Molecular Detection and Genotyping of Toxoplasma gondii from Pigs for Human Consumption in Zhejiang and Jiangsu Provinces,Eastern China

Abstract

Toxoplasma gondii infections are common in humans and animals worldwide. Ingestion of raw or undercooked meat containing tissue cysts of T. gondii is one major source of transmission of this parasite. It is important to guarantee the meat quality of China since our pork industry produces about half of the world's pork. In this study, a total of 746 pig samples were collected from Zhejiang and Jiangsu provinces in eastern China, and examined for T. gondii infection by PCR amplification targeting B1 gene. In this study, we found that 57 of 746 (7.6%) pigs were positive for B1 gene, with 8.5% (48/562) in Zhejiang province and 4.9% (9/184) in Jiangsu province, respectively. The positive DNA samples were further genotyped at 11 genetic markers, including SAG1, 5′-and 3′-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico through PCR–restriction fragment length polymorphism (PCR-RFLP) technology. Two genotypes (ToxoDB 9 and ToxoDB 10) of T. gondii were identified by PCR-RFLP in Zhejiang province. However, both genotypes were not determined from Jiangsu province, which is speculated on the low DNA concentration and the small number of samples. These results indicate that T. gondii infection is endemic in pigs in eastern China and may raise public food safety concerns, suggesting more interventions for T. gondii-related risks are needed in the future.

Introduction

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan that can infect almost all warm-blooded animals and humans (Dubey and Jones, 2008; Lopes et al., 2013), which infects about one-third of the world's population according to the seroprevalence investigations. Toxoplasmosis is generally subclinical or asymptomatic (Montoya and Liesenfeld, 2004), but symptoms may occur in pregnant women and immunocompromised individuals (Wang et al., 2017) sometimes according to serious disease (i.e., spontaneous abortion or death).

The felids are the only definitive hosts of T. gondii (Frenkel et al., 1970), and other animals and humans are intermediate hosts (Dubey, 2009a). Three stages of this protozoan parasite are responsible for transmitting: oocysts, bradyzoites, and tachyzoites (Dubey and Frenkel, 1976). Infection can be acquired by consumption of food and water contaminated with oocysts, by the ingestion of raw or undercooked meat from animals containing tissue cysts (bradyzoites) (Tenter et al., 2000) or vertical transmission of T. gondii tachyzoites from infected mothers to the fetus (Maani et al., 2021). Among livestock, pigs are highly susceptible intermediate hosts of T. gondii. Consumption of pork is considered to be an important food source of humans infected by T. gondii among domestic economic animals (Dubey, 2009b), since pork is the most highly consumed meat in many countries, including China (Cubas-Atienzar et al., 2018; Gui et al., 2018; Vergara et al., 2018).

Pathogenicity of T. gondii was affected with genetic background, host immune system, and the parasite genotype (Maani et al., 2021) and three major clone lineages of T. gondii (types I, II, and III) have been identified based on the pathogenicity. Type I strains (i.e., T. gondii RH and GT1 strains) are the most pathogenic, whereas genotype II and III strains (Fernández-Escobar et al., 2020; Fazel et al., 2021) are significantly less virulent (Dardé., 2008). In addition, other genotypes and atypical strains also have been reported (Ajzenberg et al., 2009; Robert-Gangneux and Dardé, 2012; Döşkaya et al., 2013), indicating a high diversity of T. gondii population. Thus, it is very important to carry overall investigations of T. gondii infection in pigs, including prevalence and genotype to analyze risk factors of this parasite. The technology of polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) based on specific markers providing a high resolution for identification is widely used to determine the strains of T. gondii (Liu et al., 2015) and the genetic characterization of T. gondii vary in different geographic regions (Sun et al., 2013).

The objective of this study was to identify the molecular prevalence and genotypes of T. gondii in pigs in Zhejiang and Jiangsu provinces (eastern China) and estimate the potential public risk of toxoplasmosis. Accumulation of the related information will be helpful for us to better control and prevent T. gondii infection in the future.

