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Research Article
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Zoonotic Chicken Toxoplasmosis in Some Egyptians Governorates |
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Ashraf Mohamed Barakat,
Lobna Mohamed Ali Salem,
Adel M. Abdel-Aziz El-Newishy,
Raafat Mohamed Shaapan
and
Ehab Kotb El-Mahllawy
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ABSTRACT
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Toxoplasmosis is one of the most common diseases prevalent in the world, caused by a coccidian parasite Toxoplasma gondii which infects humans, animals and birds. Poultry consider reliable human source of food in addition it is considered an intermediate host in transmission of the disease to humans. Trails of isolation of local T. gondii chicken strain through bioassay of the suspected infected chicken tissues in mice was carried out and the isolated strain was confirmed as being T. gondii using Polymerase Chain Reaction (PCR). Seroprevalence of antibodies against T. gondii in chicken sera in six Egyptian governorates were conducted by enzyme linked immune-sorbent assay (ELISA) using the isolated chicken strain antigen. Moreover, comparison between the prevalence rates in different regions of the Egyptian governorates were been estimated. Isolation of local T. gondii chicken strain was accomplished from chicken tissues and confirmed by PCR technique. The total prevalence rate was 68.8% comprised of 59.5, 82.3, 67.1, 62.2, 75 and 50% in El Sharkia, El Gharbia, Kafr El sheikh, Cairo, Quena and Sohag governorates, respectively. The prevalence rates were higher among Free Range (FR) (69.5%) than commercial farm Chickens (C) (68.5%); while, the prevalence rate was less in Upper Egypt than Lower Egypt governorates and Cairo. This study is the first was used antigen from locally isolated T. gondii chicken strain for the diagnosis of chicken toxoplasmosis. The higher seroprevalence particularly in free range chickens (house-reared) refers to the public health importance of chickens as source of zoonotic toxoplasmosis to human.
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Received: October 04, 2012;
Accepted: December 16, 2012;
Published: February 12, 2013
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INTRODUCTION
There is no doubt that the world suffers from the lack of nutrition especially
those of animal origin, such problem is enlarged day after day especially in
Egypt, poultry sector is one of the key elements in the economies of developed
countries as it is one of the main sources for the provision of animal protein
and meat consumed (Deyab and Hassanein, 2005). The
improvement of poultry production is an important way to compensate the deficiency
in protein requirements especially in Egypt, where Poultry production in Egypt
and other middle-income countries totaled 37.5 million tons, compared with 29.3
million tons in high-income countries and only 3.6 million tons in low-income
countries (Taha, 2003).
T. gondii is an obligate intracellular, the definitive hosts only are
domestic and wild cats; in which the sexual phase of the life cycle occurs in
the intestinal epithelium ended by the shedding of un-sporulated oocysts in
feces and the sporulation occurs outside the host. In the intermediate hosts
which are either animals, birds or man a transient acute phase caused by tachyzoites
followed by the chronic phase which is characterized by formation of dormant
tissue cysts contain bradyzoites (Dubey, 2010a). The
T. gondii infection routes to human through ingesting undercooked meat
containing the tissue cysts, consuming food or drink contaminated with oocysts
and/or accidentally ingesting oocysts from the environment (Dubey
et al., 2004).
Different serological tests were performed in many countries for serologic
prevalence in free-range or backyard chickens worldwide. Prevalence varied from
2-100% depending on the source of the chickens. Prevalence of infection was
17.9% in India (Sreekumar et al., 2003), 38%
in Brazil (Dubey et al., 2007a) and 85% in Nicaragua
(Dubey et al., 2006) using Modified Agglutination
Test (MAT). Recently, Bartova et al. (2009)
reported, T. gondii antibodies in 1 of 293 chickens from the Czech Republic,
Chumpolbanchorn et al. (2009) found T. gondii
antibodies in 64% of 303 FR chickens from Thailand using Indirect Fluorescent
Antibody Test(IFAT) and Yan et al. (2009) reported
T. gondii antibodies in 11.4% of 361 FR chickens and in 4.1% of caged
chickens in Peoples Republic of China using MAT. Few reports of chicken
toxoplasmosis from Egypt, the infection rate were 47.2% (El-Massey
et al., 2000) and 40.4% (Dubey et al.,
2003) in free range chicken using MAT and in additionally Deyab
and Hassanein, 2005, found MAT antibodies in 18.7% of 150 chickens from
slaughterhouses in Egypt.
