Abstract
Background
Toxoplasma gondii is a widely prevalent protozoan parasite that causes serious toxoplasmosis in humans and animals. The present study aimed to determine the genetic diversity of T. gondii isolates from pigs in Jiangxi, Sichuan, Guangdong Provinces and Chongqing Municipality in China using multilocous polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology.
Methods
A total of 38 DNA samples were extracted from hilar lymph nodes of pigs with suspected toxoplasmosis, and were detected for the presence of T. gondii by semi-nested PCR of B1 gene. The positive DNA samples were typed at 11 genetic markers, including 10 nuclear loci, namely, SAG1, 5′-SAG2 and 3′-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast locus Apico.
Results
Twenty-five of the 38 DNA samples were T. gondii B1 gene positive. Complete genotyping data for all loci could be obtained for 17 of the 25 samples. Two genotypes were revealed (ToxoDB PCR-RFLP genotypes #9 and #3). Sixteen samples belong to genotype #9 which is the major lineage in mainland China and one sample belongs to genotype #3 which is Type II variant.
Conclusions
To our knowledge, this is the first report of genetic typing of T. gondii isolates from pigs in Jiangxi, Sichuan Province and Chongqing Municipality, and the first report of ToxoDB #3 T. gondii from pigs in China. These results have implications for the prevention and control of foodborne toxoplasmosis in humans.
Similar content being viewed by others
Background
Toxoplasma gondii is an obligate intracellular protozoan parasite, infecting all warm-blooded animals and human beings worldwide [1–5]. It is transmitted to humans through consumption of undercooked meat containing T. gondii tissue cysts or food or water contaminated by oocysts shed in the feces of infected cats. Toxoplasmosis is one of the main illnesses related to foodborne hospitalizations and deaths [6]. One third of the world population is chronically infected with this parasite [7]. Although T. gondii infection in most people appears to be asymptomatic, it may result in life-threatening illness in some immuno-compromised individuals [8].
In China, pigs are the primary livestock raised for human consumption, and they are commonly susceptible to T. gondii. This provides the parasite a potential route to transmit the infection via consumption of raw or undercooked meat. T. gondii may cause serious diseases such as blindness through consuming raw pork [9].
According to genotypic analysis based on multi-locus approaches, such as PCR-RFLP and microsatellite typing, T. gondii strains isolated from humans and animals in North America and Europe have been sorted into 3 clonal lineages, referred to as type I, II, and III [10–12]. A fourth clonal lineage (type 12) in North America was identified in wildlife recently [13]. However, T. gondii isolates from South America are characterized with higher genetic diversity [14–16].
China is a big country, but only limited reports concerning genetic characterization of T. gondii isolates from pigs are available [17–19]. Such information is still not available for some regions of China especially Jiangxi, Sichuan Provinces and Chongqing Municipality, where swine industry plays an important role in agricultural economy. Thus, the objective of this study is to better understand the genetic diversity of T. gondii isolates from pigs in these localities of China.
Methods
Sample collection
Hilar lymph nodes were collected, post slaughter, from 38 dead pigs (36 slaughter pigs and 2 sows) with suspected toxoplasmosis during May 2010-March 2013. No ethical approval is required. These pigs came from different geographic regions of China, including 33 from Jiangxi Province, 3 from Sichuan Province, 1 from Chongqing Municipality, and 1 from Guangdong Province, and they all exhibited typical symptoms of toxoplasmosis, which manifest primarily as high fever, dyspnea, subcutaneous hemorrhage, abortion, enlargement and necrosis of liver and spleen.
Genomic DNA extraction
Genomic DNA was extracted from these hilar lymph nodes using TIANamp Genomic DNA kit (TianGen™, Beijing, China) according to manufacturer’s recommendations. In brief, 30 mg of the hilar lymph nodes were treated with sodium dodecyl sulphate/proteinase K at 56°C for overnight digestion in a thermostat water bath. DNA samples were prepared after purification by silica gel column chromatography and eluted into 50 μl elution buffer. Then, a semi-nested PCR was performed to detect the T. gondii B1 gene following the previously described protocol [20].
Genetic characterization of T. gondii isolates
Multilocous polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method [21] was employed to genetically characterize the T. gondii isolates from pigs. Firstly, pre-amplification was carried out using a set of mixed external primers in a single reaction. Then 1 μl of the products served as template DNA for nested PCR with internal primers for each marker, respectively. The nested PCR products were digested with restriction endonucleases. The restriction fragments were resolved in 2.5%-3% agarose gel to display single nucleotide polymorphisms (SNPs) using a gel document system (UVP GelDoc-It™ Imaging System, Cambridge, U.K.), and the genotypes of T. gondii isolates were finally revealed.
