Abstract Examinations were carried out in 46 intensive farms in northern China to investigate avian Chlamydophila psittaci. Five hundred and twenty-five avian sera were selected for examining antibodies to C. psittaci by ELISA. One hundred and fifty-five clinical samples from throat swabs and oviduct tissues were tested for the presence of chlamydial antigen using IDEIA TM PCE chlamydia dual amplification immunoassay, and 60 samples were tested by ompA gene-based PCR. C. psittaci antibodies were detected in 387 (77.8%) out of 525 serum samples, with seroprevalences ranging from 50% to 100%. Among the tested samples, 98/150 (65.3%) in broilers, 173/210 (82.3%) in ducks, and 116/165 (70.3%) in laying hens were detected to be positive, respectively. Using PCE-ELISA test kits, in 91 out of 155 clinical samples the presence of antigen was confirmed, while 64 samples were negative. Forty-three PCR&apos;s were tested as positive out of 60 samples, while 17 samples were confirmed to be negative. Both higher positive antibodies and the presence of antigens were found in avian flocks associated with typical clinical signs suggestive of chlamydiosis. This study showed a severe prevalence of C. psittaci among different species of domestic birds in China

AND CHENG HE Jianming Yang

Junjing Yang

Q I Yang

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Bull Vet Inst Pulawy 51, 347-350, 2007 
 
PREVALENCE OF AVIAN CHLAMYDOPHILA PSITTACI 
IN CHINA 
 
JUNJING YANG, QI YANG, JIANMING YANG, AND CHENG HE 
 
College of Veterinary Medicine, China Agricultural University, 
Beijing 100094, China 
hehonest@yahoo.com.cn 
 
Received for publication April 30, 2007 
 
 
 
Abstract 
 
Examinations were carried out in 46 intensive farms 
in northern China to investigate avian Chlamydophila psittaci. 
Five hundred and twenty-five avian sera were selected for 
examining antibodies to C. psittaci by ELISA. One hundred 
and fifty-five clinical samples from throat swabs and oviduct 
tissues were tested for the presence of chlamydial antigen 
using IDEIATM PCE chlamydia dual amplification 
immunoassay, and 60 samples were tested by ompA 
gene-based PCR. C. psittaci antibodies were detected in 387 
(77.8%) out of 525 serum samples, with seroprevalences 
ranging from 50% to 100%. Among the tested samples, 98/150 
(65.3%) in broilers, 173/210 (82.3%) in ducks, and 116/165 
(70.3%) in laying hens were detected to be positive, 
respectively. Using PCE-ELISA test kits, in 91 out of 155 
clinical samples the presence of antigen was confirmed, while 
64 samples were negative. Forty-three PCR’s were tested as 
positive  out of 60 samples, while 17 samples were confirmed 
to be negative. Both higher positive antibodies and the 
presence of antigens were found in avian flocks associated 
with typical clinical signs suggestive of chlamydiosis. This 
study showed a severe prevalence of C. psittaci among 
different species of domestic birds in China. 
 
  Key words: poultry, Chlamydophila psittaci, 
chlamydiosis, antibodies, immunodiagnosis, China. 
 
 
Avian chlamydiosis (AC) is caused by the 
bacterium Chlamydophila psittaci（C. psittaci). It has 
been isolated from a wide range of avian species (1, 3). 
Chlamydiae are responsible for a variety of infections in 
birds and are important aetiological agents in respiratory 
diseases. Moreover, this pathogen is known to be 
transmissible to humans causing significant zoonotic 
infection (7, 8). C. psittaci infection in turkey has been 
present in the US already for more than 50 years, and 
has become prevalent in turkey farms in Europe today, 
resulting in economical problems (3, 12). There are no 
commercial vaccines available for chlamydiosis control 
in poultry. Antibiotics are the only current means of 
control; however, C. psittaci is becoming resistant to a 
number of antibiotics due to their administrative abuse 
(7). Hence, it is important for scientists to control avian 
chlamydioses and the cross-species transmission. 
Since 1980s, outbreaks of respiratory diseases 
in poultry were recorded in ducks, pigeons, imported 
flamingo, and black swan in China. Later on, avian C. 
psittaci strains were isolated and identified in laying 
hens and broilers (4, 9). Chlamydial infection was 
ignored in recent years due to the outbreak of avian 
influenza in China and there are no recent data on the 
prevalence of chlamydial infection in Chinese poultry. 
Hence, the study was carried out in order to determine 
the prevalence of chlamydial infection in poultry flocks 
from different regions of China.  
 
