J. Parasitol., 94(1), 2008, pp. 68–71 䉷 American Society of Parasitologists 2008
SEROPREVALENCE AND ISOLATION OF TOXOPLASMA GONDII FROM FREE-RANGE CHICKENS IN GHANA, INDONESIA, ITALY, POLAND, AND VIETNAM J. P. Dubey, Lam Thi Thu Huong*, B. W. L. Lawson†, D. T. Subekti‡, P. Tassi§, W. Cabaj㛳, N. Sundar, G. V. Velmurugan, O. C. H. Kwok, and C. Su# U.S. Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, Maryland 20705-2350. e-mail:
[email protected] ABSTRACT:
The prevalence of Toxoplasma gondii in free-ranging chickens (Gallus domesticus) is a good indicator of the prevalence of the parasite’s oocysts in soil because chicken feed from the ground. The prevalence of T. gondii in free-range chickens from Ghana, Indonesia, Italy, Poland, and Vietnam was determined using the modified agglutination test (MAT). Antibodies to T. gondii were found in 41 (64%) of 64 chickens from Ghana, 24 (24.4%) of 98 chickens from Indonesia, 10 (12.5%) of 80 chickens from Italy, 6 (30%) of 20 chickens from Poland, and 81 (24.2%) of 330 chickens from Vietnam. Hearts and brains of chickens were bioassayed for T. gondii. Viable T. gondii was isolated from 2 chickens from Ghana, 1 chicken from Indonesia, 3 chickens from Italy, 2 chickens from Poland, and 1 chicken from Vietnam. Toxoplasma gondii isolates from 9 chickens were genotyped using 10 PCR-RFLP markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico. A total of 7 genotypes was identified; the 3 isolates from chickens from Italy were clonal type II, and the others were nonclonal. This is the first report of genetic characterization of T. gondii isolates from animals from these countries. chickens in Kumasi during August 2006 and January 2007 and sent to Beltsville in 4 batches. Kumasi is the second largest city of Ghana, with a population of about 3 million. It is located between 6⬚30⬘ and 7⬚00⬘N, and 1⬚30⬘ and 2⬚00⬘W, at an altitude of 287 m above sea level. It is a humid area (relative humidity ranges between 53 and 93%). Chickens from Indonesia were obtained from 19 properties in 12 towns, killed 7–13 July 2003 and sent as a single batch. Chickens from Italy were from 16 rural livestock farms located in the southern part of the country, and the sampling was from November 2003 to 2004; 5 chickens were obtained from each of the 16 farms. Chickens from Poland were from 2 small farms near Kartuzy (54⬚20⬘N, 18⬚11⬘E) in the Pomeranian Province (the northern part of Poland). Hearts and sera from 20 chickens from Poland were received in February 2005. Chickens from Vietnam were from rural Dong Nai, Ben Tre, Tien Giang, Long An, Tra` Vinh, and Vinh Long and were purchased during April–September 2003 from 38 properties that were at least 1 km apart.
Toxoplasma gondii infections are widely prevalent in humans and other animals worldwide (Dubey and Beattie, 1988). Humans become infected postnatally by ingesting tissue cysts from undercooked meat, consuming food or drink contaminated with oocysts, or by accidentally ingesting oocysts from the environment. Felids are the most important host in the life cycle of T. gondii because they excrete environmentally resistant oocysts. Cats become infected with T. gondii by eating infected tissues from intermediate hosts. Birds and rodents are considered an important source of T. gondii infection for cats. We have initiated a worldwide study of T. gondii population structure (Dubey, Gennari et al., 2006, and references contained; Lehmann et al., 2006). For this purpose, we chose the free-range chicken as the best indicator for soil contamination with T. gondii oocysts because they feed from the ground. In the present study, we report seroprevalence of T. gondii in chickens from Ghana, Indonesia, Italy, Poland, and Vietnam, and results of our effort to isolate viable T. gondii from chickens.
