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Israel Journal of Veterinary Medicine  Vol. 70 (2)  June 2015 9 Toxoplasma gondii in Ducks
Preliminary Studies on the Relevance of Antibodies and Bioassay to
Experimental Infection with Toxoplasma gondii In Ducks
Zhao, G.W.,
1#
Wang, S.,
2#
Hassan, I.A.,
2
Wang, W.,
3
Song, Z.H.,
1
Yang, L.W.
1
and Yang, X.W.
1
*
1
Department of Veterinary Medicine, Southwest University, Rongchang, Chongqing, 402460, PR China
2
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, PR China
3
Institute of Neuroscience, Soochow University, Suzhou, Jiangsu, 215123, PR China
#
Tese authors contributed equally to this paper.
*
Corresponding Author: Dr. Yang Xiaowei, Department of Veterinary Medicine, Southwest University, Rongchang, Chongqing, 402460, PR China;
Tel/Fax.: +86-23 -46751041. Email address: yangxiaowei396@163.com.
ABSTRACT
Little is known about the relationship between antibody dynamics and positive bioassay with Toxoplasma
gondii (T. gondii) infection in ducks. In this study, 42 ducks were allocated randomly into groups A, B and
C with 14 birds of 14 days of age in each group. Tree groups were infected intravenously with 10
5
, 10
6
and
10
8
of T. gondii tachyzoites, respectively. Blood samples were collected on day 7, 14, 21, 28, 42, 56, 70 post-
infection (p.i.). Sera was prepared from each group to evaluate the anti-T. gondii antibodies by the modifed
agglutination test (MAT); from each group 2 duck samples were used to bioassay T. gondii in mice. Te
T. gondii were identifed by PCR and nucleic acids sequencing. In group A, the parasite was successfully
bioassayed from day 7, 14 and 21 in post-infected ducks, with the MAT titers of 1:2, 1:5 and 1:80, respectively.
However, in ducks with MAT titers 1:80, 1:40, 1:20 and 1:4 on the following days 28, 42, 56 and 70 p.i., the
bioassay was negative. In group B, T. gondii was successfully biossayed at all the time points, regardless of
the seropositivity or seronegativity for T. gondii antibodies. All birds of group C died before day 4 p.i, and
therefore they were not bioassayed. PCR and sequencing results confrmed that all the isolates belong to T.
gondii. Tis study demonstrated that successful bioassay does not completely correlate with positive MAT
diagnosis. Te existence of T. gondii and the concentration of the parasite may be the key parameters during
the bioassay studies.
Keywords: Duck; Toxoplasma gondii; Antibody; Bioassay; Artifcial Infection
INTRODUCTION
Toxoplasma gondii is an important intracellular protozoan
parasite, widely prevalent in humans and animals and also
birds throughout the world (1-3). Humans can be infected
by ingestion of raw, or undercooked meat from the infected
animals, and by consuming food or water contaminated with
oocysts excreted by cats (4). Ducks are an important interme-
diate host for T. gondii. Te consumption of raw duck meat
or improperly cooked duck meat is a risk factor for T. gondii
infection in humans and other animals (3, 5).
In recent years, seroprevalence studies of T. gondii in ducks
have had been conducted extensively in various parts of the
world (3, 6-8), and T. gondii strains have been constantly
isolated from this host (7-9). It has been already shown that
successful bioassay of T. gondii in ducks is crucial to the further
research about this parasite. So far, to the best of our knowl-
edge, most of the isolated strains of T. gondii from ducks were
based on the serologic diagnosis with a modifed agglutination
test MAT (7, 8). However, to date, little is known about the
relationship between the dynamics of antibodies and successful
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (2)  June 2015 Zhao, G.W. 10
T. gondii isolation. Terefore, the present study has been de-
signed to evaluate the relevance and relationship of serological
response to T. gondii and its bioassay in ducks.
MATERIALS AND METHODS
Ethics statement
In this research, all the studies using ducks were submit-
ted as protocols approved by the Animal Care and Ethics
Committee of Southwest University (Approval No.
201209025).
Mice and parasites
ICR mice of 4-6 weeks of age were obtained from Center
of Laboratory Animals, Chongqing Medical University
(Chongqing, PR China). All animals were reared under
specifc-pathogen-free conditions at Southwest University,
and commercial basal diet and tap water was provided ad
libtum. Mice were infected or challenged intraperitoneally
(i.p.) with the highly virulent RH strain of T. gondii main-
tained in the laboratory. Te tachyzoites for infection were
obtained from peritoneal washings and diluted in phosphate
bufer saline (PBS) to obtain the doses of 1×10
5
, 1×10
6
and
1×10
8
in 0.5 ml of inoculum.
