Introduction
Trichinellosis is a food-borne zoonotic parasitic disease caused by ingestion of Trichinella sp. infective larvae via consuming raw or undercooked meat (Pozio, Reference Pozio2015). The genus Trichinella consists of 13 taxa (10 species and 3 genotypes) distributed into 2 clades: encapsulated and nonencapsulated (Pozio, Reference Pozio2021). To date, 4 species have reportedly been isolated in China: T. spiralis, T. nativa, T. pseudospiralis and T. papuae (Bai et al., Reference Bai, Hu, Liu, Tang and Liu2017).
Pork meat and its products are China's largest source of human Trichinella infection (Cui et al., Reference Cui, Wang and Kennedy2006; Pozio, Reference Pozio2014). During 2009–2020, up to 87.5% (7/8) of outbreaks of human trichinellosis were caused by the consumption of raw or undercooked pork (Zhang et al., Reference Zhang, Wang and Cui2022). Other cases of human trichinellosis were shown to be sporadic infections resulting from the ingestion of meat from dogs, wild boar and other game (Wang et al., Reference Wang, Cui and Xu2006; Bai et al., Reference Bai, Hu, Liu, Tang and Liu2017).
Wild boar meat and meat-derived products are considered a further important source of Trichinella sp. infection in humans (Pozio, Reference Pozio2015; Rostami et al., Reference Rostami, Gamble, Dupouy-Camet, Khazan and Bruschi2017). Approximately two million commercial wild boars are reared on farms for trade in China every year. Most of them are kept under non-controlled conditions. Improper manage of farmed wild boars increases the risk of occurrence of trichinellosis in humans. Trichinella sp. infection in hunted wild boars has been widely reported in Europe (Vieira-Pinto et al., Reference Vieira-Pinto, Fernandes, Santos and Marucci2021), South America (Ribicich et al., Reference Ribicich, Fariña, Aronowicz, Ercole, Bessi, Winter and Pasqualetti2020), the Middle East (Haim et al., Reference Haim, Efrat, Wilson, Schantz, Cohen and Shemer1997) and Southeast Asia (Yera et al., Reference Yera, Bory, Khieu and Caron2022). However, the prevalence of Trichinella sp. infection in farmed wild boars is unknown in China.
The current study aimed to determine the presence of Trichinella infections in farmed wild boars (Sus scrofa) in northeastern China, which would increase awareness of the prevalence of Trichinella sp. in farmed wild boars and encourage surveillance measures.
Materials and methods
Study area and animals
The present study was conducted between August 2017 and December 2020 to investigate the presence of Trichinella infections in wild boars that could freely forage on small farms from Gongzhuling, Fusong and Jilin cities in Jilin Province, northeastern China (Fig. 1). The seroprevalence of Trichinella infections in pigs in northeastern China was reported to vary from 0.02% to below 4% (Cui et al., Reference Cui, Wang and Kennedy2006; Liu and Boireau, Reference Liu and Boireau2002). With a confidence level of 95% (z = 1.96) and an allowed deviation of the true prevalence of 5% (d), the projected seroprevalence is 4% (P). Consequently, 59 was used to calculate the sample size [according to n = P (1 − P)z 2/d 2 ] (Zhang et al., Reference Zhang, Zhang, Zhou, Huang, Tian, Yang, Zhao and Zhu2015). Information regarding geographical position, age and sex is provided in Table 1.
Figure 1. Map of China indicating the locality investigated in this study. The area marked in red represents the location of the investigated region.
Table 1. Information about farmed wild boars used in this study from 3 cities of Northeast China
Muscle samples
Diaphragm muscles were collected from 761 farmed wild boar carcasses for post-mortem inspection. The diaphragms (60 g–120 g from each boar) were trimmed of fat and fascia and stored at −15°C for pending testing by artificial digestion.
Magnetic stirrer artificial digestion
Diaphragm muscles were digested by the magnetic stirrer method utilizing HCl-pepsin to examine the presence of muscle larvae (Gamble et al., Reference Gamble, Bessonov, Cuperlovic, Gajadhar, Van Knapen, Noeckler, Schenone and Zhu2000). Each muscle sample was initially cut into 5 g pieces to create a pool of 50 g mixture for screen. Subsequently, all samples of positive pools were digested individually weighing 97 ± 6 g to identify Trichinella larvae. Briefly, the minced muscle sample was mixed with a solution containing 1% pepsin (1: 10 000 NF, Sigma, USA), 1% HCl and 0.9% NaCl. This mixture was stirred using a magnetic stirrer at 45 °C for 1 h. The homogenized digestive fluid was settled in a 2-litre separatory funnel for 1 h. The Trichinella larvae were obtained from the bottom of the settling funnel and kept in 90% ethyl alcohol after being washed 5 times using 0.9% NaCl. The parasite load was calculated as larvae per gram (lpg) ± s.d..
