Genetic characterization of Toxoplasma gondii isolates from China

Parasitology International 58 (2009) 193–195

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Genetic characterization of Toxoplasma gondii isolates from China Peng Zhou a, Han Zhang b, Rui-Qing Lin a, De-Lin Zhang c, Hui-Qun Song a, Chunlei Su d, Xing-Quan Zhu a,⁎ a

College of Veterinary Medicine, South China Agricultural University, 483 Wushan Street, Tianhe District, Guangzhou, Guangdong Province 510642, PR China Guangdong Institute for Animal Disease Control and Surveillance, 113 Wanshou Street, Haizhu District, Guangzhou, Guangdong Province 510230, PR China c Department of Parasitology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 1 Xujiaping, Yanchangbu, Lanzhou, Gansu Province 730046, PR China d Department of Microbiology, The University of Tennessee, Knoxville, TN. 37996, USA b

a r t i c l e

i n f o

Article history: Received 18 November 2008 Received in revised form 20 December 2008 Accepted 24 January 2009 Available online 1 February 2009 Keywords: Toxoplasma gondii Population structure Polymorphism Genotyping Multilocus PCR–RFLP China

a b s t r a c t Toxoplasma gondii infections are prevalent in humans and animals worldwide. In North America and Europe, T. gondii is highly clonal, consisting of three distinct lineages (Types I, II and III), whereas in South America, T. gondii is highly diverse with a few lineages expanded in the population. However, there is limited data on the diversity of T. gondii in Asia. Here we report the genetic characterization of T. gondii isolates from different hosts and geographical locations in China using the multilocus PCR–RFLP. A total of 17 T. gondii isolates from humans (3 strains), sheep (1 strain), pigs (5 strains) and cats (8 strains) were typed at 10 genetic markers including 9 nuclear loci SAG1, SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2 and an apicoplast locus Apico. Four genotypes were revealed, including three previously reported and one new genotype. Three isolates belong to the clonal Type I lineage, one isolate belongs to the clonal Type II lineage, and the rest 13 isolates are grouped into two genotypes. This is the first report of genetic typing of T. gondii isolates from different hosts and geographical locations in China using a number of genetic markers, which has implications for the studies of population genetic structures of T. gondii, as well as for the prevention and control of T. gondii infections in humans and animals in China. © 2009 Elsevier Ireland Ltd. All rights reserved.

Toxoplasma gondii infections are widely prevalent in human beings and animals worldwide [1]. Humans become infected post-natally by ingesting tissue cysts from undercooked meat, consuming food or water contaminated with oocysts, or by accidentally ingesting oocysts from the environment [1]. Most T. gondii isolates from humans and animals in North America and Europe have been grouped into three clonal lineage types (I, II, III) by multi-locus enzyme electrophoresis, PCR–RFLP and microsatellite typing [2–4]. Even though the differences at the genomic level among the three dominant lineages are less than 1%, the virulence phenotypes in mice are strikingly different, with archetypal I strains are uniformly lethal (LD100 = 1) in mice; by contrast, archetypal II and III strains are significantly less virulent (LD100 ≥ 103) [5]. Type II lineage is the predominant type causing human toxoplasmosis. Type I and atypical strains are not found in asymptomatic or benign congenital toxoplasmosis but have been found in severe toxoplasmic retinochoroiditis in human patients and acute disseminated toxoplasmosis in immunocomprimised patients [4]. Type III strains are found in patients with ocular toxoplasmosis and in animals [3,6]. These three clonal lineage types can induce genotype-specific antibody responses in infected patients [7]. Recent studies showed that T. gondii isolates from Brazil are biologically and genetically different from those in North America and

⁎ Corresponding author. Tel./fax: +86 20 85283730. E-mail address: [email protected] (X.-Q. Zhu). 1383-5769/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2009.01.006

