Epidemiological Investigation and Genotype of Chlamydia Exposure in Pigeons in Three Provinces in Northern China

Abstract

Chlamydia is considered as one of the most widely prevalent zoonotic pathogens. It can spread from infected birds to human beings through direct or indirect contact with fecal shedding of Chlamydia. However, data concerning prevalence and genotypes of Chlamydia in pigeons are limited. In the present study, a total of 963 serum samples was collected from Jilin Province, Liaoning Province, and Inner Mongolia Autonomous Region (IMAR) in China between August 2015 and December 2016 and the seroprevalence for Chlamydia was analyzed by indirect hemagglutination assay test. The seroprevalence of Chlamydia was 20.4% (215/963) in total, at the cutoff 1:16, with the titers of 1:16 in 109, 1:64 in 49, 1:256 in 38, and 1:1024 in 18. Samples from all six administrative cities were detected Chlamydia-seropositive, ranging from 19.0% to 25.0%. Adult pigeons (23.5%) have a significant higher seroprevalence than juveniles (15.2%). Four PCR-positive samples represented Chlamydia psittaci genotype B. This is the first report of Chlamydia infection in pigeons in Liaoning Province and IMAR. The occurrence of C. psittaci genotype B in the droppings of pigeons suggests potential environmental contamination with C. psittaci and may raise a public health concern.

Introduction

Psittacosis, caused by Chlamydia , is prevalent in poultry and birds, and is also responsible for great economic losses in the bird and poultry industry (Cong et al. 2014). Chlamydia is one of the most important zoonotic pathogens that can infect a wide range of animals, including humans and pigeons (Cong et al. 2013). Chlamydia psittaci is the most important Chlamydia sp. (Zhang et al. 2015), which can spread from infected birds to humans through direct or indirect contact with fecal shedding of C. psittaci (Zhang et al. 2015). Chlamydia infection in humans or animals may lead to high fever, difficult breathing, encephalitis, myelitis, respiratory tract infection, meningitis, and other diseases (Qin et al. 2014).

In view of such severe background, investigation of the Chlamydia prevalence is quite urgent. Recently, C. psittaci prevalence in birds has been reported around the world. Dickx et al. (2013) found 58.0% prevalence in feral Canada geese (Branta canadensis) in Belgium by PCR. In China, pigeons were recognized as the popular birds for a long time. With the development of living condition, more and more people pay attention to the improvement of nutrition. Pigeon meat and eggs possess high nutritional value, so they are widely recognized by people. More and more people start to raise pigeons because the trade of pigeon production represents a lucrative business for owners and shops. According to some statistics, annual growth rate of 10–15% has occurred in pigeon industry in China. The “company & peasant household” mode has higher adoption rate in Chinese pigeon industry. An increasing number of people are in close contact with pigeons, which may be a potential source of C. psittaci. However, data concerning prevalence and genotypes of Chlamydia in pigeons are scarce (Cong et al. 2013). Therefore, 963 pigeons were examined in the present study to investigate the Chlamydia seroprevalence in northern China and to estimate the potential risk of C. psittaci infection in humans.

Materials and Methods

Ethics approval and consent to participate

All operations were handled in strict accordance with the Good Animal Practice requirements of the Animal Ethics Procedures and Guidelines of the People's Republic of China. This study was approved by the Animal Ethics Committee of Heilongjiang Bayi Agricultural University.

The investigated sites

The investigation was carried out in six cities in northern China. Changchun City (43°05′–45°15′ N, 124°18′–127°05′ E), Yanji City (129°01′–129°48′ E, 42°50′–43°23′ N), and Jilin City (42°31′–44°40′ N, 125°40′–127°56′ E) are located in Jilin Province. Jilin Province has a warm temperate continental monsoon climate, and has four seasons. The average precipitation of Jilin is 650–750 mm per year, with an average annual temperature of 3.9°C. The large population is ethnic Korean in Yanji City. Shenyang City (48.8° N, 123.4° E) and Jinzhou City (climate of Middle Latitude Zone) are located in Liaoning Province. Liaoning Province has a continental monsoon climate, and more rainfall than other provinces in northern China. Tongliao City (119°15′–123°43′ E, 42°15′–45°41′ N) is located in the eastern part of Inner Mongolia Autonomous Region. The average annual temperature of Tongliao is 5.8°C, with 0–6°C average annual temperature, and 350–400 mm annual precipitation. All of those cities have the most important status for transaction of agricultural products in north China.