 
Material and Methods 
 
Serum and clinical samples. The 
examinations were performed on 525 blood samples 
from 46 farms, representing 7 main intensive poultry 
areas in China, 1 farm of specific pathogen free (SPF) 
hens, 5 farms of commercial ducks, 6 farms of breeder 
ducks, 10 farms of parent laying hens, and 24 farms of 
laying hens. The blood samples were collected from 
birds between 20 and 40 weeks of age. Serum samples 
were kept frozen at -20°C until testing. 
One hundred and fifty-five clinical samples 
were collected from suspected birds, including 10 throat 
swabs from SPF hens. Ten oviduct membranes from 
laying hens with cystic oviduct, 30 throat swabs from 
boilers, 50 throat swabs from laying hens, and 55 throat 
swabs from laying ducks. Approximately 100 mg of 
minced tissues from cystic oviduct were suspended in 
Eagle’s minimal essential medium (MEM). The samples 
were vortexed with 3-mm-diameter glass beads, 
centrifuged at 500 x g for 5 min at 4°C, and the 
supernatants were collected for the detection of 
chlamydial antigens. Throats samples for the antigen test 
and PCR were placed in chlamydial transport medium 
and stored at -20C° until use (1). 
Detection of chlamydial antibodies and 
antigens. Sera were tested for the presence of antibodies 
 348 
using RIDASCREEN Chlamydia psittaci ELISA 
(R-Biopharm, Darmstat, Germany) according to 
manufacturer’s recommendation. The collected tissues 
samples and throats were screened using IDEIATM PCE 
chlamydia dual amplification immunoassay (Dako 
Cytomation Ltd., UK) following the manufacturer’s 
procedure. IDEIATMPCE-ELISA is based on the 
identical monoclonal antibody for binding and capturing 
Chlamydia from a sample matrix. The value of optical 
density (OD) below 0.119 were considered as negative, 
while values above 0.147 as positive. Positive values 
were subdivided into weakly (OD=0.147-0.222), 
moderately (OD=0.230-0.310), and strongly positive 
(OD=0.320). This allowed discrimination between farms 
with more recent and severe chlamydial infection. 
PCR analysis. Genomic DNA of autopsy 
samples was prepared from purified Chlamydia using 
the DNeasy Tissue Kit (Qiagen) following the 
manufacturer’s instructions. The PCR procedure was 
performed using a pair of oligonucleotide primers 
CTU/CTL (CTU, 5'-ATG AAA AAA CTC TTG AAA 
TCG G-3', CTL, 5'-CAA GAT TTT CTA GAC TTC 
ATT TTG TT-3') chosen in the highly conserved regions 
of the published omp1 sequences (5,6,11). One reference 
strain of the genomic DNA was Chlamyidophila psittaci 
ISN 1528 from Dr David Longbottom (Moredun 
Research Institute, UK). A non-infected McCoy cells 
preparation was included as a negative control. PCR 
products were separated by 2.5% or 1.0% agarose gel 
(Sigma) electrophoresis and visualised by ethidium 
bromide staining. The DNA molecular weight marker 
(Boehringer Mannheim) was used as a size marker.  
 
