Abstract
Toxoplasma gondii is prevalent in humans and animals worldwide. The present study aimed to determine the genetic diversity of T. gondii isolates from pigs in Jilin province, northeastern China. A total of 100 DNA samples were extracted from the hilar lymph nodes of slaughtered pigs, and 9 (9.0%, 95% confidence interval: 3.4–14.6%) were detected positive for T. gondii B1 gene by a nested polymerase chain reaction (PCR). The positive DNA samples were typed at 11 genetic markers, including 10 nuclear loci (SAG1, 5′-SAG2, and 3′-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, and PK1) and an apicoplast locus (Apico) using the multilocus PCR–restriction fragment length polymorphism technology. Only three isolates were completely typed at all loci, showing that they all belonged to the clonal type I. One isolate was typed at five loci, including 5′ +3′-SAG2, SAG2, SAG3, GRA6, and L358, revealing the possible clonal type I. This is the first report of the genetic characterization of T. gondii isolates in pigs in Jilin province, northeastern China, which has implications for better understanding the population structure of T. gondii infection in China.
Introduction
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Most of the T. gondii isolates in Europe and North America are classified into three clonal lineages (type I, II, and III) based on multilocus enzyme electrophoresis, polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), and microsatellite analysis (Darde et al., 1992; Howe and Sibley, 1995; Ajzenberg et al., 2002). The fourth clonal lineage (ToxoDB #12) has recently been found prevalent in wildlife in North America (Khan et al., 2011). In contrast, T. gondii isolates in South America are more genetically diverse (Dubey et al., 2002; Lehmann et al., 2006; Dubey et al., 2008).
Consequence of T. gondii infection varies with parasite genotypes. Type I lineage in mice is uniformly lethal (LD100 = 1), while type II and III lineages are significantly less virulent to mice (LD100 ≥ 103) (Sibley and Boothroyd, 1992). Thus, it is necessary to determine the genetic characterization of T. gondii isolates from different sources in China.
Eating undercooked or raw pork or pork products is considered an important route of human infection with T. gondii. Several reports concerning the genetic chacterization of T. gondii isolates from pigs are available in China (Zhou et al., 2009, 2010, 2011b; Jiang et al., 2013). Type I has been identified in pigs in the provinces of Henan, Hunan, Jiangsu, and Hubei, and ToxoDB#9 is predominant in pigs in the provinces of Jiangxi and Yunnan. In Jilin province of northeastern China, pork is responsible for 50% of animalborne food consumed by humans. There were 22 million pigs on hand and 12 million slaughtered in Jilin province in 2007, and the pig population was annually increased by 10% between 2004 and 2007 (Zhang et al., 2011). The pork could meet the needs of the people in Jilin province, with a small amount transported to other provinces. Seroprevalence of T. gondii infection has been detected in 19.1% of pigs in Jilin province using an indirect hemagglutination assay (Xu et al., 2015), but the genotypic characterization remains unclear. This study, therefore, was conducted to determine the genetic diversity of T. gondii isolates in pigs in Jilin province, northeastern China.
Materials and Methods
Sample collection
The hilar lymph nodes were collected from 100 slaughtered pigs in Jilin province between January and July, 2013. Of these, 90 were selected from 10 counties, including Panshi (9), Songyuan (10), Jiutai (10), Longjing (10), Qian'an (10), Meihekou (9), Fusong (10), Jiangyuan (10), Tongyu (10), and Zhenlai (2). Unfortunately, geographical origin of the other 10 samples was missing. The collected hilar lymph nodes were ground by liquid nitrogen and stored at −20°C until used.
Genomic DNA extraction
Genomic DNA was extracted from the tissue samples using a commercially available kit (TianGen™, Beijing, China). Briefly, 30 mg of the ground hilar lymph nodes was treated with digestion by sodium dodecyl sulfate/proteinase K overnight and subsequent column purification according to manufacturer's recommendations (Jiang et al., 2013). DNA samples were diluted into 50-μL elution buffer. The seminested PCR targeting T. gondii B1 gene was performed to detect possible infection as described previously (Hill et al., 2006). DNA samples positive for B1 gene were used for genotyping.
Genetic characterization of T. gondii isolates
Genetic characterization of T. gondii isolates from pigs was performed by the PCR-RFLP analyses of 11 genetic markers, including 10 nuclear loci (SAG1, 5′-SAG2 and 3′-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, and PK1) and an apicoplast locus (Apico) as described previously (Su et al., 2010). Reference T. gondii strains, including GT1 (type I), PTG (type II), CTG (type III), MAS (ToxoDB#17), TgCgCa1 (ToxoDB#66), TgCatBr5 (ToxoDB#19), TgWtdSc40 (type 12), TgCatBr64 (ToxoDB#111), and TgRsCr1 (ToxoDB#52) were used as controls for genotyping using the primers (Table 1). The PCR reactions were conducted as described previously (Su et al., 2010). First, the target DNA sequences for all 11 markers were pre-amplified in a single reaction using a set of external primers. Pre-amplified PCR products (1 μL) then served as template DNA for nested PCR to amplify each individual marker with individual internal primers. To reveal single nucleotide polymorphisms at all loci, the nested PCR products were digested with appropriate restriction endonucleases (Su et al., 2010). The enzyme-digested products were resolved in 2.5–3% agarose gels stained with ethidium bromide, visualized under ultraviolet light, and photographed using a gel documentation system (UVP GelDoc-It™ Imaging System, Cambridge, UK). The results were compared and matched to those identified RFLP genotypes listed in the ToxoDB genotyping database (
F represents forward primer; R represents reverse primer.