Serological examination Sera of chickens were tested for T. gondii antibodies using the modified agglutination test (MAT) as described by Dubey and Desmonts (1987). Bioassay of chicken tissues for T. gondii
MATERIALS AND METHODS Naturally infected chickens
Tissues of chickens were bioassayed in mice or cats for T. gondii either individually for each chicken or in groups. Tissues were homogenized, digested in an acidic pepsin solution, neutralized, and centrifuged (Dubey, 1998), and the homogenate was inoculated subcutaneously into 4–5 outbred female Swiss Webster (SW) mice (Taconic Farms, Germantown, New York) as described by Dubey et al. (2002). The recipient mice were examined for T. gondii infection. Tissue imprints of mice that died were examined for T. gondii tachyzoites or tissue cysts. Survivors were bled on days 40–42 postinoculation (PI) and a 1:25 dilution of serum from each mouse was tested for T. gondii antibodies with the MAT. Mice were killed 45–48 days PI and brains of all mice were examined for tissue cysts as described (Dubey and Beattie, 1988). The inoculated mice were considered infected with T. gondii when tachyzoites or tissue cysts were found in tissues. For further propagation of T. gondii, tissues from some SW infected mice were inoculated in to interferon gamma gene knock out (KO) mice; the KO mice are highly susceptible to protozoa because they lack the cytokines needed for protection against T. gondii (Dubey et al., 2005). For bioassay in cats, tissues from chickens were fed to T. gondii– free cats (Dubey et al., 2002). Feces of cats were examined for shedding of T. gondii oocysts 3–14 days postingestion of chicken tissues. Fecal floats were incubated for 1 wk at room temperature to allow sporulation of oocysts and were bioassayed in mice (Dubey and Beattie, 1988). The inoculated mice were examined for T. gondii infection.
Free-range chickens were obtained from the countries listed in Table I. Chickens were purchased, killed by cervical dislocation, and samples of brain, whole heart, and blood were collected from each chicken, kept at 4 C until sent with cold packs by air to Beltsville, Maryland. Two to 10 days elapsed between killing of chickens and receipt of samples at Beltsville. Chickens from Vietnam, Indonesia, and Ghana had autolyzed by the time they were received at Beltsville. Chickens from Ghana were purchased from the market for free-range Received 20 June 2007; revised 9 July 2007; accepted 10 July 2007. * Department of Parasitology and Pathobiology, Faculty of Veterinary Medicine, University of Agriculture and Forestry, Ho Chi Min City, Vietnam. † Department of Theoretical and Applied Biology, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana, West Africa. ‡ Research Institute for Veterinary Science, Martadinata 30, Bogor 30 Bonor 16114, Indonesia. § Dipartimento di Sanita´ e Benessere degli Animali, Universita` di Bari, Strada Prov. Per Casamassima, km 3, 70010 Valenzano, Italy. 㛳 Witold Stefanski Institute of Parasitology, Polish Academy of Sciences, 00-818 Warszawa, Twarda 51/55, Poland. # Department of Microbiology, The University of Tennessee, 1414 West Cumberland Ave., Room 409, Knoxville, Tennessee 37996-0845. 68
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TABLE I. Seroprevalence of Toxoplasma gondii antibodies in chickens from different countries.
Country
No. of chickens
Ghana Italy Indonesia Poland Vietnam
64 80 90 20 330
No. of chickens with MAT* titers of
No. positive (%) 41 11 24 6 80
(64) (13.7) (26.6) (30) (24.2)
1:5
1:10
1:20
1:40
1:80
1:160
1:320
1:640
9 4 11 2 34
15 3 5 3 22
1 1 3 0 13
4 0 1 0 1
3 2 2 0 1
4 0 2 1 2
1 1 0 0 7
4 0 0 0 0
* MAT, modified agglutination test.
Genetic characterization of T. gondii isolates Toxoplasma gondii DNA was extracted from the tissues of infected mice and strain typing was performed using PCR-RFLP genetic markers SAG1, SAG2, SAG3, BTUB, GRA6 c22-8, c29-2, L358, PK1, and Apico (Dubey, Patitucci et al., 2006; Su et al., 2006; Dubey, Sundar et al., 2007).
RESULTS Toxoplasma gondii prevalence Ghana: Tissues of 32 of the 41 seropositive chickens were bioassayed in mice. Toxoplasma gondii was isolated from tissues (brain and heart mixed) from 2 chickens (nos. 54 and 55). Chicken 54 had a MAT titer of 1:160, and chicken 55 had a MAT titer of 1:40. Only a few tissue cysts were found in the brains of the 4 mice killed 43 day after inoculation with tissues of chicken no. 54. To propagate and cryopreserve this isolate, brain of each SW mouse was subinoculated into an interferon gamma gene KO mouse; all 4 KO mice inoculated with isolate TgCkGh1 died of acute toxoplasmosis 15 day PI, and tachyzoites were found in lungs of the mice. The T. gondii strain from chicken 54 was designated TgCkGh1. Several tissue cysts were found in the brains of the 4 SW mice inoculated with brain and heart of chicken 55; this isolate was designated as TgCkGh2. Indonesia: Toxoplasma gondii antibodies were found in 24 of 94 chickens received in 1 batch. Tissues of all chickens were bioassayed. Tissues from seronegative birds were mixed and fed to cat; the cat did not shed oocysts. Toxoplasma gondii was isolated from tissues (brain and heart mixed) from 1 chicken; this chicken had a MAT titer of 1:40 and came from Province