 
Results 
 
Antibodies against Chlamydia in poultry 
flocks. Out of 525 blood samples, 387 (73.7%) revealed 
a significant level of C. psittaci antibodies, while 138 
(26.3%) were negative. With respect to regions, there 
was 74.5%, 84%, 70%, 78%, 61.3%, 53.3%, and 73.3% 
positive, respectively, in different provinces. The highest 
percentage of seropositive birds was found in both 
breeder hens and SPF laying hens from Beijing (Table 1). 
Sera from laying hens in the Shanxi province had the 
lowest incidence of positive results (53.3%). It is 
obviously evident that parent flocks have a higher 
positive rate than commercial flocks. Regarding species, 
positive rates were 65.3%, 70.3%, and 82.3% in broilers, 
laying hens, and ducks, respectively (Table 2).  
Antigen detection in throat swabs and 
oviduct tissues in birds with clinical signs. Using 
PCE-ELISA kits, 91 out of 155 clinical samples were 
detected to be positive (58.7%), while 64 samples were 
negative (Table 3). It was interesting to find that laying 
hens with oviduct cysts revealed more positive results 
(70%). The highest positive rate was observed in duck 
flocks (93.3%) with a lower egg production, while 
73.3% positive results were detected in laying hens with 
similar clinical signs. As antigen presence is concerned, 
broilers with a severe pneumonia revealed 60% positive 
results, as compared to 30.0% in the control group. In 
this epidemiological survey, in clinically healthy SPF 
hens a lower incidence of antigen presence was detected. 
Analysis of avian clinical samples by PCR 
test. As expected, PCR reactions using genomics DNA 
purified from clinical samples as templates with 
CTU/CTL primers generated amplication products of an 
average size of 1200-bp as well as a slightly smaller 
band. Forty-three out of 60 (71.6%) tissues samples 
were PCR positive, while 17 (28.4%) were negative 
(Table 4). Particularly, 9 out of 10 samples taken from 
laying hens with severe cystic oviduct form were PCR 
positive, while 8 of 10 samples were ELISA positive in 
laying hens with a lower egg production. Higher positive 
rate was also found in duck samples (90%) as well as in 
broilers with clinical signs (70%). PCR analysis 
revealed 1 positive result in 10 throat swabs in SPF 
hens. 
 
 
Discussion 
 
In the present study, ELISA, PCE-ELISA, 
and PCR were used to examine the prevalence of avian 
chlamydiosis in China. In average, all of the 46 poultry 
farms examined were 77.8% positive for Chlamydia 
antibodies by ELISA, 57.4% positive for antigen by 
PCE-ELISA, and 71.6% positive for antigen detected by 
PCR. Between 1990 and 2000, positive antibodies to C. 
psittaci were found in 21.0% of laying hens and 20.6% 
of broilers by indirect haemagglutination inhibition (IHI) 
test (9). Recently, a report showed that an average of 
24.9% positivity was confirmed using IHI test kits (2). 
In the current study, 77.8% positivity was detected by 
ELISA kits. In contrast to the serological results, 57.4% 
of 155 clinical samples were antigen positive. This is the 
first report on the presence of chlamydial antigen using 
PCE-ELISA and PCR. The evidence shows that avian 
chlamydiosis is prevalent in laying hens, ducks, and 
broilers.  
In European countries, avian chlamydiosis 
has also been increasing in recent years. In Belgium in 
2002, 188 out of 200 (94%) turkey sera reacted 
positively, compared to 175 of 200 (87.5%) in 1992 (13). 
In Bosnia and Herzegovina, and in Macedonia, 45.5% of 
intensively bred chickens, 12%-32.8% of free living 
pigeons, and 8% of parrots investigated with 
PCE-ELISA were positive to C. .psittaci antigen (10). 
Laying hens with poor egg production revealed the 
presence of antigens in 22 out of 28 (78.5%) triple 
swabs (conjunctiva, pharynx, and cloaca) (8). Therefore, 
a higher presence of antigens and seropositivity in avian 
species demonstrates again that a potential relationship 
between chlamydial infection and avian health should be 
considered. 
 