Multiplex polymerase chain reaction (PCR) assays of Trichinella larvae
The species of collected muscle larvae were identified by multiplex PCR as previously described (Zarlenga et al., Reference Zarlenga, Chute, Martin and Kapel1999). Briefly, extracted DNA from single Trichinella larvae (Pozio and La Rosa, Reference Pozio and La Rosa2003) was subjected to amplification of specific regions (ESV, ITS1 and ITS2) of the ribosomal DNA repeats using 5 primer sets (Zarlenga et al., Reference Zarlenga, Chute, Martin and Kapel1999). The species-specific DNA banding pattern was visualized on 1% (w/v) agarose gels stained with GoldenViewTM.
Sequencing analysis
The 5S rDNA inter-genic spacer region of Trichinella was amplified as previously described for phylogenetic analysis (Fu et al., Reference Fu, Liu, Yao, Li, Li, Wang, Wu, Zhang, Cai, Blaga and Boireau2009). Briefly, the PCR system in a volume of 25 μL contained 2 μL of DNA, 12.5 μL of 2 × UniqueTM Taq MasterMix (Novogene, China) and each primer at 10 μ m. The cycling conditions were 35 cycles of 94°C for 1 min, 55.8°C for 1 min and 72°C for 1 min. The PCR products were directly sequenced by Sangon Biotech (Shanghai) Co., Ltd., China.
Statistical analysis
Exact binomial 95% confidence intervals (CIs) were established for the rate of infection in this study. The t-test was used to assess statistical differences of infection rates according to the animals' sex, with values of P < 0.05 considered as statistically significant. Statistical analyses were performed by using the online software (Epitools-Epidemiological Calculators, http://epitools.ausvet.com.au).
Results
The overall prevalence of Trichinella in farmed wild boars was 0.53% [95% confidence interval (CI): 0.51–0.55]. Of the 4 positive results, 1 sample came from a male (1/109, 0.92%), and the positive rate in males was higher than that in females (3/652, 0.46%), but the difference was not statistically significant (P > 0.05, t = 0.047). Three out of the 4 Trichinella-positive wild boars were from Fusong city, and the other positive boar was from Jilin city. A higher prevalence of Trichinella infection in wild boars at the age > 66 days, compared to those at 22–66 days (P > 0.05, t = 0.013). The average parasite burden was 0.076 ± 0.025 larvae per gram (lpg), and the highest burden was 0.21 lpg detected in a female boar (age >66 days) from Fusong city.
The multiplex PCR allowed to identify all the larvae collected from the farmed wild boars as belonging to T. spiralis species (Fig. 2). The 5S rDNA obtained sequence (GenBank accession no. OQ725583) showed a 100% similarity with the corresponding sequence of the T. spiralis HLJ isolate (accession no. MH289505).
Figure 2. Identification of Trichinella isolates by PCR. M: DL2000 DNA marker; line 1: Multiplex PCR amplification of single larvae of Trichinella isolates in this study; line 2: Multiplex PCR amplification of single larvae of the T. spiralis Henan strain; line 3: Blank control.
Discussion
Wild boar meat is the second largest source of human infections of Trichinella worldwide (Pozio, Reference Pozio2015; Rostami et al., Reference Rostami, Gamble, Dupouy-Camet, Khazan and Bruschi2017). An increasing number of wild boars are being kept in captivity because of the rising demands for consumable wild boar meat in China. The present survey aimed to investigate the risk of human exposure to these products intended for human consumption. The overall prevalence of Trichinella in the present study was lower than that revealed by similar studies in Argentina (11.4%) (Cohen et al., Reference Cohen, Costantino, Calcagno, Blanco, Pozio and Venturiello2010), northwest Vietnam (3.2%) (Thi et al., Reference Thi, Nguyen, Praet, Claes, Gabriël, Huyen and Dorny2014), Latvia (2.2%) (Kirjušina et al., Reference Kirjušina, Deksne, Marucci, Bakasejevs, Jahundoviča, Daukšte, Zdankovska, Bērziņa, Esīte, Bella, Galati, Krūmiņa and Pozio2015), Laos (2.1%) (Conlan et al., Reference Conlan, Vongxay, Khamlome, Gomez-Morales, Pozio, Blacksell, Fenwick and Thompson2014), Chile (1.8%) (Hidalgo et al., Reference Hidalgo, Villanueva, Becerra, Soriano, Melo and Fonseca-Salamanca2019), Romania (1.66%) (Nicorescu et al., Reference Nicorescu, Ionita, Ciupescu, Buzatu, Tanasuica and Mitrea2015), and Estonia (0.9%) (Kärssin et al., Reference Kärssin, Häkkinen, Vilem, Jokelainen and Lassen2021) but was higher than that in Hungary (0.0077%) (Széll et al., Reference Széll, Marucci, Ludovisi, Gómez-Morales, Sréter and Pozio2012), Italy (0.01%) (Sgroi et al., Reference Sgroi, D'Alessio, Marucci, Pacifico, Buono, Deak, Anastasio, Interisano, Fraulo, Pesce, Toscano, Romano, Toce, Palazzo, De Carlo, Fioretti and Veneziano2022), Croatia (0.17%) (Balić et al., Reference Balić, Marucci, Agičić, Benić, Krovina, Miškić, Aladić and Škrivanko2020), and some other countries, where Trichinella parasites were not detected (Hatta et al., Reference Hatta, Imamura, Yamamoto, Matsubayashi, Tsuji and Tsutsui2017; Dimzas et al., Reference Dimzas, Chassalevris, Ozolina, Dovas and Diakou2021). The different prevalence levels of Trichinella infection in wild boars could be ascribed to differences in management conditions, food source, geographic region, climatic conditions, living environments, parasite species and animal welfare. Furthermore, the possibility that low infection could have passed unnoticed because the tested frozen meat drastically reduce the larvae recovery and easily lead to false negatives, although we attempted to increase the weight of frozen samples along with the sedimentation time to compensate reduction in test sensitivity (Gajadhar et al., Reference Gajadhar, Noeckler, Boireau, Rossi, Scandrett and Gamble2019).