Central Java, subdistrict Muntilan (109⬚57⬘0⬙E, 7⬚45⬘0⬙S). Tissue cysts were found in the brains of 3 of the 5 mice killed on day 73 after inoculation with tissues of chicken 12. Italy: Antibodies to T. gondii were found in 11 of 80 chickens (Table I); 8 of these 11 seropositive chickens were from 2 farms. All 11 seropositive chickens were bioassayed for T. gondii. In the first batch, there were 5 seropositive chickens from Farm A located in Gioia del Colle (40⬚44⬘775⬙N, 16⬚48⬘537⬙E). Tissues of all 5 chickens were bioassayed individually in mice, and T. gondii was isolated from 2 chickens. These 2 chickens had titers of 1:80 and 1:320, and the isolates were designated TgCkIt1 and TgCkIt2. In the second batch of 20 chickens, 6 were seropositive, and tissues of all 20 chickens were bioassayed for T. gondii. For this bioassay, tissues of all 6 seropositive chickens were fed to 6 cats individually, and tissues of the 14 seronegative chickens were mixed and fed to 1 cat. One cat fed tissues of a chicken with MAT titer of 1:5 shed T. gondii oocysts; that chicken was from Farm G located in Santeramo. Oocysts of this T. gondii isolate were infective to mice; mice fed oocysts remained asymptomatic, and tissue cysts were found in the brains of the mice killed 31 day PI. This isolate was designated TgCkIt3. Thus, all 3 isolates from chickens from Italy were avirulent for mice. Poland: Six of 20 chickens had T. gondii antibodies, and tissues of all 20 birds were bioassayed. Hearts of 15 seronegative chickens were mixed and fed to 1 cat; the cat did not shed oocysts. Hearts of 5 seropositive chickens were bioassayed individually in mice. Toxoplasma gondii was isolated from 2 chickens (birds 9 and 18). One of the 5 mice inoculated with the heart of bird 9 (MAT titer 1:5) and 1 of the 5 mice inocu-
TABLE II. Genotyping of Toxoplasma gondii isolates from chickens.
Country Ghana Indonesia Italy
Poland Vietnam
Genotyping
T. gondii isolate designation
SAG1
SAG2
New SAG2
SAG3
BTUB
GRA6
c22-8
c29-2
L358
PK1
Apico
TgCkGh1 TgCkGh2 TgCkId1 TgCkIt1 TgCkIt2 TgCkIt3 TgCkPo1 TgCkPo2 TgCkVn1
u-1 II or III I II II II u-1 u-1 u-1
II III I II II II I I II
II III I II II II II II nd*
III III III II II II III III III
III II III II II II III III nd
II II III II II II III III nd
II II II II II II III III II
III III III II II II I I nd
II III III II II II I I nd
III II III II II II III III nd
I III III I II II I I nd
* nd, No data because the quantity of DNA was insufficient.
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THE JOURNAL OF PARASITOLOGY, VOL. 94, NO. 1, FEBRUARY 2008
lated with heart of chicken 18 became infected with T. gondii, and many tissue cysts were found in mouse brains when killed day 49 PI; these isolates were designated TgCkPo1 and TgCkPo2. Vietnam: Tissues of 289 of 330 chickens were bioassayed for T. gondii. Tissues of 80 seropositive chickens were bioassayed in mice individually, and T. gondii was not isolated from any of these chickens. Tissues of 205 chickens with titers of 1:5 or less were mixed into 7 pools and fed to 7 cats. One cat fed tissues of 15 chickens shed oocysts; mice fed oocysts were killed 5 days later, and T. gondii tachyzoites were found in their mesenteric lymph nodes. Pathogenicity of T. gondii isolates to mice All isolates of T. gondii from these chickens were avirulent for mice because none of the infected mice died of toxoplasmosis. Genotyping of T. gondii Genotyping revealed that the 3 isolates from Italy were clonal type II with difference at locus Apico; both TgCkIt1 and TgCkIt2 have type I allele, whereas TgCkIt3 has a type II allele (Table II). The isolates from Poland, Ghana, Indonesia, and Vietnam were nonclonal. Both genotypes from the 2 Ghana isolates (TgCkGh1, TgCkGh2) are unique and have not been previously identified. The genotype of the isolate (TgCkId1) from Indonesia is also unique. The 2 isolates (TgCkPo1, TgCkPo2) share a genotype that is identical to a sheep isolate, CASTELLS, from Uruguay (Su et al., 2006). Genotyping results for the only chicken isolate (TgCkVn1) from Vietnam were incomplete, but the available data suggest it may be identical to dog isolates studied previously (Dubey, Huong et al., 2007). DISCUSSION The low isolation rate of T. gondii from chickens from Ghana, Indonesia, and Vietnam is probably due to autolysis of tissues during delay in transport