Chickens rarely have clinical toxoplasmosis (Dubey, 2010b).
First reported of toxoplasmosis was confirmed in a flock of 40 White Leghorn
hens from Norway by Erichsen and Harboe (1953) such
an outbreak has not been reported in the literature in the last 50 years. Two
reports of clinical toxoplasmosis were detected chickens from the United States;
1st from Georgia, peripheral neuritis in three chickens (Goodwin
et al., 1994) and 2nd in Illinois, 3 birds died suddenly out of a
group of 14 backyard chickens with an unusual finding was the presence of numerous
tissue cysts and tachyzoites in the lesions; the protozoa in the brain reacted
positively with T. gondii antibodies and the remaining 11 chickens remained
asymptomatic, though all contained viable T. gondii (Dubey
et al., 2007b).
Many serological tests were reported in the diagnosis of chicken toxoplasmosis;
DT, like the CFT, does not detect antibodies to T. gondii in chicken
sera or had only low titers even sera obtained from experimentally infected
chickens with T. gondii (Dubey et al., 1993).
The IHAT is an insensitive test, in 13 experimentally infected chickens, its
sensitivity was 46% and specificity of a negative test was 25% (Frenkel,
1981). There is limited information on the efficacy of IFAT test for the
detection of T. gondii antibodies in chicken sera, viable T. gondii
isolated from tissues of 9 of 15 (60%) chickens with IFAT titers of 1:16 or
higher and from 2 of 13 (15.3%) sero-negative free-range chickens from Brazil
(Brandao et al, 2006). ELISA, 1st studied in
chickens fed oocysts, using the soluble fraction of tachyzoites, the chicken
sera were diluted in buffered 7% NaCl (instead of 0.85%) aqueous solution (saline)
because most chicken immunoglobulin precipitate when diluted with phosphate
buffered saline (Biancifiori et al., 1986).
Although, the sensitivity and specificity of the MAT in naturally exposed chickens
is under investigation in my laboratory using isolation of the parasite as a
standard, MAT is efficient in detecting T. gondii antibodies in chickens
(Dubey, 2010b).
T. gondii infected chickens has important role in the epidemiology of
toxoplasmosis in humans. Therefore, the main objectives of this study are to
isolate the isolate local T. gondii chicken strain through bioassay of
the suspected infected chicken tissues in mice and confirm it by PCR. Also,
to estimate the seroprevalence of T. gondii infection among chicken from
free range and commercial farm in some Egyptian governorates using ELISA. Moreover,
compare between the prevalence rates results in different regions of the Egyptian
governorates.
MATERIALS AND METHODS
Experimental animals: This research study was conducted from 5-2012
to 12-2012. Swiss albino laboratory breed mice about 1 month-old were used for
bioassay of the suspected infected chicken tissues and maintenance of isolated
T. gondii local strain in the lab. All mice were kept in clean cages,
fed on pelleted food and clean water supply with multivitamins.
Chicken tissue samples: Meat and tissue samples were obtained from slaughtered
chicken at different regions, in Egypt. Pooled tissue sample, about 25-50 g,
from heart, brain and pectoral muscles were prepared as described by Shaapan
and Ghazy, 2007, the tissues and organs was cut into small cubes about 5x5x5
cm and stored at 4°C for few hours until used for bioassay in mice.
Isolation of local T. gondii chicken strain: Tissue samples were
digested by pepsin according to method of Sharma and Dubey
(1981), followed by microscopically examined at low and high powers for
the presence of T. gondii tissue cysts containing the bradyzoites. Bioassay
of the digested positive tissues in mice according method mentioned by Dubey
(2010a), in order to obtain the tachyzoites stage which maintained in the
laboratory through repeated intra peritoneal mouse inoculation in mice (Johnson
et al., 1979).