Results and discussion
Twenty-five of the 38 DNA samples were T. gondii B1 gene positive, including 23 from slaughter pigs and 2 from sows. Due to low DNA concentration, only 17 DNA samples presented complete genotyping data. The results of genotyping of these isolates and 9 references were summarized in Table 1. Two genotypes were revealed (ToxoDB PCR-RFLP genotypes #9 and #3). It is interesting that only one genotype (ToxoDB#9) was identified from all 12 T. gondii isolates from different districts of Jiangxi Province, which suggests that this genotype is predominantly prevalent in Jiangxi Province and the genetic diversity of T. gondii may be low in pigs in this province, although further studies using more samples collected from much broader geographical localities of this province are warranted before a valid conclusion can be drawn. Moreover, ToxoDB#9 was also identified in 4 isolates from 3 localities (Zigong and Ziyang in Sichuan and Rongchang in Chongqing) near the border of Sichuan Province and Chongqing Municipality. This same genotype was previously identified in cats in Beijing Municipality, Guangdong and Anhui Provinces [22–24], and it was also founded in pigs in Guangdong, Henan, Yunnan and Anhui Provinces [17–19, 25]. Based on these data, ToxoDB#9 was a predominant lineage prevalent in Mainland China. Interestingly, ToxoDB#9 has been identified in North and Latin America [15, 26–28], as well as other Asian countries, such as Sri Lanka, Vietnam [29, 30], indicating that it has a worldwide distribution. In the near future, it would be interesting to genotype T. gondii isolates from humans in the same localities to see if humans and animals share the same genotypes.
In this study, another genotype ToxoDB#3 (the type II variant) was identified in a pig in Zhongshan, Guangdong Province. This was the fourth time that ToxoDB#3 was identified in China. Previously, this type was founded from sheep in Qinghai Province [17], from birds in Xinjiang Uygur Autonomous Region [31] and from sparrow in Lanzhou, Gansu Province [32]. This is also the first report of ToxoDB#3 from pigs in China, which indicated that ToxoDB#3 is also a major lineage prevalent in Mainland China.
For Chinese people especially the Han ethnic, pork is the main meat of choice. With the development of the economy in China, the standard of life has greatly improved in recent years. As a result, the pig industry and pork products have been driven by increasing consumption for high quality animal protein. Unlike herbivorous cattle and sheep, pigs are omnivores, they feed on a variety of animal meat and vegetable matter, which increases the chance of contact with cat feces and exposure to T. gondii, especially for those free-range pigs. Thus, pigs pose a risk for transmission of toxoplasmosis to human beings.
Conclusions
The present study has genetically characterized T. gondii isolates from pigs in Jiangxi, Sichuan, Guangdong Provinces and Chongqing Municipality. Two genotypes were revealed (ToxoDB PCR-RFLP genotypes #9 and #3), with the genotype #9 as the major lineage in mainland China. To our knowledge, this is the first report of genetic typing of T. gondii isolates from pigs in these localities, and the first report of ToxoDB genotype #3 from pigs in China. These findings not only enrich genetic diversity of T. gondii, but also have implications for the prevention and control of foodborne toxoplasmosis in humans.
References
Dubey JP: Toxoplasmosis of animals and humans. 2010, Boca Raton, Florida: CRC Press, 313-2
Nardoni S, Angelici MC, Mugnaini L, Mancianti F: Prevalence of Toxoplasma gondii infection in Myocastor coypus in a protected Italian wetland. Parasit Vectors. 2011, 4: 240-10.1186/1756-3305-4-240.
Chen J, Xu MJ, Zhou DH, Song HQ, Wang CR, Zhu XQ: Canine and feline parasitic zoonoses in China. Parasit Vectors. 2012, 5: 152-10.1186/1756-3305-5-152.
Tian YM, Dai FY, Huang SY, Deng ZH, Duan G, Zhou DH, Yang JF, Weng YB, Zhu XQ, Zou FC: First report of Toxoplasma gondii seroprevalence in peafowls in Yunnan Province, Southwestern China. Parasit Vectors. 2012, 5: 205-10.1186/1756-3305-5-205.