Materials and Methods

Ethics statement

The Zhejiang Academy of Agricultural Sciences Institutional Animal Care Committee in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Ethics protocol No. 2020001) approved all protocols.

Sample collection

The samples (blood, lymph node, intestine, liver, lung, and aborted fetus) were collected during August 2019 to September 2021. A total of 746 sick pigs in intensive farms from 30 regions of 14 cities in Zhejiang and Jiangsu provinces, eastern China, were used (Fig. 1) to investigate the presence of T. gondii infection.

FIG. 1.

Pig samples were collected from Zhejiang province, including cities from Northern (Huzhou, Jiaxing, Hangzhou, and Shaoxing), Central (Jinhua), Western (Quzhou), Eastern (Zhoushan, Ningbo, and Taizhou), and Southern (Lishui and Wenzhou) regions; samples from cities of Nanjing, Xuzhou, and Nantong in Jiangsu province were also detected. (A) map of China, (B) map of Zhejiang province, (C) map of Jiangsu province.

Briefly, each sample was placed into a clean polyethylene glove, and the background information (e.g., sex, age, and physical condition) of each sample was recorded, and the samples' information is shown in Table 1. All samples were stored at −20°C until used.

Table 1.

Samples Information

Tissue/blood	No. of examined (%)^a	No. of positive	Prevalence (%)	Age of pigs
Blood	26 (3.5)	1^b	3.8	Fattening pigs^b (n = 6), Breeding pigs (n = 20)
Lymph node	369 (49.5)	37	10.0	Fattening pigs (n = 369)
Intestine	30 (4.0)	1	3.3	Fattening pigs (n = 30)
Liver	222 (29.8)	16	7.2	Fattening pigs (n = 222)
Lung	96 (12.9)	2	2.1	Fattening pigs (n = 96)
aborted Fetus	3 (0.4)	0	0.0	Fetus (n = 3)
Total	746	57	7.6

The percent accounts for the total pigs.

The Toxoplasma gondii—positive blood was from a fattening pig.

DNA extraction, nested PCR

The genomic DNA was extracted from the blood, lymph node, intestine, liver, lung, and aborted fetus using the Genomic DNA Kit (GENFINE, Beijing, China, FD202-02) according to the manufacturer's recommendations, and the DNA samples were stored at −20°C until used. A nested PCR was performed to detect infection with T. gondii targeting the B1 gene as described elsewhere (Jones et al., 2000). The first round PCR was performed on the T. gondii genomic DNA samples using the B1 gene primers 5′-GGAACTGCATCCGTTCATGAG-3′ and 5′-TCTTTAAAGCGTTCGTGGTC-3′ and the nested amplification was performed using 1 μL of the amplicons resulting from the first round PCR using specific B1 gene primers 5′-GGAACTGCATCCGTTCATGAG-3′ and 5′-TCTTTAAAGCGTTCGTGGTC-3′. PCR for the first round PCR was denaturation at 93°C for 5 min, followed by 40 cycles at 93°C for 10 s, 57°C for 10 s, 72°C for 30 s, and 72°C for 10 min.

The condition for the nested PCR was denaturation at 93°C for 5 min, followed by 40 cycles of 93°C for 10 s, 62.5°C for 10 s, and extension at 72°C for 15 s, with a final extension of 72°C for 10 min. Samples were run in triplicate and both positive (DNA extracted from T. gondii RH strain tachyzoites) and negative controls (sterile distilled water) were included in each run.

Genetic characterization of T. gondii

The B1 gene-positive DNA samples were used for genotyping by multilocus PCR-RFLP with 11 markers (i.e., SAG1, SAG2, alter.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) (Su et al., 2006, 2010), and the samples were amplified separately for each marker. PCR amplification was performed in a volume of 25 μL containing 17.25 μL of nuclease-free water, 2.5 μL of Buffer (Mg²⁺ plus), 2 μL of dNTP, 1 μL of forward primer, 1 μL of reverse primer, 0.25 μL of TaKaRa rTaq (1.25 U/μL), and 1 μL of DNA template. The amplification procedure was performed as follows: all B1 gene-positive samples were incubated at 95°C for 5 min to activate the DNA polymerase, followed by 35 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min 30 s, and final extension at 72°C for 10 min. Then the PCR products were diluted at a ratio of 1:1 in sterile water and used as template DNA for the nested PCR amplification with internal primers. The amplification condition was similar for the first round, except the annealing temperature of Apico marker was 55°C for 1 min, other genetic markers were amplified with an annealing temperature of 60°C for 1 min.