Ducks, experimental design, nutrition and management
A total of 42 ducks were obtained from a commercial farm
in Rongchang county of Chongqing, China. Blood samples
were collected for sera from all ducks before infection. MAT
and PCR were performed to confrm the negativity of the
ducks for T. gondii.
At 14 days of age, the ducks were allocated randomly into
three groups (A, B and C) with 14 birds in each group. Ducks
were infected intravenously as shown in Table 1. On days 7,
14, 21, 28, 42, 56, 70 post infection (p.i.), two ducks in each
group were bled, and the sera were prepared by centrifugation
and stored in –20℃ until further use.
During the experimental phase, tap water and duck for-
mula feed (Chia Tai Co., Ltd, Chongqing, China) sterilized
with high pressure steam before use, were provided to all
ducks.
Clinical examinations
Infected ducks were observed throughout the experimental
period for any clinical manifestations.
Serological examination
Antibodies to T. gondii were determined in duck sera
by the modifed agglutination test (MAT) as described
previously (7, 10, 11). In brief, sera were added to the
“U” bottom of 96 well microtiter plates, and diluted two-
fold starting from 1:5 to 1:160. Briefy, killed T. gondii
tachyzoites were used as antigen and serum samples
were treated with 0.2M 2-mercapthetanol to destroy
the IgM class antibodies. Positive and negative controls
were included in each plate. Tose sera with suspicious
results were retested, and antibodies to T. gondii were
considered seropositive with MAT ≥1:5.
Bioassay in mice for T. gondii isolation
On days 7, 14, 21, 28, 42, 56, 70 post-infection (p.i.), brains,
hearts, spleens, lungs, livers and kidneys of two ducks in each
group were bioassayed for T. gondii as previously described
(12). Briefy, samples were pooled, homogenized in fve
volumes of 0.85% NaCl solution, mixed with fve volumes
of acid-pepsin solution and the mixture was incubated in a
shaker water bath at 37℃ for 60 min. Te digest was fltered
through gauze, centrifuged at 1200×g for 10 min, neutral-
ized with 1.2% sodium bicarbonate, mixed with 1000 U/ml
ampicillin (Amresco, Solon, USA) and 100 µg/ml genta-
mycin sulfate (Amresco, Solon, USA), and immediately the
homogenate of tissues was inoculated intraperitoneally into
fve mice. Tissue imprints of lungs, ascites and brains of dead
mice were examined for T. gondii tachyzoites or tissue cysts.
Survivors were blind passaged 14 days after the inoculation
and again at 7 days intervals. After 4 times of blind passage,
the mice were killed and all brains were examined for tissue
cysts as described (13). Mice were considered infected by T.
gondii if peritoneal exudates or tissue imprints were positive
for tachyzoites or tissue cysts.
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 (10). In brief, PCR reactions (25 uL)
were performed in 2.5 mM of MgCl
2
, 0.4 µM of each primer,
2.5 µL 1×rTaq bufer, 0.25 mM of each desoxyribonucleotide,
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (2)  June 2015 11 Toxoplasma gondii in Ducks
0.625 U of rTaq DNA polymerase (TaKaRa), and 3 µL of
DNA sample in a thermocycler (Biometra) under the follow-
ing conditions: after an initial denaturation at 94℃ for 5 min,
then 35 cycles of 94℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s,
followed by a fnal extension at 72℃ for 7 min. Te positive
control of T. gondii DNA and negative control (non-DNA)
were included in all amplifcation runs. Each amplicon (13
µL) was examined by agarose gel electrophoresis to validate
amplifcation efciency.
RESULTS
Clinical examinations
Ducks in groups A and B appeared clinically normal dur-
ing the experimental period. In group C, all the 14 ducks
displayed typical clinical signs of toxoplasmosis, such as
diarrhea, anorexia and apathy, and they died before day 4 p.i.
Serological examination
Te specifc anti-T.gondii antibodies at titers between 1:5 and
1:320 were detected in all infected ducks of groups A and
B from day 14 to day 56 p.i. using the MAT (Table 1). On
the days 7 and 70 p.i., the titers of these two groups were all
lower than 1: 5 and were therefore regarded as seronegative.
In group C, no samples could be collected as all the ducks
had died before day 4 p.i.
Isolation of T. gondii from the ducks
All the living ducks (Table 1) were used for T. gondii
bioassay and the results were shown in Table 2. In group
A, T. gondii were successfully bioassayed from day 7 p.i.,
14 p.i. and 21 p.i. ducks, with the MAT titers nega-
tive, 1:5 and 1:80, respectively. However in ducks with
MAT titers of 1:80, 1:40, 1:20 and negative titers on
the following days 28, 42, 56 and 70 the bioassay was
negative. Whereas regarding group B, T. gondii were
successfully bioassayed at all the time points, regardless
of the seropositivity or seronegativity for T.gondii.
