吉林省鸽子鹦鹉热衣原体的分子流行病学调查和基因型分布研究
姚秋霞1,2, 张晓轩2,3, 陈凯2, 马剑刚2,3, 郑文斌2,3, 许小琴1, 朱兴全2
1.扬州大学兽医学院,扬州 225009
2.中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,兰州 730046
3.吉林农业大学动物科学与技术学院,长春 130118
通讯作者:许小琴,Email: xuxq@yzu.edu.cn;朱兴全, Email: xingquanzhu1@hotmail.com
摘要

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。

关键词: 鹦鹉热衣原体; 鸽子; 基因型; 吉林省
中图分类号:R374 文献标志码:A 文章编号:1002-2694(2017)02-0104-06 doi: 10.3969/j.issn.1002-2694.2017.02.003
Prevalence and genotypes of Chlamydia psittaci in pigeons in Jilin Province, Northeastern China
YAO Qiu-xia1,2, ZHANG Xiao-xuan2,3, CHEN Kai2, MA Jian-gang2,3, ZHENG Wen-Bing2,3, XU Xiao-qin1, ZHU Xing-quan2
1. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009 China
2. State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences, Lanzhou 730046 China
3. College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118 China
Correspondence authors:Xu Xiao-qin, Email: xuxq@yzu.edu.cn;Zhu Xing-quan, Email: xingquanzhu1@hotmail.com
Abstract

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.

Keyword: Chlamydia psittaci; pigeon; genotypes; Jilin Province

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.

Materials and methods
The study site

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.

Collection and preparation of samples

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.

Tab.1 C.prevalence in pigeons in different farms in Jilin Province, northeastern China
Tab.2 Factors associated with prevalence of chlamydiosis pigeons in Jilin Province, northeastern China

Fig.1 Phylogenetic analyses of C.psittaci based on the 1 019 bp sequence of the ompA gene.
The numbers at nodes indicate bootstrap values. The C. psittaci isolates identified in this study are underlined.

DNA testing and sequencing

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.

Results and discussion

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.

Conclusion

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.