Finally, all PCR products were analyzed using electrophoresis on 2.5% agarose gel. The genotypes were then determined by RFLP method based on appropriate restriction enzymes for digesting the amplification products according to the previous method (Su et al., 2006). The total reaction volume and procedure were conducted according to the manufacturer's protocol (New England Biolabs, America), and then the digestion fragments were separated using 3% agarose gel electrophoresis. Finally, the genotypes of T. gondii isolates were distinguished as described previously (Su et al., 2010; Abdoli et al., 2017)), and the results were determined according to genotypes present in ToxoDB (http://toxodb.org/toxo).

Statistical analysis

Statistical analysis was performed using SAS 9.3 statistical packages, differences in the prevalence rates were determined by the chi-squared test, p value of <0.05 was considered as a significant difference.

Results and Discussion

In total, out of the 746 examined pig samples, 57 (7.6%) samples (blood = 1, lymph node = 37, intestinal = 1, liver = 16, lung = 2) were tested positive for the T. gondii B1 gene by PCR amplification (Table 1), all of these positive samples came from fattening pigs (57/723) and B1 gene was not amplified in breeding pigs (n = 20) and aborted fetus (n = 3). This study demonstrated that 64.9% (37/57) of T. gondii infection-positive samples were from lymph node, which was higher than that in liver (28.1%, 16/57). T. gondii infection was detected in only one blood (1.8%, 1/57), one intestinal (1.8%, 1/57), and two lung (3.5%, 2/57) samples. The differences in lymph node and other tissues were statistically significant, but this analysis maybe influenced by the differences of the samples size from different tissues.

We found a wide distribution of T. gondii in Zhejiang province where the prevalence rates varied from 10.5% (22/210), 5.8% (6/103), 13.5% (5/37), 7.4% (7/94) and 6.8% (8/118) in Northern Zhejiang, Central Zhejiang, Western Zhejiang, Eastern Zhejiang, and Southern Zhejiang, respectively, but the differences in these five regions were not statistically significant (p > 0.05). Interestingly, no positive results were obtained from pigs in Jiaxing, probably due to the small number of samples or the low prevalence. We also found that 7.4% (8/108) of pigs were positive for T. gondii infection in Nantong, Jiangsu province, which was significantly higher than that in Nanjing (0.0%, 0/52) and Xuzhou (4.2%, 1/24) (p < 0.05).

In this study, the overall prevalence of T. gondii in pigs in Zhejiang and Jiangsu provinces (eastern China) was 7.6%, with 8.5% (48/562) in Zhejiang province and 4.9% (9/184) in Jiangsu province (Table 2). The different detection rates of T. gondii in Zhejiang and Jiangsu provinces may be related to the sampling times, and number of samples. Our findings were similar to those of Zhou et al. (2010) who confirmed a 7.8% (34/434) prevalence in Hubei and Henan provinces in central China, and was lower than that in Hunan (10%, 34/339, the southeastern of China) (Gui et al., 2018), Yunnan (13.6%, 56/412, the southwestern of China) (Zhou et al., 2011), Jilin, and Liaoning provinces (18.3%, 34/186, the northeastern of China) (Zhang et al., 2018). In other countries, the prevalence of T. gondii infection in pigs was found to be 6.7% (54/810) in North India (Thakur et al., 2019), 19.8% (125/632) in Yucatan, Mexico (Cubas-Atienzar et al., 2018), and 31.3% (66/211) in Benue state, Nigeria (Nzelu et al., 2021).

Table 2.