Zhou P, Chen Z, Li HL, Zheng H, He S, Lin RQ, Zhu XQ: Toxoplasma gondii infection in humans in China. Parasit Vectors. 2011, 4: 165-10.1186/1756-3305-4-165.
Dubey JP: Toxoplasmosis-a waterborne zoonosis. Vet Parasitol. 2004, 126: 57-72. 10.1016/j.vetpar.2004.09.005.
Weiss LM, Dubey JP: Toxoplasmosis: a history of clinical observations. Int J Parasitol. 2009, 39: 895-901. 10.1016/j.ijpara.2009.02.004.
Montoya JG, Liesenfeld O: Toxoplasmosis. Lancet. 2004, 363: 1965-1976. 10.1016/S0140-6736(04)16412-X.
Choi WY, Nam HW, Kwak NH, Huh W, Kim YR, Kang MW, Cho SY, Dubey JP: Foodborne outbreaks of human toxoplasmosis. J Infect Dis. 1997, 175: 1280-1282. 10.1086/593702.
Ajzenberg D, Banuls AL, Tibayrenc M, Darde ML: Microsatellite analysis of Toxoplasma gondii shows considerable polymorphism structured into two main clonal groups. Int J Parasitol. 2002, 32: 27-38. 10.1016/S0020-7519(01)00301-0.
Darde ML, Bouteille B, Pestre-Alexandre M: Isoenzyme analysis of 35 Toxoplasma gondii isolates and the biological and epidemiological implications. J Parasitol. 1992, 78: 786-794. 10.2307/3283305.
Howe DK, Sibley LD: Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J Infect Dis. 1995, 172: 1561-1566. 10.1093/infdis/172.6.1561.
Khan A, Dubey JP, Su C, Ajioka JW, Rosenthal BM, Sibley LD: Genetic analyses of atypical Toxoplasma gondii strains reveal a fourth clonal lineage in North America. Int J Parasitol. 2011, 41: 645-655. 10.1016/j.ijpara.2011.01.005.
Dubey JP, Graham DH, Blackston CR, Lehmann T, Gennari SM, Ragozo AM, Nishi SM, Shen SK, Kwok OC, Hill DE, Thulliez P: Biological and genetic characterisation of Toxoplasma gondii isolates from chickens (Gallus domesticus) from Sao Paulo, Brazil: unexpected findings. Int J Parasitol. 2002, 32: 99-105. 10.1016/S0020-7519(01)00364-2.
Dubey JP, Sundar N, Hill D, Velmurugan GV, Bandini LA, Kwok OC, Majumdar D, Su C: High prevalence and abundant atypical genotypes of Toxoplasma gondii isolated from lambs destined for human consumption in the USA. Int J Parasitol. 2008, 38: 999-1006. 10.1016/j.ijpara.2007.11.012.
Lehmann T, Marcet PL, Graham DH, Dahl ER, Dubey JP: Globalization and the population structure of Toxoplasma gondii. Proc Natl Acad Sci USA. 2006, 103: 11423-11428. 10.1073/pnas.0601438103.
Zhou P, Zhang H, Lin RQ, Zhang DL, Song HQ, Su C, Zhu XQ: Genetic characterization of Toxoplasma gondii isolates from China. Parasitol Int. 2009, 58: 193-195. 10.1016/j.parint.2009.01.006.
Zhou P, Nie H, Zhang LX, Wang HY, Yin CC, Su C, Zhu XQ, Zhao JL: Genetic characterization of Toxoplasma gondii isolates from pigs in China. J Parasitol. 2010, 96: 1027-1029. 10.1645/GE-2465.1.
Zhou P, Sun XT, Yin CC, Yang JF, Yuan ZG, Yan HK, Zhu XQ, Zou FC: Genetic characterization of Toxoplasma gondii isolates from pigs in southwestern China. J Parasitol. 2011, 97: 1193-1195. 10.1645/GE-2851.1.
Hill DE, Chirukandoth S, Dubey JP, Lunney JK, Gamble HR: Comparison of detection methods for Toxoplasma gondii in naturally and experimentally infected swine. Vet Parasitol. 2006, 141: 9-17. 10.1016/j.vetpar.2006.05.008.
Su C, Shwab EK, Zhou P, Zhu XQ, Dubey JP: Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii. Parasitology. 2010, 137: 1-11. 10.1017/S0031182009991065.
Dubey JP, Zhu XQ, Sundar N, Zhang H, Kwok OC, Su C: Genetic and biologic characterization of Toxoplasma gondii isolates of cats from China. Vet Parasitol. 2007, 145: 352-356. 10.1016/j.vetpar.2006.12.016.