Chicken blood collection and sera preparation: Blood samples were collected
in dry, sterile screw capped tubes of 15 cm from 439 apparently healthy chickens
(free range and commercial farms) from six governorates include El Gharabia,
Kafr El-shiekh, El Sharkia, Sohag Quena and Cairo. Samples were centrifuged
at 3000 rpm for 15 min and stored at -20°C until used in serological investigation.
Serological survey for detection of T. gondii antibodies: The
collected chicken sera were examined serologically for detection of T. gondii
antibodies using the Enzyme Linked Immuno-sorbant Assay (ELISA), the whole soluble
tachyzoites antigens were prepared as described by Waltman
et al. (1984) and the procedures adopted according to Lind
et al. (1997).
Polymerase chain reaction (PCR): In this study, PCR was adopted for
confirmation that tachyzoites as previously isolated from chicken tissues and
used in serological diagnosis (as local strain) belongs to T. gondii
strain. Genomic DNA was extracted from tested T. gondii local and RH
(positive control) strains using a commercially available kit (Dneasy blood
and Tissue kit, Qiagen Co., Cat. No. 69504) with modifications to the manufacturers
protocols (Howe et al., 1997). Primers used for
PCR were targeting the repetitive 35-fold B1 gene. The amplified product was
analyzed on 1% agarose gel and stained with Ethidium bromide. Every PCR run
included controls (Burg et al., 1989).
RESULTS
Morphological studies of the isolated T. gondii developmental stages:
Two developmental infective stages of T. gondii were isolated; trachyzoites
and bradyzoites.
Tachyzoites: Tachyzoites were obtained from the peritoneal exudates
of previously inoculated mice with infected digested chickens
tissues 6-8 post inoculation. Tachyzoites were found inside leucocytes (lymphocytes
and macrophages) or free in the peritoneal exudates after rupture of leucocytes.
The Giemsa stained isolated tachyzoites were often crescent in shape or banana
shape pointed at one end and rounded or blunt at the other one, their size were
2x7 μm. It showed pale blue cytoplasm with reddish purple nucleus which
centrally located or near the blunt end. The leucocytes appear as dark blue
color (Fig. 1).
Tissue cysts containing bradyzoites: The tissue cysts of isolated T.
gondii strain were noticed in the fresh smears after digestion of heart
and brain tissues.
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Fig. 1: |
Giemsa stained T. gondii tachyzoites (X400) |
The tissue cyst is the resting stage of parasite within the host, they were
usually sub-spherical to spherical in shape and its cyst wall was thin elastic
and will defined enclosing up several hundreds of crescent shaped Bradyzoites
(Fig. 2).
Confirmatory PCR results: As number of base pairs (94 pb) of the isolated
T. gondii strain obtained from chicken tissues (lane, 3 and 4) was the
same of compared positive control RH strain (lane, 2) with the presence of negative
control sample (lane,1) and matched with standard marker (lane, M) (Fig.
3). So the PCR results confirm that tachyzoites which previously isolated
is belong to T. gondii.
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Fig. 2: |
Giemsa stained T. gondii tissue cysts (X1000) |
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Fig. 3: |
Detection of T. gondii by PCR. Lane 1: Negative control,
Lane 2: Positive control (RH stain), M: MW marker (100 bp ladder) and Lanes
3 and 4: Positive PCR samples of isolated chicken strain at 94 bp |
Table 1: |
Seroprevalence of T. gondii in six governorates |
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C: Commercial forms, FR: Free range |
Serological investigation of T. gondii antibodies in chicken sera:
Examination of the total 439 serum samples of chickens by ELISA revealed that
302 sample (68.8%) had antibodies against T. gondii which was considered
the percentage of infection. The obtained results indicate that prevalence rate
was slightly higher, (69.5%) in free range chicken (FR) than that (68.5%) in
chicken from commercial farms (C) in the most of 6 examined governorate and
also results refer to that the prevalence rates was less in Upper Egypt than
governorates than that of Lower Egypt and Cairo (Table 1).
