃, and 1 min at 72 ℃, and terminated at 72 ℃ for 10 min. The PCR products were electrophoresed in 1% agarose gels containing 0.5 μ g/mL GoldViewTM (Solarbio, China) and were observed under UV light.
To determine the genotypes of C. psittaci, a 1 000 bp fragment of the ompA gene was amplified using a pair of primers, namely FOMPF1/ FOMPF2, according to previous studies[18, 19]. The positive PCR products were sent to Genscript Company (Nanjing, China) for sequencing. All the obtained sequences were then aligned with reference sequences of Chlamydia available in GenBank using the software of Clustal 2.0 and BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). Neighbor-joining (NJ) method (Kimura 2-parameter model) with 1 000 bootstrap replicates was used to analyze the evolutionary relationships using the Mega 6.0 software.
In the present study, of 399 pigeons, 20(5.01%) samples were detected Chlamydia-positive, with 9 (3.19%) in Changchun and 11 (9.04%) in Jilin. Prevalence of Chlamydia in different farm groups ranged from 0% to 15%. Boiler pigeons has a 7.49% prevalence Chlamydia, whereas no racing pigeon was detected positive for Chlamydia. In addition, Chlamydia prevalence in young and adult pigeons was 6.38% and 4.83%, respectively. Sequences analysis of the ompA gene indicated that all 20 Chlamydia isolates were C. psittaci genotype B. The representative sequences from the present study were deposited in the GenBank database with accession nos of KU937362 and KU937363.
The overall Chlamydia prevalence (5.01%, 20/399) in this study was lower compared with the 19.4%-95.6% seroprevalence rates in pigeons in Europe[10], 10% seroprevalence in racing pigeons in Beijing (37/370)[15], and 31.09% seroprevalence in pigeons in north-western China[14]. Furthermore, it is also lower than that in pigeons in Belgium (6.3%)[11], Poland (7.6%)[20], but higher than that in pigeons in Switzerland (3.2%, 3.3%)[21, 22]. Feeding and living conditions, different detection methods, socioeconomic and ecological conditions may contribute to these differences[14].
Transmission of C. psittaci occurs mainly through the respiratory tract[5], so higher breeding density in Jilin City is possibly one of the most important reasons why pigeons in Jilin City (9.40%) has a significantly higher C. psittaci prevalence than that from Changchun (3.19%). Moreover, the different raising conditions and individual health status may also be related to the difference. Probably due to good animal husbandry practice in racing pigeon industry, no racing pigeon was tested Chlamydia-positive, but higher prevalence was detected in boiler pigeons, the difference was significant statistically (P=0.001 3).Moreover, no significant difference was found among different age groups (P=0.65), which was different to a report in pet birds in northwest China[19].
Many gene loci, such as the inclusion membrane protein A gene (IncA)[23], ribosomal RNA genes (16S-23S), and the ompA gene[24, 25], have been used to detect Chlamydia infection previously. In the present study, the genotypes of Chlamydia were determined by nested PCR amplification of the ompA gene, one of the most commonly used loci for identifying the Chlamydia genotypes[10]. Although genotypes A, B, C, D, E and E/B have been recorded in pigeons previously[26], only genotype B was identified in this study. The results further confirmed that genotype B was the most prevalent Chlamydia genotype in pigeons[27, 28]. In addition to pigeons, genotype B was also reported in a range of other animals and humans around the world[8, 15], which indicates that pigeons may be an important resource for human infection. Moreover, it is also reported in adult chickens, ducks and pigeons in northeastern China[14], which suggests that transmission of C. psittaci genotype B might occur in these areas, which raises public health concern. Interestingly, the genotype B identified in this study was also found in pigeons in USA[8], Sweden[29], Poland[20] and several European countries[6, 28], indicating a worldwide distribution.
The present study revealed the occurrence of Chlamydia infection in pigeons in Jilin Province for the first time. DNA sequence analysis indicates that all the isolates represent C. psittaci genotype B. Moreover, this study provides important base-data for designing and executing strategies and measures for controlling C. psittaci infection in pigeons and humans in the examined areas.
The authors have declared that no competing interests exist.
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