Europe [8–14], suggesting that the overall diversity of T. gondii population is high. At present, there are limited reports on the genetic diversity of T. gondii from different hosts and geographical locations in Asia. A preliminary study identified two genotypes by typing 17 T. gondii isolates from cats in Guangzhou, China [15]. Extending this previous investigation, the objective of the present study was to genotype 17 T. gondii isolates originating from different hosts including human patients, pigs, cats and sheep from different geographically distant locations in China. For the present study, 17 strains of T. gondii isolated from 9 locations were investigated. Eight strains were isolated from cats, 5 strains from pigs, 3 strains from human patients and 1 strain from sheep (Table 1) by following essentially the procedures described previously [8,14]. T. gondii strains RH88, GT1, PTG, CTG, COUG (a.k.a. TgCgCa1), MAS and TgCatBr5 were used as references for genotyping (Table 1, see [16]). T. gondii tachyzoites of each isolates were collected from mice that were experimentally infected with those T. gondii isolates, following the procedures described previously [15]. Genomic DNA was extracted from the T. gondii tachyzoites by sodium dodecyl sulphate/proteinase K treatment, column-purified (Wizard™ DNA Clean-UP, Promega, Madison) and eluted into 40 μl of H2O according to the manufacturer's recommendations. Genetic characterization of T. gondii isolates was carried out by the multilocus PCR–RFLP method described recently [9,15,16]. In brief, the target DNA sequences were amplified by multiplex PCR using external primers for all 10 markers, including SAG1, SAG2, SAG3, BTUB, GRA6,

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Table 1 Summary of genotyping of T. gondii from different hosts and geographical locations in China. Isolate ID GT1 RH88 PTG CTG MAS TgCatBr5 COUG SH CN NT QHO TgC1 TgC2 TgC3 TgC4 TgC5 TgC6 TgC7 TgC8 GYS PYS ZC ZS ZS1 a b

Host

Human Pig Pig Sheep Cat Cat Cat Cat Cat Cat Cat Cat Pig Pig Pig Human Human

Location

Shanghai Changning, Hunan Tangshan, Nanjing Huzhu, Qinghai Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Guangzhou, Guangdong Jingyuan, Gansu Panyu, Guangdong Zengcheng, Guangdong Guangzhou, Guangdong Zhejiang

SAG1 I I II/III II/III u-1a I I I I I II u-1a u-1a u-1a u-1a u-1a u-1a u-1a u-1a u-1a u-1a u-1a u-1a u-1a

5′+3′

Alternative

SAG2

SAG2

I I II III I III I I I I II II II II II II II II II II II II II II

I I II III II III II I I I II II II II II II II II II II II II II II

SAG3

BTUB

GRA6

c22-8

c29-2

L358

PK1

Apico

Genotype

I I II III III III III I I I II III III III III III III III III III III III III III

I I II III III III II I I I II III III III III III III III III III III III III III

I I II III III III II I I I II II II II II II II II II II II II II II

I I II III u-1a I II I I I II II II II II II II II II II II II II II

I I II III I I u-1a I I I II III III III III III III III III III III III III II

I I II III I I I I I I II II II II II II II II II II II II II II

I I II III III u-1a u-2a I I I II II II II II II II II II II II II II ndb

I I II III I I I I I I I I I I I I I I I I I I I I

Reference (Type I) Reference (Type I) Reference (Type II) Reference (Type III) Reference Reference Reference #1 (Type I) #1 (Type I) #1 (Type I) #2 (Type II) #3 #3 #3 #3 #3 #3 #3 #3 #3 #3 #3 #3 #4

u-1 and u-2 represent unique RFLP genotypes, respectively. nd represents no data.