Prevalence of Toxoplasma gondii Infection in Pigs in Zhejiang and Jiangsu Provinces, China

Province	Cities	No. of examined (%)^a	No. of positive	Prevalence (%)
Zhejiang	Huzhou	64 (8.6)	8	12.5
	Jiaxing	11 (1.5)	0	0.0
	Hangzhou	73 (9.8)	3	4.1
	Shaoxing	62 (8.3)	11	17.7
	Subtotal (northern)	210	22	10.5 ^b
	Jinhua (Central)	103 (13.8)	6	5.8 ^b
	Quzhou (Western)	37 (5.0)	5	13.5 ^b
	Zhoushan	8 (1.1)	1	12.5
	Ningbo	55 (7.4)	4	7.3
	Taizhou	31 (4.2)	2	6.5
	Subtotal (Eastern)	94	7	7.4 ^b
	Lishui	33 (4.4)	3	9.1
	Wenzhou	85 (11.4)	5	5.9
	Subtotal (Southern)	118	8	6.8 ^b
	Subtotal	562 (75.3)	48	8.5
Jiangsu	Nantong	108 (14.5)	8	7.4
	Nanjing	52 (7.0)	0	0.0
	Xuzhou	24 (3.2)	1	4.2
	Subtotal	184 (24.7)	9	4.9
Total		746	57	7.6

The bold values represent samples size and prevelence of T. gondii from five regions (northern, central, western, eastern and southern) of Zhejiang province.

The percent accounts for the total pigs.

Prevalence of Toxoplasma gondii infection in northern, Central, Western, Eastern, and Southern Zhejiang, respectively.

The prevalence varies among regions and countries, which may be related to the detection methods (Qin et al., 2014; Zhang et al., 2018), the type of samples, the number of samples, and the climate of the study area, but the most important is the feeding methods and farm management (Havelaar et al., 2012). It is conceivable that T. gondii prevalence in free-ranging domestic pigs would be higher than that in intensively farmed pigs. In addition, the asymptomatic infection and incorrect diagnosis usually lead to the underestimation of the disease control, resulting in a source of foodborne toxoplasmosis in the fresh pork market.

In the present study, due to the low DNA concentration, only one sample presented complete genotyping data, which was identified as ToxoDB genotype 9, and this result was consistent with previous studies as the main isolate strain of T. gondii found in China (Zhou et al., 2010; Wang et al., 2012; Jiang et al., 2013; Zhang et al., 2018). We found one sample that was genotyped at 9 loci, which represented ToxoDB genotype 10; another two samples showed alleles at 6 loci left the genotypes uncertain because of some missing genetic markers (Table 3), and the rest B1 gene-positive samples showed fewer genetic loci (data not shown). Regrettably, T. gondii genotype in pigs was not successfully identified in Jiangsu in this study, probably due to the low DNA concentration.

Table 3.

Genetic Characterization of Toxoplasma gondii Infection in Pigs in Zhejiang and Jiangsu Provinces, Eastern China

Isolate ID	Host	Tissue	Location	SAG1	5′+3′ SAG2	Alternative SAG2	SAG3	BTUB	GRA6	c22-8	c29-2	L358	PK1	Apico	Genotype
GT1	Goat		United States	I	I	I	I	I	I	I	I	I	I	I	Reference, ToxoDB 10
PTG	Sheep		United States	II/III	II	II	II	II	II	II	II	II	II	II	Reference, ToxoDB 1
CTG	Cat		United States	II/III	III	III	III	III	III	III	III	III	III	III	Reference, ToxoDB 2
MAS	Human		France	u-1^a	I	II	III	III	III	u-1^a	I	I	III	I	Reference, ToxoDB 17
TgCgCa1	Cougar		Canada	I	II	II	III	II	II	II	u-1^a	I	u-2^a	I	Reference, ToxoDB 66
TgCatBr5	Cat		Brazil	I	III	III	III	III	III	I	I	I	u-1^a	I	Reference, ToxoDB 19
TgWtdSc40	WTD		USA	u-1^a	II	II	II	II	II	II	II	I	II	I	Reference, ToxoDB 5
TgCatBr64	Cat		Brazil	I	I	u-1	III	III	III	u-1	I	III	III	I	Reference, ToxoDB 111
TgRsCr1	Toucan		Costa Rica	u-1^a	I	II	III	I	III	u-2	I	I	III	I	Reference, ToxoDB 52
ZJP1		Lymph node	Zhejiang, China	u-1^a	II	II	III	III	II	II	III	II	II	I	ToxoDB 9
ZJP2		Liver	Zhejiang, China	nd	I	I	I	nd	I	I	I	I	I	I	ToxoDB 10(?)
ZJP3		Lymph node	Zhejiang, China	nd	I	I	I	I	nd	nd	I	I	nd	nd	nd
ZJP4		Lymph node	Zhejiang, China	u-1^a	II	III	III	I	nd	nd	nd	nd	I	nd	nd