DISCUSSION
The high prevalence rate of chicken toxoplasmosis (68.8%) in the present study
which comprised 6 examined Egyptian governorates; refer to the importance of
chickens meat as important source of infection among Egyptian consumers
which agreed with that previously detected in Egypt by El-Massey
et al. (1990), who reported that sheep, goat and chicken are important
intermediate hosts for T. gondii and their meat are main source of infection
to man if un-hygienically consumed.
However, present survey showed that seroprevalence rates was higher in Free-Range
(FR) (69.5%) than commercial farm Chicken (C) (68.5%) indicated that the distribution
of T. gondii in chicken might be related to the cats that disseminated
the oocysts in the soil (Asgari et al., 2006).
On the other hand, cats could be infected from eating chicken meat, wild birds
and rodents infected with T. gondii (Hassanain et
al., 2008). Therefore, T. gondii infection in chickens
is epidemiologically significant and people have the habit of eating under-cooked
chicken meat, should be aware of the risk of encountering T. gondii infection.
Little is known concerning the validity of the serologic tests for the detection
of T. gondii antibodies in avian sera. Viable T. gondii was isolated
from up to 100% of free-range chickens bio-assayed in mice (Dubey
et al., 2007b). The present study, ELISA test used for seroprevalence
of 439 chicken blood samples collected from six Egyptian governorates. The ELISA
test has the advantage that it specific and of great sensitivity, objective,
quantitative, low coast and can be automated and is convenient for large-scale
surveys of toxoplasmosis in human and animals but it need a refinement in the
procedures and standardization (Shaapan et al., 2008).
Many surveys detecting T. gondii infection in FR chickens have been
conducted in a number of countries and the prevalence rates in most of these
studies ranged between 17.9% and 85% (Deyab and Hassanein,
2005; Sreekumar et al., 2003; Dubey
et al., 2007a; Dubey et al., 2006;
Yan et al., 2009; El-Massey
et al., 2000; Dubey et al., 2003).
The seroprevalence of T. gondii in domestic birds varies within
countries due to the testing methods, the number of examined animals, type and
hygiene of animal breeding Dubey (2010a). The total
seroprevalence T. gondii among chicken in our study (68.7%) was lower
than some results other results (Sreekumar et al.,
2003; Dubey et al., 2007a; Yan
et al., 2009; El-Massey et al., 2000)
similar to that (64%) in FR chicken in Thailand (Chumpolbanchorn
et al., 2009), While lower than 85% obtained in Nicaragua (Dubey
et al., 2006). Reasons for this variability could be many, including
the age of the chicken, number examined, type of serological test used and the
tissues bio-assayed. Overall, prevalence of viable T. gondii in chickens
raised indoors was low (Dubey et al., 2007a).
Concerning PCR results, the matched sequence of base pairs with the stander
for T. gondii confirm that PCR assay is a specific, speedy, sensitive
and cost-effective method for detecting T. gondii DNA in chickens. The
choice of using the fragment of 94 bp from the B1 gene as target to PCR amplification
was based on the observations made by other studies (Howe
et al., 1997; Dubey et al., 2005).
CONCLUSION
In considering the above-mentioned findings, it could be concluded that T.
gondii infection was spread widely in Egyptian poultry, so it give aware
of the public health danger of poultry as an intermediate host which transmit
the infection to man. The significantly higher infection rate than previous
studies indicate that ELISA is more sensitive method for the diagnosis of toxoplasmosis.
Moreover, this study is the first was used antigen from locally isolated T.
gondii chicken strain for the diagnosis of chicken toxoplasmosis.