c22-8, c29-2, L358, PK1 and Apico. The multiplex PCR reaction was carried out in 25 µl of volume containing 10 mM Tris–HCl (pH 8.4), 50 mM KCl, 2 mM MgCl2, 200 µM each of the dNTPs, 0.1 µM each of the forward and reverse primers, 0.5 units of HotStart DNA polymerase (TaKaRa, Dalian) and 1.5 μl of DNA. The reaction mixture was treated at 95 °C for 4 min, followed by 25 cycles of 94 °C for 30 s, 55 °C for 1 min and 72 °C for 1.5 min. Multiplex PCR-amplified products were 1:1 diluted in water and then used for nested PCR with internal primers for each marker separately. The nested PCR was carried out in 25 µl of volume containing 10 mM Tris–HCl (pH 8.4), 50 mM KCl, 2 mM MgCl2, 200 µM each of the dNTPs, 0.3 µM each of the forward and reverse primers, 0.5 units of HotStart DNA polymerase (TaKaRa, Dalian) and 1.5 μl of diluted multiplex PCR products. The reaction mixture was treated at 95 °C for 4 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 1 min and 72 °C for 2 min. The nested PCR products were digested by restriction enzymes to reveal their genotypes by the method described previously [9,15,16]. The restriction fragments were resolved in 2.5% agarose gel, stained with ethidium bromide and photographed using a gel documentation system (UVI Tec Ltd, Cambridge). Genotyping results of 17 T. gondii isolates from China are presented in Table 1. Genotyping of the 17 T. gondii isolates identified 4 genotypes. The genotype #3 was identified from pigs in Jingyuan, Gansu Province, from pigs in Panyu and Zengcheng, Guangdong Province, from Human patient in Guangzhou, Guangdong Province, and from cats in Guangzhou, Guangdong Province. This same genotype was previously identified in 88% (15/17) T. gondii strains isolated from cats in Guangzhou, Guangdong Province, China [15]. These data suggest that genotype #3 is a major lineage prevalent in Mainland China. Interestingly, genotype #3 has also been identified from Colombia, Sri-Lanka, Vietnam, and the USA, indicating that it is widespread in Asia, and South and North America [10,15,17–19]. In this study, both clonal Type I and II lineages were identified in China (genotype #1 and #2 in Table 1). Although no data were obtained for the PK1 locus for isolate ZS1 (genotype #4) due to failed PCR amplification, Genotype #4 differs from Genotype #3 at the locus c29-2, suggesting they are closely related (Table 1). Combining data from this study of 17 isolates from different hosts with 17 cat isolates previously reported [15], a total of 5 genotypes was identified,

indicating limited diversity of the parasite in China, which is in sharp contrast to South America where a variety of parasite lineages are transmitted in the environment [9–11]. The clonal Type I and II lineages were identified in China, but in contrast to North American and Europe, they are not the predominant genotypes in the region. In China, T. gondii infection in humans and animals is widespread, and toxoplasmosis is considered an important food-borne parasitic zoonosis [20]. For instance, the infection rate in humans is 7.9% nationwide [20]. The prevalence of T. gondii in pigs was 32.8% in Yunnan province and 33.1% in Hebei province [20]. The infection rate of T. gondii in dogs was 13.2% in Beijing, 14.6% in Yunnan, and 26.9% in Hebei, respectively [20]. Cats, the definitive host for T. gondii, had a prevalence of up to 79.4% in Guangzhou [20]. The present genetic characterization of T. gondii isolates from human, pig, cat and sheep revealed four different genotypes, which has implications for further studies of population genetics of T. gondii in China, as well as for the prevention and control of T. gondii infections in humans and animals. Acknowledgements Project support was provided in part by grants from the National Basic Research Program of China (Grant No. 2007CB116301), the National Special Research Program for Non-Profit Trades (Agriculture) (Grant No. 200803017), the Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT0723), and the Key Research Programs in Natural Sciences for Institutions of Higher Education in Guangdong Province (Grant No. 06Z004) to XQZ. Dr Hua-Wen Li of Sun Yat-Sen (Zhongshan) University is thanked for providing the ZS, SH, and CN strains. References [1] Dubey JP, Beattie CP. Toxoplasmosis of animals and man. Boca Raton, Florida: CRC Press, Inc.; 1988. [2] Dardé ML, Bouteille B, Pestre-Alexandre M. Isoenzyme analysis of 35 Toxoplasma gondii isolates and the biological and epidemiological implications. J Parasitol 1992;78:786–94. [3] Howe DK, Sibley LD. Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J Infect Dis 1995;172:1561–6.

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