Although no Trichinella species were detected in Gongzhuling city in this study, adequate supervisions are also necessary to control circulation of the worm from farmed wild boars to humans. The tested farmed wild boars were all reared on non-controlled, small farms. Under this type of system, the animals would be exposed to vehicles that transfer Trichinella from the sylvatic environment to the domestic cycle, such as synanthropic rodents, mustelids, or other small carnivorous and omnivorous mammals (Pozio, Reference Pozio2015).
In the present study, only T. spiralis was identified using the multiplex PCR method in the farmed wild boars. T. spiralis is the most common epidemic strain in China and is the exclusive species in pigs (Bai et al., Reference Bai, Hu, Liu, Tang and Liu2017). In this study, T. spiralis was reported for the first time in Jilin Province, where in the past T. nativa was identified in dogs (Liu and Boireau, Reference Liu and Boireau2002), demonstrating the co-presence of the 2 species in this province.
T. spiralis isolated from farmed wild boars in the present data indicates a small but uncontrollable risk associated with transfer to local residents. The low burdens of Trichinella from the diaphragm, the predilection site, of wild boars would indicate a negligible load in the rest of the carcass, contributing to the low risk for transmission. The risks come from failure to valid monitor Trichinella infection in wild boars consumed in China. No human clinical cases of trichinellosis have been reported in Jilin Province due to ingestion of wild boar meat infected by Trichinella larvae. The possible reasons are (1) the number of slaughtered farmed wild boars has not been very large in China in the last several years compared to 50 million tons of pork per year; (2) a relatively small proportion of carcasses is eaten by consumers as fresh meat because well-cooked food is more popular for Chinese individuals; and (3) asymptomatic infections due to consumption of positive meat are ignored in clinical practice. Cases of human trichinellosis in Jilin Province were reported 20 years ago (Liu et al., 2002). As the consumption of meat from farmed wild boars is predicted to increase in the future in China, an improvement in breeding conditions as well as an increase in veterinary controls is needed to reduce the risk of human infection. Also, it is suggested to implementing education of farmers and the public on good farming practices and responsible consumption.
Conclusions
In Jilin Province, China, T. spiralis was firstly detected in farmed wild boars. The prevalence rate was found to be 0.53%, indicating a low yet underestimated danger given the existing breeding conditions. This incident highlights the significance of creating unique regulations for China's processing of wild boar meat from farms. The frequency of Trichinella infection in farmed wild boars needs to be further investigated nationwide.
Author's contributions
NZZ and BQF conceived and designed the study. MW, WGC, HRZ and WYG collected and prepared samples. WHL gained the GenBank accession number. TTL performed statistical analyses. NZZ, MW and WGC prepared figures, prepared tables, wrote original drafts. TTHD, HY, NTBT and BQF edited and reviewed manuscript drafts.
Data availability statement
No additional data available.
Financial support
This study was funded by the National Key Research and Development Program of China (2023YFD1801000); Fundamental Research Funds for the Central Universities (Grant Number: lzujbky-2022-35); Science and Technology Major Project of Gansu Province (Grant Number: 22ZD6NA001); Natural Science Foundation of Gansu Province, China (Grant Number: 21JR7RA021) and Innovation Project for Scientific and Technological Youth Talents of Lanzhou City (2023-QN-21).
Competing interests
None.
Ethical standards
All procedures involving animals in the present study were approved and this study was approved by the Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Approval No. LVRIAEC2017-028).
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