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REFERENCES |
1: Asgari, Q., A. Farzaneh, M. Kalantari, F.A. Mohajeri and M. Moazeni et al., 2006. Seroprevalence of free-ranging chicken toxoplasmosis in Sub-Urban Regions of Shiraz, Iran. Int. J. Poult. Sci., 5: 262-264. CrossRef | Direct Link |
2: Bartova, E., K. Sedlak and I. Literak, 2009. Serologic survey for toxoplasmosis in domestic birds from the Czech Republic. Avian Pathol., 38: 317-320. CrossRef |
3: Biancifiori, F., C. Rondini, V. Grelloni and T. Frescura, 1986. Avian toxoplasmosis: Experimental infection of chicken and pigeon. Comp. Immunol. Microbiol. Infect. Dis., 9: 337-346. CrossRef | Direct Link |
4: Brandao, G.P., A.M. Ferreira, M.N. Melo and R.W.A. Vitor, 2006. Characterization of Toxoplasma gondii from domestic animals from Minas Gerais, Brazil. Parasite, 13: 143-149. PubMed | Direct Link |
5: Burg, J.L., C.M. Grover, P. Pouletty and J.C. Boothroyd, 1989. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. J. Clin. Microbiol., 27: 1787-1792. PubMed | Direct Link |
6: Chumpolbanchorn, K., P. Anankeatikul, W. Ratanasak, J. Wiengcharoen, R.C.A. Thompson and Y. Sukthana, 2009. Prevalence of Toxoplasma gondii indirect fluorescent antibodies in naturally-and experimentally-infected chickens (Gallus domesticus) in Thailand. Acta Parasitol., 54: 194-196. CrossRef |
7: Deyab, A.K. and R. Hassanein, 2005. Zoonotic toxoplasmosis in chicken. J. Egypt. Soc. Parasitol., 35: 341-350. PubMed | Direct Link |
8: Dubey, J.P., 2010. Toxoplasmosis of Animals and Humans. 2nd Edn., CRC Press Inc., Boca Raton, FL., USA., ISBN-13: 9781420092363, Pages: 336
9: Dubey, J.P., 2010. Toxoplasma gondii infections in chickens (Gallus domesticus): Prevalence, clinical disease, diagnosis and public health significance. Zoonoses Public Health, 57: 60-73. CrossRef | PubMed | Direct Link |
10: Dubey, J.P., M.D. Ruff, M.E. Camargo, S.K. Shen, G.L. Wilkins, O.C.H. Kwok and P. Thulliez, 1993. Serologic and parasitologic responses of domestic chickens after oral inoculation with Toxoplasma gondii oocysts. Am. J. Vet. Res., 54: 1668-1672. PubMed | Direct Link |
11: Dubey, J.P., D.H. Graham, E. Dahl, M. Hilali and A. El-Ghaysh et al., 2003. Isolation and molecular characterization of Toxoplasma gondii from chickens and ducks from Egypt. Vet. Parasitol., 114: 89-95. CrossRef | PubMed | Direct Link |
12: Dubey, J.P., M.Z. Levy, C. Sreekumar, O.C.H. Kwok and S.K. Shen et al., 2004. Tissue distribution and molecular characterization of chicken isolates of Toxoplasma gondii from Peru. J. Parasitol., 90: 1015-1018. CrossRef | Direct Link |
13: Dubey, J.P., B. Lopez, M. Alvarez, C. Mendoza and T. Lehmann, 2005. Isolation, tissue distribution and molecular characterization of Toxoplasma gondii from free-range chickens from Guatemala. J. Parasitol., 91: 955-957. CrossRef | Direct Link |
14: Dubey, J.P., N. Sundar, N. Pineda, N.C. Kyvsgaard and L.A. Luna et al., 2006. Biologic and genetic characteristics of Toxoplasma gondii isolates in free-range chickens from Nicaragua, Central America. Vet. Parasitol., 142: 47-53. CrossRef |
15: Dubey, J.P., N. Sundar, S.M. Gennari, A.H. Minervino and N.A. Farias et al., 2007. Biologic and genetic comparison of Toxoplasma gondii isolates in free-range chickens from the northern Para state and the southern state Rio Grande do Sul, Brazil revealed highly diverse and distinct parasite populations. Vet. Parasitol., 143: 182-188. CrossRef |
16: Dubey, J.P., D.M. Webb, N. Sundar, G.V. Velmurugan, L.A. Bandini, O.C.H. Kwok and C. Su, 2007. Endemic avian toxoplasmosis on a farm in Illinois: Clinical disease, diagnosis, biologic and genetic characteristics of Toxoplasma gondii isolates from chickens (Gallus domesticus) and a goose (Anser anser). Vet. Parasitol., 148: 207-212. CrossRef |
17: El-Massey, A.A., A.M. Abdel-Gawad and A.M. Nassar, 1990. Isolation of Toxoplasma gondii, Isospora felis and Isospora revolta from (sheep, goats, chicken) in Egypt. J. Egypt. Vet. Med. Assoc., 50: 275-284.