Qian W, Wang H, Su C, Shan D, Cui X, Yang N, Lv C, Liu Q: Isolation and characterization of Toxoplasma gondii strains from stray cats revealed a single genotype in Beijing, China. Vet Parasitol. 2012, 187: 408-413. 10.1016/j.vetpar.2012.01.026.
Chen ZW, Gao JM, Huo XX, Wang L, Yu L, Halm-Lai F, Xu YH, Song WJ, Hide G, Shen JL, Lun ZR: Genotyping of Toxoplasma gondii isolates from cats in different geographic regions of China. Vet Parasitol. 2011, 183: 166-170. 10.1016/j.vetpar.2011.06.013.
Wang H, Wang T, Luo Q, Huo X, Wang L, Liu T, Xu X, Wang Y, Lu F, Lun Z, Yu L, Shen J: Prevalence and genotypes of Toxoplasma gondii in pork from retail meat stores in Eastern China. Int J Food Microbiol. 2012, 157: 393-397. 10.1016/j.ijfoodmicro.2012.06.011.
Dubey JP, Velmurugan GV, Alvarado-Esquivel C, Alvarado-Esquivel D, Rodriguez-Pena S, Martinez-Garcia S, Gonzalez-Herrera A, Ferreira LR, Kwok OC, Su C: Isolation of Toxoplasma gondii from animals in Durango, Mexico. J Parasitol. 2009, 95: 319-322. 10.1645/GE-1874.1.
Dubey JP, Cortes-Vecino JA, Vargas-Duarte JJ, Sundar N, Velmurugan GV, Bandini LM, Polo LJ, Zambrano L, Mora LE, Kwok OC, Smith T, Su C: Prevalence of Toxoplasma gondii in dogs from Colombia, South America and genetic characterization of T. gondii isolates. Vet Parasitol. 2007, 145: 45-50. 10.1016/j.vetpar.2006.12.001.
Dubey JP, Sundar N, Gennari SM, Minervino AH, Farias NA, Ruas JL, Dos Santos TR, Cavalcante GT, Kwok OC, Su C: 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. 2007, 143: 182-188. 10.1016/j.vetpar.2006.08.024.
Dubey JP, Huong LT, Sundar N, Su C: Genetic characterization of Toxoplasma gondii isolates in dogs from Vietnam suggests their South American origin. Vet Parasitol. 2007, 146: 347-351. 10.1016/j.vetpar.2007.03.008.
Dubey JP, Rajapakse RP, Wijesundera RR, Sundar N, Velmurugan GV, Kwok OC, Su C: Prevalence of Toxoplasma gondii in dogs from Sri Lanka and genetic characterization of the parasite isolates. Vet Parasitol. 2007, 146: 341-346. 10.1016/j.vetpar.2007.03.009.
Huang SY, Cong W, Zhou P, Zhou DH, Wu SM, Xu MJ, Zou FC, Song HQ, Zhu XQ: First report of genotyping of Toxoplasma gondii isolates from wild birds in China. J Parasitol. 2012, 98: 681-682. 10.1645/GE-3038.1.
Cong W, Huang SY, Zhou DH, Zhang XX, Zhang NZ, Zhao Q, Zhu XQ: Prevalence and genetic characterization of Toxoplasma gondii in house sparrows (Passer domesticus) in Lanzhou, China. Korean J Parasitol. 2013, 51: 363-367. 10.3347/kjp.2013.51.3.363.
Acknowledgements
Project support was provided by the National Natural Science Foundation of China (Grant Nos. 31228022, 31101812, 31230073 and 31172316), and the Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006).
Author information
Authors and Affiliations
Corresponding authors
Additional information
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
XQZ and CS conceived and designed the study, and critically revised the manuscript. HHJ, SYH and SZD performed the experiments, analyzed the data and drafted the manuscript. DHZ and XXZ helped in study design, study implementation and manuscript revision. All authors read and approved the final manuscript.
Rights and permissions
Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
About this article
Cite this article
Jiang, HH., Huang, SY., Zhou, DH. et al. Genetic characterization of Toxoplasma gondii from pigs from different localities in China by PCR-RFLP. Parasites Vectors 6, 227 (2013). https://doi.org/10.1186/1756-3305-6-227
Received:
Accepted:
Published:
DOI: https://doi.org/10.1186/1756-3305-6-227