Collection and preparation of serum samples

A total of 963 pigeons (273 from Changchun City, 152 from Shenyang City, 139 from Yanji City, 188 from Jilin City, 137 from Jinzhou City, and 74 from Tongliao City) were randomly selected between August 2015 and December 2016. Blood samples were collected from the wing vein of pigeons using blood lancets. Serum samples were separated from blood samples in local veterinary stations and then were taken to the laboratory and stored at −20°C until test. Biometric data regarding gender, age, and geographic origin of pigeons were acquired from owners.

Serological tests

A commercially available Indirect Hemagglutination Assay (IHA) Kit (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) was used to detect the antibodies against Chlamydia, and the test operations were carried out according to the manufacturer's description as previously reported (Cong et al. 2013, 2014, Qin et al. 2014, Zhang et al. 2015), at the cutoff of 1:16. In brief, serum samples were added to 96-well V-bottomed polystyrene plates, which were diluted 4-fold serially from 1:4 to 1:1024. Serum samples having positive reaction at dilutions of 1:16 or higher dilutions were considered positive for Chlamydia antibodies. Sensitivity and specificity of the IHA Kit is 100% and 95%, respectively (Qin et al. 2014). Positive, negative, and blank controls were provided by kits and included in each test. The titer between 1:4 and 1:16 were dubious and must be retested.

DNA testing and sequencing

For DNA extraction from droppings of seropositive pigeons, genomic DNA was extracted using the Stool DNA Kit (Omega Bio-Tek, Inc., Norcross, GA), and all the operations were conducted according to the previous study (Zhang et al. 2015). To determine the genotype of Chlamydia, ∼1000-bp fragment of the ompA gene was amplified using nested PCR with previously reported primers, FOMPF1/FOMPF2 (Zhang et al. 2015). The PCR products were tested using 1% agarose gel containing 0.5 μg/mL GoldView (Solarbio, Beijing, China) and were observed under ultraviolet light. The positive PCR products were then sequenced by the Sangon Biotech Company (Shanghai, China). The obtained sequences were aligned with reference sequences to determine the Chlamydia genotypes/species using the BLAST (www.ncbi.nlm.nih.gov/BLAST).

Statistical analysis

Differences in Chlamydia seroprevalence in pigeons of different geographical locations, gender groups, and age groups were analyzed by the chi-square test using SPSS version 20.0 (IBM SPSS, Inc., Chicago, IL) (Wu et al. 2017). Results were considered statistically significant if p < 0.05. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) were also provided.

Results

In the present study, 214 (22.22%, 95% CI 19.60–24.85) out of 963 pigeons were detected as seropositive for Chlamydia antibodies, at the cutoff 1:16, with titers of 1:16 in 109, 1:64 in 49, 1:256 in 38, and 1:1024 in 18 pigeons (Table 1). Samples from all seven administrative cities have been detected seropositive for Chlamydia antibodies, ranging from 16.55% (Yanji City) to 35.14% (Tongliao City) (Table 1). Seroprevalence of Chlamydia in male pigeons was 25.11% (95% CI 21.17–29.04) and 19.52% (95% CI 16.03–23.00) in female pigeons (Table 1). Chlamydia seroprevalence in juvenile and adult pigeons was 18.79% (95% CI 14.24–23.35) and 23.64% (95% CI 20.45–26.83), respectively (Table 1). Moreover, 9/40 fecal samples were Chlamydia positive examined by nested PCR amplification of the ompA gene. However, because of the low DNA concentrations of the other five samples, only four of them were successfully sequenced, and they represented C. psittaci genotype B. Sequence analysis showed that the identified Chlamydia isolates were 100% identical to that of the Chlamydiaceae strain isolated from canary in Iran (GenBank accession no. HQ845544).