18: El-Massry, A.A., O.A. Mahdy, A. El-Ghaysh and J.P. Dubey, 2000. Prevalence of Toxoplasma gondii antibodies in sera of turkeys, chickens and ducks from Egypt. J. Parasitol., 86: 627-628. CrossRef | PubMed | Direct Link |
19: Erichsen, S. and A. Harboe, 1953. Toxoplasmosis in chickens: I. An epidemic outbreak of toxoplasmosis in a chicken flock in Southeastern Norway. Acta Pathol. Microbiol. Scand., 33: 56-71. CrossRef |
20: Frenkel, J.K., 1981. False-negative serologic tests for Toxoplasma in birds. J. Parasitol., 67: 952-953. PubMed |
21: Goodwin, M.A., J.P. Dubey and J. Hatkin, 1994. Toxoplasma gondii peripheral neuritis in chickens. J. Vet. Diagn. Invest., 6: 382-385. CrossRef | Direct Link |
22: Hassanain, M.A., A.M. Barakat, H.A. Elfadaly, N.A. Hassanain and R.M. Shaapan, 2008. Zoonotic impact of Toxoplasma gondii Sero-prevalance in naturally infected Egyptian kittens. J. Arab Soc. Med. Res., 3: 243-248. Direct Link |
23: Howe, D.K., S. Honore, F. Derouin and L.D. Sibley, 1997. Determination of genotypes of Toxoplasma gondii strains isolated from patients with toxoplasmosis. J. Clin. Microbiol., 35: 1411-1414. Direct Link |
24: Johnson, A.M., P.J. McDonald and S.H. Neoh, 1979. Kinetics of the growth of Toxoplasma gondii (RH strain) in mice. Int. J. Parasitol., 9: 55-56. CrossRef | Direct Link |
25: Lind, P., J. Haugegaard, A. Wingstrand and S.A. Henriksen, 1997. The time course of the specific antibody response by various ELISAs in pigs experimentally infected with Toxoplasma gondii. Vet. Parasitol., 71: 1-15. CrossRef | PubMed | Direct Link |
26: Shaapan, R.M. and A.A. Ghazy, 2007. Isolation of Toxoplasma gondii from horse meat in Egypt. Pak. J. Biol. Sci., 10: 174-177. CrossRef | PubMed | Direct Link |
27: Shaapan, R.M., F.A. El-Nawawi and M.A.A. Tawfik, 2008. Sensitivity and specificity of various serological tests for the detection of Toxoplasma gondii infection in naturally infected sheep. Vet. Parasitol., 153: 359-362. CrossRef | Direct Link |
28: Sharma, S.P. and J.P. Dubey, 1981. Quantitative survival of Toxoplasma gondii tachyzoites and bradyzoites in pepsin and in trypsin solutions. Am. J. Vet. Res., 42: 128-130. PubMed | Direct Link |
29: Sreekumar, C., D.H. Graham, E. Dahl, T. Lehmann and M. Raman et al., 2003. Genotyping of Toxoplasma gondii isolates from chickens from India. Vet. Parasitol., 118: 187-194. CrossRef |
30: Taha, A.F., 2003. The poultry sector in middle-income countries and its feed requirements: The case of Egypt. Agriculture and Trade Reports, Electronic Outlook Report from the Economic Research Service, United States Department of Agriculture, USA.
31: Waltman, W.D., D.W. Dreesen, M.D. Prickett, J.L. Blue and D.G. Oliver, 1984. Enzyme-linked immunosorbent assay for the detection of toxoplasmosis in swine: Interpreting assay results and comparing with other serologic tests. Am. J. Vet. Res., 45: 1719-1725. PubMed | Direct Link |
32: Yan, C., C.L. Yue, Z.G. Yuan, Y. He and C.C. Yin et al., 2009. Toxoplasma gondii infection in domestic ducks, free-range and caged chickens in southern China. Vet. Parasitol., 165: 337-340. CrossRef |
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