 349
 
Table 1  
Seroprevalence of C. psittaci in different regions 
Regions Species Numbers  Positive Negative Positive (%) 
Breeder hensa 30 27 3 90.0 
Ducksb 30 25 5 83.3 
Broilersc 40 20 20 50.0 
Beijing 
SPF hensd 10 10 0 100.0 
Laying hensb 45 35 10 77.7 Hebei 
 Breeder ducksb 80 70 10 87.5 
Tianjin Broilersc 50 35 15 58.3 
Inner Mongolia Ducksb 100 78 22 78.0 
Shandong Laying hensb 50 28 22 56.0 
 Broilersc 30 21 9 70.0 
Shanxi Laying hensb 30 16 14 53.3 
Henan Broilersc 30 22 8 73.3 
Total  525 387 138 73.7 
a Birds with a oviduct cyst; b Birds with an unknown egg drop; c Broilers with a sever pneumonia; d SPF laying hens 
with a clinical health 
 
Table 2 
Seroprevalence of avian C. psittaci in different poultry flocks 
Species Number of birds Positive Negative Positive (%) 
Broilers 150 98 52 65.3 
Laying hens 165 116 49 70.3 
Ducks 210 173 37 82.3 
 
Table 3 
Detection of Chlamydia antigen in avian clinical samples by PCE-ELISA 
Species Numbers of birds  Positive  Negative Positive (%) 
Breeder hens a  10 7 3 70.0 
Laying hens b 30  22 8 73.3 
Control hens c 20  3 20 15.0 
Ducks b 30  28 4 93.3 
Control ducks c 25  15 10 60.0 
Broilers d 20  12 8 60.0 
Control broilers e 10  3 7 30.0 
SPF hens e 10  1 9 10.0 
Total 155 91 64 58.7 
a Breeder hens with oviduct cyst; b Birds with a lower egg production (≤50%); c Birds with a higher egg production 
(≥85%); d Broilers with a severe pneumonia; e Clinically healthy birds 
 
Table 4 
Detection of C. psittaci in avian clinical samples by PCR 
Species Numbers of birds  Positives   Negatives Positive (%) 
Breeder hens a 10 9 1 90.0 
Laying hens b 10 8 2 80.0 
Ducks b 20 18 2 90.0 
Broilers c 10 7 3 70.0 
SPF chickens d 10 1 9 10.0 
Total 60 43 17 71.6 
a Birds with cystic oviducts; b Birds with lower egg production (≤50%); c Broilers with a severe pneumonia; d Clinically 
healthy birds  
 350 
Interestingly, swab samples revealed 10% 
antigen positivity, while their corresponding serum 
showed 100% seropositivity. SPF eggs are widely used 
for growing attenuated vaccine and purifying bio-drugs 
for humans. Once SPF chickens are infected, chlamydia 
will be transmitted directly to each vaccinated chicken 
and in consequence to human beings. Whether it is true 
or not, further studies will be done to assess the 
morbidity of SPF chickens and potential risk for human 
health in the future. It is high time that attention will be 
paid to Chlamydophila psittaci infection and its 
implications. 
A complement fixation test (CFT) and cell 
cultures are considered as a golden standard (1, 3). 
However, CFT is unsuitable for other poultry species 
(turkey, duck, and chickens) except for pigeon, due to 
poor cross-species sensitivity and reactivity. Moreover, 
it is time-consuming and needs standard test reagents. In 
contrast to CFT, PCE-ELISA and PCR, based on ompA 
gene, are reliable methods to identify the typical 
inclusion bodies in suspected samples. Current PCR 
tests for the detection of C. psittaci target the omp1 gene. 
As far as avian chlamydial diagnosis is concerned, 
tracheal swabs extracts used in this study provided a 
suitable material for analysis. PCR, with the use of 
CTU/CTL primers, performed on extracted DNA was 
more sensitive than PCE-ELISA. The test was also 
useful when isolation of chlamydia was a less 
favourable method, and when the bacterium cannot be 
cultured from pathological samples due to the need for 
rapid diagnosis (5, 6). In the present study and earlier 
studies, the question arises of the diagnostic and 
economic significance of C. psittaci as a causative agent 
of pneumonia, oviduct cysts, and poor laying 
performance. Additional investigations will focus on the 
transmission route and efficient measure for control of 
avian chlamydial infection. 
 
Acknowledgments: This study was 
supported by a grant from the Beijing Municipal Science 
and Technology Commission contract 6052014. 
 
 
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PREVALENCE OF AVIAN CHLAMYDOPHILA PSITTACI IN CHINA

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PREVALENCE OF AVIAN CHLAMYDOPHILA PSITTACI IN CHINA

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