A Seroepidemiological Survey of Toxoplasma gondii Infection in Free-Range and Caged Ducks in Southwest China

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Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 41 Toxoplasma gondii in Ducks in Southwest China
A Seroepidemiological Survey of Toxoplasma gondii Infection in
Free-Range and Caged Ducks in Southwest China
Zhao, G.,
1
Song, Z.,
1
Wang, S.,
2
Hassan, I.A.,
2
Wang, W.,
2
Cheng, F.
1
and Yang, X.
1
*
1
Department of Veterinary Medicine, Southwest University, Chongqing, 402460, PR China.
2
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, PR China.
*
Corresponding Author: Dr. X. Yang, Department of Veterinary Medicine, Southwest University, Chongqing, 402460, PR China. Tel.: +86 23 46751041,
Fax: +86 23 46751041. E-mail: yangxiaowei396@163.com
ABSTRACT
Toxoplasma gondii is widely distributed in humans and other animals including domestic poultry throughout
the world, but data on prevalence of T. gondii in ducks in People’s Republic of China (PRC), especially in the
southwest China are limited. In the present study, the seroprevalence of T. gondii infection in free-range (FR) and
caged ducks was investigated in Chongqing municipality (southwest China) during the period from February
to July 2013. A total of 1162 serum samples including 635 FR ducks and 527 caged ducks from 8 municipal
districts/counties including Yongchuan, Dazu, Rongchang, Hechuan, Liangping, Wanzhou, Yunyang and
Kaixian were collected and assayed for T. gondii antibodies using modifed agglutination test (MAT) technique.
Partial seropositive samples were validated for T. gondii by bioassay in mice. Te prevalence in FR and caged
ducks was 13.23% and 6.64% respectively. Statistical analysis showed that FR group was signifcantly higher
than caged group (p<0.05). From 12 FR ducks in Rongchang, one strain of T. gondii was successfully isolated
and the isolation rate was 8.33% (1/12). Tis is the frst report of isolation of T. gondii from FR ducks in China.
Keywords: Duck; Toxoplasma gondii; Prevalence; China
INTRODUCTION
Toxoplasma gondii is an important intracellular protozoan
parasite, widely prevalent in humans and animals, includ-
ing ducks throughout the world (1-4). Felids are the de-
fnitive host of this parasite and almost all warm-blooded
animals including humans can act as intermediate hosts
(5). Humans can be infected by ingesting tissue cysts from
under-processed infected animal meat or from food or drink
contaminated with oocysts shed in cat feces (6). Some early
studies demonstrated that meat from T. gondii-infected
poultry (including chickens, ducks, geese, and pigeons) was
the primary risk factor for humans’ toxoplasmosis (1, 7, 8).
Ducks (free-range (FR) and caged), are consumed widely in
many countries, including China, especially in Chongqing,
southwest China. In addition individuals have the habit of
eating under-cooked duck intestines called “chafng dish”.
Tis further emphasizes the need to study the prevalence
of T. gondii in ducks.
In recent years sero-prevalence studies of T. gondii in
ducks have been conducted extensively in various parts
of the world (9-11). Surveys have also been conducted
in the mainland China including northeast China (8),
southeast China (12) and northwest China (13). However,
little is known about the prevalence of T. gondii in ducks
in southwest China. Here, we report for the frst time T.
gondii sero-prevalence in FR and caged ducks in Chongqing
municipality, southwest China.
MATERIALS AND METHODS
Te study area
Te study was conducted in Chongqing municipality which
is located in the upper reaches of the Yangtze River in the
Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 Zhao, G. 42
southwest part of mainland China, covering an area of 82,000
km
2
and a population of approximately 2.94 million. Its geo-
graphical position is at east longitude 105°11’ - 110°11’ and
at north latitude 28°10’ - 32°13’. Te area has a subtropical
humid climate, a long winter and summer, with a brief spring
and autumn. Te average annual temperature is 18.0°C (in
winter the average is 6-8°C, in summer the average is above
35°C), with a mean annual rainfall of 1000-1450mm. Tere
are 19 municipal districts and 19 municipal counties distrib-
uted in the Chongqing municipality.
Eight municipal districts or counties including
Yongchuan, Dazu, Rongchang, Hechuan, Liangping,
Wanzhou, Yunyang and Kaixian, located in the western and
eastern parts of Chongqing municipality, were selected for
sample collections. All of the above locations are main suppli-
ers of duck meat to Chongqing and the neighboring regions.
Blood samples
A total of 1162 blood samples from adult ducks, 635 blood
samples from free-range (FR) ducks and 527 blood samples
from caged ducks were collected from the above eight places
in Chongqing between February and July 2013. Te blood
samples were sent to the laboratory for serological examina-
tion and centrifuged at 3,000 rpm for 10 minutes and the sera
were stored at −20°C until tested for antibodies to T. gondii.