Results
In total, there were nine DNA samples that tested positive for the T. gondii B1 gene by the nested PCR, suggesting a T. gondii prevalence of 9% (9.0%, 95% confidence interval: 3.4–14.6%) in hilar lymph nodes of slaughtered pigs from Jilin province of northeastern China.
Three DNA samples of T. gondii isolates TgPJL1-3 from Meihekou (one) and Jiutai (two) had complete genotyping results, and the DNA sample of T. gondii isolate TgPJL4 without geographical location information presented genotyping data at 5′ +3′-SAG2, SAG2, SAG3, GRA6, and L358 loci. Other DNA samples were genotyped at less than five loci, due to the low DNA concentrations of T. gondii in the samples (data not shown). Genotyping results of the four isolates TgPJL1–-4 and nine reference T. gondii strains are summarized in Table 2. Isolates TgPJL1-3 were identified as type I. Since data for SAG1, BTUB, c22-8, c29-2, PK1, and Apico loci were not obtained, the genotype of TgPJL4 could not be determined.
u-1 and u-2 represent unique restriction fragment length polymorphism genotypes, respectively.
WTD, white-tailed deer; nd, no data.
Discussion
In the present study, a T. gondii prevalence of 9% was found in hilar lymph nodes of slaughtered pigs from Jilin province of northeastern China. Previously, the overall prevalence of 65.8% in hilar lymph nodes from pigs in south China (Jiang et al., 2013), 7.8% in hilar lymph nodes from pigs in central China (Zhou et al., 2010), and 53.9% in lung from Microtus fortis in Jilin province of northeastern China (Zhang et al., 2013) were reported. In other countries, the infection was detected in 4.7% in slaughtered pigs in Ireland (Halova et al., 2013), 7.1% in slaughtered pigs in Portugal (Esteves et al., 2014), 12.5% in slaughtered pigs in Brazil (Samico Fernandes et al., 2012), 50.8% in fattening pigs in Mexico (Ortega-Pacheco et al., 2013), and 57.1% in free-range, organic pigs in Italy (Bacci et al., 2015). The significant difference in prevalences of T. gondii infection may come from the geographical distribution, animal species, or detection methods.
T. gondii infection poses a great health threat to both humans and animals. To date, several studies have been conducted on the genetic characterization of T. gondii isolates from pigs in China (Zhou et al., 2009, 2010, 2011b; Jiang et al., 2013). A total of four genotypes, including type I (ToxoDB#10), type II (ToxoDB#1), type II variant (ToxoDB#3), and one additional atypical genotype (ToxoDB#9) have been found in pigs in different regions in China. However, the genetic data of T. gondii isolates in pigs in Jilin province, northeastern China remain unclear.
In this study, the clonal type I was identified from three T. gondii isolates in Jilin province, northeastern China. Although data were not obtained at some genetic markers, isolate TgPJL4 showed type I patterns at 5′ +3′-SAG2, SAG2, SAG3, GRA6 and L358 loci, which were identical to that of TgPJL1-3, suggesting the possible genotype being type I. Similarly, type I has been identified in T. gondii isolates from pigs in provinces of Henan, Hunan, Jiangsu, and Hubei (Zhou et al., 2009, 2010). However, the results of the present study are different from other studies, which have shown that the atypical genotype (i.e., ToxoDB#9) is predominant in pigs in the provinces of Jiangxi and Yunnan (Zhou et al., 2011b; Jiang et al., 2013). The difference may be attributed to pigs living in different geographical locations and environments. Distinct areas varying in the foreign trade of pigs may also in part account for the differences among different studies. The present results indicate that type I is the prevalent T. gondii genotype in pigs in Jilin province, and the genetic diversity may be low in pigs in this area, which is also supported by the genotype and its variant present in bats, cattle, and free-living Microtus fortis in Jilin province (Ge et al., 2014; Qin et al., 2014; Zhang et al., 2014). To draw a valid conclusion, however, more samples should be collected from much broader geographical regions of Jilin province for further studies.
In addition to pigs, type I has also been reported in cats, humans, tree sparrows, and plateau pika in China (Zhou et al., 2009; Huang et al., 2012; Wang et al., 2013; Zhang et al., 2013), suggesting it is a widespread genotype circulating in China. In contrast to North America and Europe, where type I is the predominant genotype, the atypical genotype ToxoDB#9 dominantly circulates in mainland China (Qian et al., 2012; Jiang et al., 2013; Wang et al., 2013).
T. gondii infection in pigs has great public health significance. Pork is widely consumed in China, including Jilin province, but there is no legal requirement to inspect pork for T. gondii infection before it enters the market. If pork infected with tissue cysts is undercooked and consumed, humans will be at risk of infection with T. gondii. Hence, it is urgent to implement good management measures on pig farms to reduce the T. gondii infection in pigs, and strengthen compulsory quarantine of T. gondii in pork.
In conclusion, the present study identified the clonal type I in pigs in Jilin province. To our knowledge, this is the first report of genetic characterization of T. gondii isolates from pigs in Jilin province, northeastern China, which provides new genetic data of T. gondii from pigs and has implications for better understanding the population structure of T. gondii infection in China.
Footnotes
Acknowledgments
Project support was provided by the National Natural Science Foundation of China (31228022 and 31372430), the Special Fund for Agro-scientific Research in the Public Interest (201303042), the Open Funds of State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (SKLVEB2013KFKT006), and the Science Fund for Creative Research Groups of Gansu Province (1210RJIA006).
Disclosure Statement
No competing financial interests exist.