u-1 and u-2 represent unique RFLP technology (RFLP genotypes), respectively.

Nd, no data; RFLP, restriction fragment length polymorphism; WTD, White-tailed Deer.

To date, series of genotypes have been reported in China from a large number of hosts, including genotypes ToxoDB 225, ToxoDB 3, ToxoDB 213, ToxoDB 9, ToxoDB 292, and ToxoDB 10 (Wang et al., 2012; Tian et al., 2014; Jiang et al., 2016; Yang et al., 2017; Gui et al., 2018; Su et al., 2019); of these, ToxoDB 9 and ToxoDB 10 were two predominant lineages prevalent in China, especially in pigs (Gui et al., 2018; Zhang et al., 2018).

In fact, ToxoDB 9 has also been reported in other countries, including United States, Sri Lanka, and Vietnam (Dubey et al., 2007a, b; Gui et al., 2018), suggesting a widespread distribution of this strain. It is worth noting that toxoDB 10 is not only popular in animals from China, but also can infect humans. One T. gondii isolate from a dead human fetus was identified as type I (toxoDB 10) (Hou et al., 2018), indicating that type I strains are more pathogenic or more likely to cause infection in pregnant women or immunocompromised patients. It is necessary to analyze the genotype of T. gondii from humans and other hosts in Zhejiang and Jiangsu provinces for further researches, which will be helpful for public health.

The European Food Safety Authority recognized T. gondii as one of the public hazards in pigs to be assessed during meat inspection (van Asseldonk et al., 2017), and pork is an important risk causing two-thirds of the disease burdens, including toxoplasmosis (Kijlstra et al., 2004). In China, pork is one of the most popular meats. Chinese pork industries produce and consume about half of the world's pork. Humans become infected with T. gondii by consumption of raw or undercooked pork, as well as personnel engaged in the pork product industry (Dubey et al., 2012), raising concerns of possible risk to humans. Nzelu et al. (2021) reported that the T. gondii infection rate in pregnant women was 25.3% in Benue state, Nigeria; intriguingly, higher prevalence was also found in pork (31.3%). This result indicted that pigs display a risk for transmission of T. gondii to humans (Havelaar et al., 2012; Nzelu et al., 2021).

Conclusions

In summary, the present study revealed an overall T. gondii prevalence of 7.6% in pigs from Zhejiang and Jiangsu provinces, eastern China. Two genotypes were identified (ToxoDB 9 and ToxoDB 10), with ToxoDB 9 as the major lineage in mainland China. More studies on the genetic characterization of T. gondii in pigs and other hosts need to be done in the future, and more intervention methods targeting the risks of T. gondii are needed.

Footnotes

Authors' Contributions

H.C.S and T.Y.S conceived and supported the study. H.C.S wrote the article. Y.F and X.-F.Y performed the PCR experiments. J.-X.L, L.-H.X, and J.-N.Z. performed the PCR-RFLP experiments. B.Y. and J.H. collected the samples. M.Q. analyzed the data. All authors contributed to the article and approved the submitted version.

Disclosure Statement

No competing financial interests exist.

Funding Information

This project was supported by grant from the National Natural Science Foundation of China (grant numbers 31802183, 32072883); Zhejiang Province “Sannongliufang” Science and Technology Cooperation Project (grant number 2020SNLF007).

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