Serological assay
Sera were tested for T. gondii antibodies using 2-fold serial
dilutions from 1:25 to 1:3,200 with the modifed agglutina-
tion test (MAT), as described previously (8, 14). Briefy, the
harvested parasites were kept in 6% formaldehyde solution
at 4°C overnight, and suspended in the alkaline bufer at
20,000 parasites/mL. Two-fold dilutions of sera were per-
formed using the serum diluting bufer, and agglutination
was performed in U-bottom 96-well microtiter plates using
a mixture of 50 µL antigen and 50 µL diluted sera. Te plates
were incubated at 37°C overnight. Te test was considered
positive when a layer of agglutinated parasites was formed
in wells at dilutions of 1:25 or higher; positive and negative
controls were included in each test.
Bioassay of ducks for T. gondii
Tissues for bioassay were sampled from the MAT sero-posi-
tive ducks as described previously (15). Brains, hearts, spleens,
lungs, livers and kidneys of ducks were bioassayed individu-
ally in outbred female Swiss Webster (SW) mice (20±2g)
obtained from Center of Laboratory Animals, Chongqing
Medical University. (Chongqing, PR China). In this research,
all the animals used were submitted to protocols approved
by the Animal Care and Ethics Committee of Southwest
University (Approval No. 201209025).
Identifcation of the isolate by PCR
Molecular identifcation of the isolate was per-
formed with a 341bp fragment of the internal tran-
scribed spacer 1(ITS-1) gene by a pair of primers
5’-AGTTTAGGAAGCAATCTGAAAGCACATC-3’
and 5’-GATTTGCATTCAAG AAGCGTGATAGTAT-3’
as described previously (16). Briefy, total genomic DNA
was extracted from the peritoneal washings with a commer-
cially available DNA extraction kit (Geneaid Biotech Ltd.,
New Taipei City, Taiwan) according to the manufacturer’s
recommendations. PCR reactions (25µl) were performed
in 2.5mM of MgCl
2
, 0.4µM of each primer, 2.5µl 1×rTaq
bufer, 0.25mM of each desoxyribonucleotide, 0.625 U of
rTaq DNA polymerase (TaKaRa), and 3µl of DNA sample
in a thermocycler (Biometra) under the following condi-
tions: after an initial denaturation at 94°C for 5 minutes,
then 35 cycles of 94°C for 30 s (denaturation), 55°C for 30 s
(annealing), 72°C for 30 s (extension), followed by a fnal
extension at 72°C for 7 minutes. Te positive control of T.
gondii DNA and negative control (no-DNA control) were
included in each amplifcation run. Each amplicon (13ul) was
examined by agarose gel electrophoresis to validate amplifca-
tion efciency.
Statistical analysis
Statistical analyses of T. gondii prevalence between free-range
and caged groups were performed by χ
2
-test using Excel
(Microsoft® Excel 2003).Te diferences were considered
statistically signifcant where p< 0.05.
RESULTS
Positive ratios for FR and caged ducks and the
overall ratios for the eight municipal districts/
counties in Chongqing
A total of 1162 duck serum samples including 635 FR
and 527 caged ducks from 8 municipal districts/counties
of Chongqing were tested for T. gondii antibodies by MAT
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 43 Toxoplasma gondii in Ducks in Southwest China
method. Overall, the seroprevalence of T. gondii in FR ducks
was 13.23%, which was signifcantly higher (p<0.05) than
that of the caged ducks (6.64%) (Table 1).
Seropositivity rates for the FR ducks from the 8 munici-
pal districts/counties ranged from 7.14% to 19.51% (Table
1). Four samples out of 56 tested was found positive (7.14%)
in Wanzhou which was statistically lower compared to Dazu
(p<0.05) and Hechuan (p<0.05). Te positive rates ranged
from 10.14% to 19.51% in Dazu, Hechuan, Yongchuan,
Rongchang, Liangping, Yunyang and Kaixian municipal
districts/counties. However, there were no signifcant difer-
ences between them (p>0.05).
For caged ducks, the positive rates of the 8 municipal
districts/counties ranged from 4.69% to 9.43% (Table 1).
No signifcant diferences were found between the eight
municipal districts/counties (p>0.05).
Isolation of T. gondii from the seropositive ducks
Twelve FR ducks from Rongchang with titers of 1:25 in 6
ducks, 1:50 in 3 ducks, and 1:100 in 3 ducks were used for T.
gondii isolation. At the third blind passages of the surviving
mice, one strain of T. gondii was isolated from a duck with titer
1:100. Tachyzoites were found in peritoneal washings of all of
the 4 mice inoculated (Figure 1). However, the rest of the mice
inoculated with samples from other ducks were negative for T.
gondii. Tissue cysts were not found in the brain squashes of any
of the mice. Te isolation rate was 8.33% (1/12).
Identifcation of the isolate by PCR
Te isolate was identifed by PCR using a 341bp fragment
of the ITS-1 gene as a target (16). Alignment of nucleic
acid sequences of PCR product shared 100% similarity to
RH strain of T. gondii (accession No. U16161) homologues,
confrmed that it belonged to T. gondii.