Table 1.

Seroprevalence of Chlamydia Infection in Pigeons by Indirect Hemagglutination Assay, Northern China

		No. of sera with IHA titers of:
Variable	Category	1:16	1:64	1:256	1:1024	No. of tested	No. of positive	Prevalence (%) (95% CI)
Region	Jilin City	19	9	7	4	188	39	20.75 (14.95–26.54)
	Yanji City	13	5	3	2	139	23	16.55 (10.37–22.72)
	Changchun City	27	12	7	3	273	49	17.95 (13.40–22.50)
	Tongliao City	13	5	6	2	74	26	35.14 (24.53–46.01)
	Jinzhou City	20	7	8	3	137	38	27.74 (20.24–35.23)
	Shenyang City	17	11	7	4	152	39	25.66 (18.72–32.60)
Gender	Male	53	29	16	11	466	109	23.39 (19.55–27.23)
Gender	Female	56	20	22	7	497	105	26.45 (22.11–30.79)
Age	Juvenile	23	11	6	3	282	43	15.25 (11.05–19.44)
Age	Adult	86	38	32	15	681	171	25.11 (21.85–28.37)
	Total	109	49	38	18	963	214	22.22 (19.60–24.85)

95% CI, 95% confidence interval; IHA, indirect hemagglutination assay.

Discussion

In the present study, overall 214 (22.22%, 95% CI 19.60–24.85) out of 963 pigeons were tested C. psittaci seropositive by IHA at the cutoff of 1:16, which was similar to 21.3% seroprevalence in water birds in the United States (Docherty et al. 2012), and higher than that in pet birds (10.8%) in China (Cong et al. 2014). It is also much higher than the Chlamydia prevalence in birds in Germany (19.3%) (Sachse et al. 2012), but lower than that in pigeons (31.1%) (Cong et al. 2013), domestic geese (25.6%) (Zhao et al. 2015), and parrots (35.4%) (Zhang et al. 2015) using IHA, and in pigeons (26.7%) by enzyme-linked immunosorbent assay (ELISA), and 93.8% seroprevalence among 81 feral Canada geese tested by ELISA in Belgium (Dickx et al. 2013), 58.0% prevalence among 81 feral Canada geese tested by PCR in Belgium (Dickx et al. 2013). The difference in the prevalence of Chlamydia exposure in birds in different countries could be due to several factors, for example, ecological and geographical factors, the number of the pigeons tested, sanitation, and animal welfare for birds, as well as the specificity and sensitivity of the detection methods. Due to partial pigeons being bred by the free ranging system, the relationship between environment and Chlamydia infection should be investigated in further studies.

C. psittaci-seropositive samples were distributed among all six administrative cities, indicating that psittacosis was a common disease in pigeons in northern China. Moreover, previous studies have demonstrated that the age of birds was the risk factor for C. psittaci infection (Cong et al. 2014, Zhang et al. 2015). In the present study, risk factor analysis also showed that age appeared to be the main risk factor associated with Chlamydia infection (Table 2). Adult pigeons (25.11%, 95% CI 21.85–28.37) had 1.86 times (OR = 1.86 95% CI 1.29–2.69, p = 0.0008) higher risk of being seropositive compared with juveniles (15.25%, 95% CI 11.05–19.44). The result was consistent with that of a previous study. C. psittaci seroprevalence in adult pet birds was significantly higher than that in juveniles (Cong et al. 2014), but different from Zhang et al. (2015), who reported that juvenile parrots are more susceptible than adults and subadults. Results may be due to the fact that adult pigeons had more chances to contact with C. psittaci compared with juvenile pigeons. Whether the difference of C. psittaci seroprevalence among different age groups of pigeons is a result of naive immunity or repeated exposures to Chlamydia should be further studied.

Table 2.