Identifcation of the isolate by PCR
All the isolates were identifed by PCR using a 341bp frag-
ment of the ITS-1 gene as a target. Alignment of nucleic
acid sequences of PCR products shared 100% similarity to
RH strain of T.gondii (accession No. U16161) homologues,
confrmed that they belonged to T. gondii.
DISCUSSION
MAT is regarded as a sensitive method for measuring anti-
bodies to T. gondii in ducks and is considered as an efective
serologic method as a guide to isolating T. gondii from tissues
of ducks. Utilizing this method, many T. gondii strains have
been successfully isolated from ducks (7, 8) and chickens
(14-20) in previous reports. Tus the MAT method was
used in this research to study the relevance and correlation
of serological response and bioassay.
In the present study, both groups A and B were success-
fully bioassayed for T.gondii on day 7 p.i, even though the
serological response was negative. Te reason for this might
be that during the early stages infection, antibodies were
not yet produced in spite of the parasite already existing in
the ducks. Tese fndings were in accordance with that of
Wang (21).
From day 14 to 56 p.i., all the ducks in the group A
and B were seropositive for T.gondii infection. But group
A ducks on day 28, 42 and 56 p.i. failed to bioassay. Tis
might be explained on the grounds of a low infective dos-
age of tachyzoites had been removed by the immune system
of the duck, or that the amount of tachyzoites or cysts was
too low to be bioassayed. However, the exact reason for this
failure of the bioassay still requires further research. At the
last time point of the study, T.gondii was successfully bioas-
Table 1. Antibody titers by MAT in ducks experimentally
infected with T. gondii.
Group Dose
Titers of antibody to T. gondii using MAT
7d p.i. 14d p.i. 21d p.i. 28d p.i. 42d p.i. 56d p.i. 70d p.i.
A 10
5
1:2 1:5 1:80 1:80 1:40 1:20 1:4
B 10
6
1:4 1:20 1:160 1:320 1:160 1:40 1:4
C 10
8
- - - - - - -
- ducks dying on day 4 post infection (p.i).
Titers were considered seronegative when MAT < 1:5
Table 2. T. gondii isolation in bioassayed mice from tissues of
experimentally infected ducks
Group Dose
Isolation results
7d p.i. 14d p.i. 21d p.i. 28d p.i. 42d p.i. 56d p.i. 70d p.i.
A 10
5
+ + + - - - -
B 10
6
+ + + + + + +
p.i.: post infection
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Israel Journal of Veterinary Medicine  Vol. 70 (2)  June 2015 Zhao, G.W. 12
sayed from group B ducks, regardless of the negative MAT
antibody titer. Tis indicated that even if the serodiagnosis
of T. gondii was negative, the parasite could still be possibly
isolated. On the basis of above fndings, we may cautiously
draw the conclusion that successful bioassay was not entirely
depend on the positive MAT diagnosis. Furthermore, the
existence of T. gondii and the quantity of the parasite may
be the major key parameters during the bioassay studies.
Our fndings were in accordance with those of Dubey et al.
(22) who reported that successful isolation depended on the
amount of tissue-bioassayed and the concentration of the
parasite in sampled tissues.
To date, the susceptibility of ducks to toxoplasmosis has
been seldom reported. In the present study, 108 tachyzoites
administrated intravenously ducks caused death within 4
days, with typical clinical signs of diarrhea, anorexia and
apathy. Although the usage of a lower dosage in ducks with
10
6
and 10
5
tachyzoites showed no clinical symptoms, they
all bioassayed positively for T.gondii. Tese fndings indicated
that ducks were relatively susceptible to the infection with
T. gondii. Our fndings are in agreement with the results ob-
tained by Bartova et al. (23) using T. gondii infected domestic
ducks.
In this study, ducks of group A and B presented a rela-
tive short period of seropositivity after infection. Te exact
reasons for this are not clear. However, it might in part be
due to the fact that the ducks were infected with tachyzoites
using the intravenous route which neither the natural route
of infection not the natural stage of infection. Tis experi-
ment carried out using oocysts via oral infection may have
produced diferent results.
CONCLUSIONS
Te results of the present study demonstrate that successful
bioassay of T. gondii from experimentally infected ducks with
tachyzoites was not entirely depended on the positive MAT
diagnosis. Te amount of T.gondii in the infected host may
be the key factor for the bioassay studies.
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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