Table 1: Seroprevalence of Toxoplasma gondii infection in ducks in Chongqing, southwestern China by the modifed agglutination test (MAT)
Places No tested No. with anti-T. gondii antibodies Total positive Prevalence
(%)
1:
25
1:
50
1:
100
1:
200
1:
400
Yongchuan FR
Caged
Total
77
65
142
7
4
11
1
1
2
1
0
1
0
0
0
0
0
0
9
5
14
11.69
7.69
9.86
Dazu FR
Caged
Total
82
70
152
11
5
16
2
1
3
1
0
1
1
0
1
1
0
1
16
6
22
19.51
8.57
14.47
Rongchang FR
Caged
Total
88
72
160
6
2
8
3
1
4
3
1
4
0
0
0
0
0
0
12
4
16
13.64
5.56
10.00
Hechuan FR
Caged
Total
66
53
119
4
2
6
4
1
5
2
1
3
1
1
2
1
0
1
12
5
17
18.18
9.43
14.29
Liangping FR
Caged
Total
90
89
179
8
4
12
2
1
3
2
0
2
0
0
0
0
0
0
12
5
17
13.33
5.62
9.50
Wanzhou FR
Caged
Total
56
55
111
3
3
6
1
0
1
0
0
0
0
0
0
0
0
0
4
3
7
7.14
5.45
6.31
Yunyang FR
Caged
Total
69
64
133
4
2
6
1
1
2
1
0
1
1
0
1
0
0
0
7
3
10
10.14
4.69
7.52
Kaixian FR
Caged
Total
107
59
166
5
3
8
4
2
6
2
0
2
1
0
1
0
0
0
12
5
17
11.21
8.47
10.24
Total FR
Total caged
Total
635
527
1162
48
25
73
18
8
26
12
2
14
4
0
4
2
0
2
84
35
109
13.23
6.64
9.38
FR: Free-range
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 Zhao, G. 44
DISCUSSION
Many surveys detecting T. gondii infection in FR ducks have
been conducted in a number of other countries (4, 8, 12,
17, 18), and the prevalence rates in most of these studies
ranged between 21.2% to 50.0%. In China, the T. gondii
seroprevalence in FR ducks were also performed in north-
eastern China by Yang et al. (8), in south-eastern China by
Yan et al. (12) and in northwest China by Cong et al. (13).Te
seropositive rates in that three reports were 12.3%, 16% and
13.90%, respectively. In the present study, the overall T. gondii
seroprevalence in FR ducks in Chongqing was 13.2%, which
was lower than that reported in other countries but similar
to that reported in China. Te diferences in seroprevalence
between China and other countries may due to diferences
in ecological and geographical factors.
Considering the caged ducks, the seroprevalence in the
present study was 6.64% which was also similar to that
reported in north-eastern China (7.50%) (8) and north-
western China (6.31%) (13). Tese fndings indicated that
the prevalence rates of T. gondii in caged ducks were approxi-
mately equal in various regions of China. When comparing
the caged and FR ducks in this study, the seroprevalence of
T. gondii in caged group (6.64%) was signifcantly lower than
the FR group (13.23%) (p<0.05). Te main reason for this
variability could be that the FR ducks were fed on the ground
and had more chance than the caged ducks to be infected
by ingesting T. gondii oocysts from the environment, such
as water, soil and infected tissues from intermediate hosts.
Our fndings were in agreement with the results obtained by
Dubey et al. (10, 19, 20) in T. gondii infected FR chickens.
Geographically, there are 38 municipal districts/counties
distributed in the Chongqing municipality and eight places
were selected for screening the T. gondii seroprevalence be-
cause they were the main suppliers of duck meat. Te positive
rate of FR ducks in Wanzhou (7.14%) was found statistically
lower than Dazu (19.51%) and Hechuan (18.18%). Reasons
for this diference could be varied, including the sampling
season, the quantity of T. gondii oocysts and the number of
samples. Duck meat can serve as a source of T. gondii infec-
tion for human and other animals; therefore it would be a
risk factor for toxoplasmosis of human and other animals;
awareness of this fact should be emphasized.
To validate the seropositive samples, 12 FR ducks from
Rongchang were used to bioassay the T. gondii from the
tissues and one strain was successfully isolated. To the best
of our knowledge, this is the frst report of the successful
isolation from FR ducks in China. Successful isolation of
T. gondii from tissues of asymptomatic animals may depend
on the number of mice inoculated, the amount of tissue-
bioassayed and the concentration of the parasite in sampled
tissues (21, 22). In this study, the isolation rate (8.33%) was
lower than previously reported (19, 23). Te reason may be
due to less tissue cysts in the processed samples. Furthermore,
the genetic characterization of the isolate was not performed
in this study and requires further investigation.
ACKNOWLEDGEMENTS
Tis work was supported by Fundamental Research Funds for
the Central Universities (XDJK2012C100) and Te Doctoral
Special Funding of SWU (2013Bsr09).
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