from these countries to Beltsville. Unfortunately, it was not possible to extend this study to more samples because of the devastation of the poultry industry in southeastern Asia from the epidemic of avian flu and restrictions imposed by the U.S. authorities on international transport of chicken tissues. In the present study, sera of chickens were tested starting with a low (1:5) dilution of serum because T. gondii has been isolated occasionally from chickens with low titers (Dubey et al., 2005, 2006). Generally, titers of ⬍1:25 are regarded as nonspecific, and sera are generally screened starting at a 1:20 or 1: 25 serum dilution in MAT. However, in any population, there will be a few infected individuals with a low titer, and these are likely missed in a routine serological screen. Therefore, Table I has data inclusive of all titers. It is of interest that T. gondii was isolated from 2 chickens from Poland with MAT titers of 1:5 and 1:10; these sera were of good quality, and there was no prozone. The genotypes of the 2 isolates (TgCkGh1 and TgCkGh2) from Ghana and the genotype of isolate (TgCkId1) from Indonesia are unique and were not identified from isolates collected from a variety of hosts in South America and Asia, in-
cluding Brazil, Colombia, Chile, Costa Rica, Nicaragua, Guiana, Vietnam, China, and Sri Lanka. The limited typing result from the Vietnam chicken isolate TgCkVn1 suggests it may be identical to a genotype previously identified in dogs in that area (Dubey, Huong et al., 2007). The 3 isolates from Italy are identical to the clonal type II lineage that is predominant in North America and Europe. Interestingly, the 2 isolates (TgCkPo1 and TgCkPo2) from Poland are identical to a sheep isolate CASTELLS from Uruguay in South America (Su et al., 2006). Toxoplasma gondii isolates have been classified into 3 genetic types (I, II, III) based on restriction fragment length polymorphism (RFLP). The parasite used to be considered clonal, with very low genetic variability. However, most of the previous information was derived from isolates from Europe and North America. Based on newer markers for genetic characterization and using recently isolated strains from Brazil and French Guiana, a higher genetic variability has been revealed than previously reported (Ajzenberg et al., 2004; Lehmann et al., 2006; Dubey, Applewhaite et al., 2007). Most of the isolates from animals and humans from Europe were type II (Owen and Trees, 1999; Ajzenberg, Ban˜uls et al., 2002; Ajzenberg, Cogne´ et al., 2002; Jungersen et al., 2002; Ajzenberg et al., 2004; de Souza et al., 2005; Dubey et al., 2005; Dubey, Vianna et al., 2006; Dumetre et al., 2006). Similarly, all 3 isolates from Italy in the present study were type II. It is also of interest that the 2 isolates from Polish birds were nonclonal and identical to a sheep isolate from Uruguay in South America. New, unique genotypes identified from Ghana and Indonesia warrant further study in Africa and Asia to more completely identify T. gondii genetic diversity in these regions. LITERATURE CITED AJZENBERG, D., A. L. BAN˜ULS, M. TIBAYRENC, AND M. L. DARDE´. 2002. Microsatellite analysis of Toxoplasma gondii shows considerable polymorphism structured into two main clonal groups. International Journal for Parasitology 32: 27–38. ———, ———, C. SU, A. DUME`TRE, M. DEMAR, B. CARME, AND M. L. DARDE´. 2004. Genetic diversity, clonality and sexuality in Toxoplasma gondii. International Journal for Parasitology 34: 1185– 1196. ———, N. COGNE´, L. PARIS, M. H. BESSIE`RES, P. THULLIEZ, D. FILISETTI, H. PELLOUX, P. MARTY, AND M. L. DARDE´. 2002. Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings. Journal of Infectious Diseases 186: 684–689. DE SOUSA, S., D. AJZENBERG, N. CANADA, L. FREIRE, J. M. CORREIA DA COSTA, M. L. DARDE´, P. THULLIEZ, AND J. P. DUBEY. 2006. Biologic and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal. Veterinary Parasitology 135: 133–136. DUBEY, J. P. 1998. Refinement of pepsin digestion method for isolation of Toxoplasma gondii from infected tissues. Veterinary Parasitology 74: 75–77. ———, L. APPLEWHAITE, N. SUNDAR, G. V. VELMURUGAN, L. A. BANDINI, O. C. H. KWOK, R. HILL, AND C. SU. 2007. Molecular and biological characterization of Toxoplasma gondii isolates from freerange chickens from Guyana, South America identified several unique and common parasite genotypes. Parasitology 134: 1–7. ———, AND C. P. BEATTIE. 1988. Toxoplasmosis of animals and man. CRC Press, Boca Raton, Florida. 220 p. ———, AND G. DESMONTS. 1987. Serological responses of equids fed Toxoplasma gondii oocysts. Equine Veterinary Journal 19: 337– 339. ———, R. EDELHOFER, P. MARCET, M. C. B. VIANNA, O. C. H. KWOK, AND T. LEHMANN. 2005. Genetic and biologic characteristics of
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