Odds Ratios for Age of Pigeons are Taken as Risk Factors for Chlamydia Seroprevalence in Pigeons

Factor	Category	No. of tested	No. of positive	Prevalence (%)	OR (95% CI)	p
Age	Juvenile	282	43	15.25 (11.05–19.44)	Reference	0.0008
Age	Adult	681	171	25.11 (21.85–28.37)	1.86 (1.29–2.69)	0.0008

OR, odds ratio.

To determine the genotype of Chlamydia, nested PCR amplification of the ompA gene was used in this study. As expected, C. psittaci genotype B was shown to be shed in the feces of seropositive pigeons in the present study, which is in agreement with previous studies showing that C. psittaci genotype B was the major genotype associated with pigeons (Van Lent et al. 2012). The obtained C. psittaci genotype B sequences of the identified Chlamydia isolates were identical to that of the Chlamydiaceae strain isolated from canary in Iran (GenBank accession no. HQ845544), suggesting little variation of this prevalent genotype in the world. This result also suggests that pigeons represent a potential risk for Chlamydia infection for other animals and humans. The result may not reflect the seasonal seroprevalence in pigeons and it needs to be further studied.

Conclusions

In summary, the present study demonstrated the existence (22.22%) of C. psittaci infection in pigeons in northern China, for the first time. The age of pigeons was the significant risk factor associated with C. psittaci seroprevalence based on logistic regression analysis. The occurrence of C. psittaci genotype B in the droppings of pigeons suggests potential environmental contamination with C. psittaci and may raise a public health concern. The result will provide orientation data for prevention and control of C. psittaci infection in pigeons, other animals, and humans in China.

Footnotes

Acknowledgment

Project support was provided by the Science and Technology Research Project of the Heilongjiang Province Education Department (grant no. 12541586).

Author Disclosure Statement

No competing financial interests exist.

References

Cong

, Huang

, Zhang

, Zhou

, et al. Chlamydia psittaci exposure in pet birds. J Med Microbiol, 2014; 63:578–581.

Cong

, Huang

, Zhang

, Zhou

, et al. Seroprevalence of Chlamydia psittaci infection in market-sold adult chickens, ducks and pigeons in north-western China. J Med Microbiol, 2013; 62:1211–1214.

Dickx

, Kalmar

, Tavernier

, Vanrompay

. Prevalence and genotype distribution of Chlamydia psittaci in feral Canada geese (Branta canadensis) in Belgium. Vector Borne Zoonotic Dis, 2013; 13:382–384.

Docherty

, Franson

, Brannian

, Long

, et al. Avian botulism and avian chlamydiosis in wild water birds, Benton Lake National Wildlife Refuge, Montana, USA. J Zoo Wildl Med, 2012; 43:885–888.

Qin

, Yin

, Cong

, Zhou

, et al. Seroprevalence and risk factors of Chlamydia abortus infection in Tibetan sheep in Gansu Province, Northwest China. ScientificWorldJournal, 2014; 2014:193464.

Sachse

, Kuehlewind

, Ruettger

, Schubert

, et al. More than classical Chlamydia psittaci in urban pigeons. Vet Microbiol, 2012; 157:476–480.

Van Lent

, Piet

, Beeckman

, van der Ende

, et al. Full genome sequences of all nine Chlamydia psittaci genotype reference strains. J Bacteriol, 2012; 194:6930–6931.

, Wang

, Yang

, Li

, et al. Seroprevalence and risk factors of Toxoplasma gondii in slaughter pigs in Shaanxi Province, Northwestern China. Vector Borne Zoonotic Dis, 2017; 17:517–519.

Zhang

, Zhang

, Zhou

, Huang

, et al. Seroprevalence and genotype of Chlamydia in pet parrots in China. Epidemiol Infect, 2015; 143:55–61.

10.

Zhao

, Rong

, Zhou

, Hou

. Seroprevalence and risk factors of Chlamydia psittaci infection in domestic geese Anser domestica, in Hainan province of China. Acta Trop, 2015; 145:23–25.