参考文献
[1] Szeredi L, Bacsadi A. Detection of Chlamydophila (Chlamydia) abortus and Toxoplasma gondii in smears from cases of ovine and caprine abortion by the streptavidin-biotin method[J]. Comp Pathol, 2002, 127: 257-263. [本文引用:1]
[2] Pantchev A, Sting R, Bauerfeind R, et al. Detection of all Chlamydophila and Chlamydia spp. of veterinary interest using species-specific real-time PCR assays[J]. Comp Immunol Microbiol Inf Dis, 2010, 33: 473-484. DOI: DOI:10.1016/j.cimid.2009.08.002 [本文引用:1]
[3] Kaleta EF, Taday EM. Avian host range of Chlamydophila spp. based on isolation, antigen detection and serology[J]. Avian Pathol, 2003, 32: 435-461. [本文引用:1]
[4] Beeckman, DSA, Vanrompay, DCG. Zoonotic Chlamydophila from a clinical perspective[J]. Clin Microbiol Infect, 2009, 15: 11-17. DOI: DOI:10.1111/j.1469-0691.2008.02669 [本文引用:2]
[5] Stewardson AJ, Grayson ML. Psittacosis[J]. Infect Dis Clin North Am, 2010, 24: 7-25. [本文引用:2]
[6] Geens T, Desplanques A, Van Loock M, et al. Sequencing of the Chlamydophila psittaci ompA gene reveals a new genotype, E/B, and the need for a rapid discriminatory genotyping method[J]. J Clin Microbiol, 2005, 43: 2456-2461. [本文引用:2]
[7] Sachse K, Laroucau K, Vorimore F, et al. DNA microarray-based genotyping of Chlamydophila psittaci strains from culture and clinical samples[J]. Vet Microbiol, 2009, 135: 22-30. DOI: DOI:10.1016/j.vetmic.2008.09.041 [本文引用:1]
[8] Van Lent S, Piet JR, Beeckman D, et al. Full genome sequences of all nine Chlamydia psittaci genotype reference strains[J]. J Bacteriol, 2012, 194: 6930-6931. DOI: DOI:10.1128/JB.01828-12 [本文引用:3]
[9] Heddema ER, ter Sluis S, Buys JA, et al. Prevalence of Chlamydophila psittaci in fecal droppings from feral pigeons in Amsterdam, The Netherland s[J]. Appl Environ Microb, 2006, 72: 4423-4425. [本文引用:2]
[10] Magnino S, Haag-Wackernagel D, Geigenfeind I, et al. Chlamydial infections in feral pigeons in Europe: review of data and focus on public health implications[J]. Vet Microbiol, 2009, 135: 54-67. DOI: DOI:10.1016/j.vetmic.2008.09.045 [本文引用:4]
[11] Dickx V, Beeckman DSA, Dossche L, et al. Chlamydophila psittaci in homing and feral pigeons and zoonotic transmission[J]. J Med Microbiol, 2010, 59: 1348-1353. DOI: DOI:10.1099/jmm.0.023499-0 [本文引用:3]
[12] Pinkerton H, Swank RL. Recovery of virus morphologically identical with psittacosis from thiamin-deficient pigeons[J]. Exp Biol and Med, 1940, 45: 704-706. [本文引用:1]
[13] Geigenfeind I, Haag-Wackernagel D. Detection of Chlamydophila psittaci from feral pigeons in environmental samples: problems with currently available techniques[J]. Integr Zool, 2010, 5: 63-69. DOI: DOI:10.1111/j.1749-4877.2010.00187 [本文引用:1]
[14] Cong W, Huang SY, Zhang XY, et al. Seroprevalence of Chlamydia psittaci infection in market-sold adult chickens, ducks and pigeons in north-western China[J]. J Med Microbiol, 2013, 62: 1211-1214. DOI: DOI:10.1099/jmm.0.059287-0 [本文引用:4]
[15] Ling Y, Chen H, Chen X, et al. Epidemiology of Chlamydia psittaci infection in racing pigeons and pigeon fanciers in Beijing, China[J]. Zoonoses Public Hlth, 2015, 62: 401-406. DOI: DOI:10.1111/zph.12161 [本文引用:3]
[16] Buxton D, Rae A, Maley SW, et al. Pathogenesis of Chlamydia psittaci infection in sheep: detection of the organism in a serial study of the lymph node[J]. J Comp Pathol, 1996, 114: 221-230. [本文引用:1]
[17] De Lima VY, Langoni H, Da Silva AV, et al. Chlamydophila psittaci and Toxoplasma gondii infection in pigeons (Columba livia) from São Paulo State, Brazil[J]. Vet Parasitol, 2011, 175: 9-14. DOI: DOI:10.1016/j.vetpar.2010.10.006 [本文引用:1]
[18] Herrmann B, Persson H, Jensen JK, et al. Chlamydophila psittaci in fulmars, the Faroe Island s[J]. Emerg Infect Dis, 2006, 12: 330-332. [本文引用:1]
[19] Zhang NZ, Zhang XX, Zhou DH, et al. Seroprevalence and genotype of Chlamydia in pet parrots in China[J]. Epidemiol Infect, 2015, 14: 55-61. DOI: DOI:10.1017/S0950268814000363 [本文引用:2]
[20] Stenzel T, Pestka D, Choszcz D. The prevalence and genetic characterization of Chlamydia psittaci from domestic and feral pigeons in Poland and the correlation between infection rate and incidence of pigeon circovirus[J]. Poultry Sci, 2014, 93: 3009-3016. DOI: DOI:10.3382/ps.2014-04219 [本文引用:2]
[21] Zweifel D, Hoop R, Sachse K, et al. Prevalence of Chlamydophila psittaci in wild birds—potential risk for domestic poultry, pet birds, and public health[J]. Eur J Wildlife Res, 2009, 55: 575-581. [本文引用:1]
[22] Geigenfeind I, Vanrompay D, Haag-Wackernagel D. Prevalence of Chlamydia psittaci in the feral pigeon population of Basel, Switzerland [J]. J Med Microbiol, 2012, 61: 261-265. DOI: DOI:10.1099/jmm.0.034025-0 [本文引用:1]
[23] Ménard A, Clerc M, Subtil A, et al. Development of a real-time PCR for the detection of Chlamydia psittaci[J]. J Med Microbiol, 2006, 55: 471-473. [本文引用:1]
[24] Smith KA, Bradley KK, Stobierski MG, et al. Compendium of measures to control Chlamydophila psittaci (formerly Chlamydia psittaci) infection among humans (psittacosis) and pet birds[J]. J Am Vet Med Assoc, 2005, 226: 532-539. [本文引用:1]
[25] Binet R, Maurelli AT. Frequency of development and associated physiological cost of azithromycin resistance in Chlamydia psittaci 6BC and C. trachomatis L2[J]. Antimicrob Agents Chemother, 2007, 51: 4267-4275. [本文引用:1]
[26] Heddema ER, Van Hannen EJ, Duim B, et al. Genotyping of Chlamydophila psittaci in human samples[J]. Emerg Infect Dis, 2006, 12: 1989-1990. [本文引用:1]
[27] Dolz G, Solórzano-Morales Á, Angelova L, et al. Chlamydia psittaci genotype B in a pigeon (Columba livia) inhabiting a public place in San José, Costa Rica[J]. Open Vet J, 2013, 3: 135-139. [本文引用:1]
[28] Madani SA, Peighambari SM. PCR-based diagnosis, molecular characterization and detection of atypical strains of avian Chlamydia psittaci in companion and wild birds[J]. Avian Pathol, 2013, 42: 38-44. DOI: DOI:10.1080/03079457.2012.757288 [本文引用:2]
[29] Rehn M, Ringberg H, Runehagen A, et al. Unusual increase of psittacosis in southern Sweden linked to wild bird exposure[J]. Euro Surveillance, 2013, 18: 13-20. [本文引用:1]