Introduction
Pigs serve as the intermediate hosts of the zoonotic cestodes Taenia solium and Taenia asiatica, the former being endemic in many developing regions, while the latter is restricted to some Asian countries (Michelet & Dauga, Reference Martin, Meek and Willeberg2012). Humans can acquire taeniasis, the establishment of an adult tapeworm in the small intestine, by eating raw or undercooked infected pig products: pork (T. solium) or pig offal (T. asiatica). Human infections with T. asiatica are restricted to taeniasis, yet, when humans ingest eggs of T. solium, they may also acquire human cysticercosis, the development of the T. solium metacestode larval stage (cysticercus) in tissues such as muscles, eyes and brain, the latter referred to as neurocysticercosis (NCC). Infections with T. solium cause a high burden in many developing countries, including an estimated 28,000 deaths globally in 2010 (WHO, 2015), and as a consequence, T. solium was ranked first on the list of foodborne parasites of concern (FAO/WHO, 2012).
Vietnam is recognized as endemic for zoonotic tapeworms (World Health Organization, 2014), and taeniasis and cysticercosis are considered to be important public health problems in the country (Trung et al., Reference Trieu2013). Human taeniasis and cysticercosis have been reported in 50 out of 63 provinces, with a prevalence of cysticercosis ranging between 0% in Hai Duong and 13% in Ha Giang provinces, both in northern Vietnam; and a prevalence of taeniasis between 0.11% in Hoa Binh (in the north) and 10% in Kon Tum (in the central highlands) (De et al., Reference De, Le, Lien and Eom2014; Ng-Nguyen et al., Reference Nga, Ninh, Hung and Lapar2017). Among the three species that cause taeniasis – T. solium, Taenia saginata and T. asiatica – T. asiatica was the most recent species to be confirmed in Vietnamese people (Le et al., Reference Le, Do, Nguyen, Nguyen and Do2002).
Pork is the most popular meat type in the country; about 3.8 million tons of pork are produced annually (Vietnam Livestock Production, Reference Vien, Thach, Thuan, Dung, Tan, Duc and Tuyet2018). Small-scale traditional pig farms are still predominant, contributing to at least 80% of the total pork production (Lapar et al., Reference Lapar, Toan, Staal, Minot, Tisdell, Que and Tuan2012; Nga et al., Reference Muigg, Ruf and Schwarzkopf2014). The common habit of eating raw or undercooked pork and organs is one of the main risk factors for acquiring pig-related taeniasis in Vietnam (Somers et al., 2007; Ng-Nguyen et al., Reference Nga, Ninh, Hung and Lapar2017). Nevertheless, data on porcine cysticercosis are scarce. The prevalence of porcine T. solium cysticercosis was estimated at 0.04%, 0.03% and 0.09% in Hanoi, northern and southern provinces, respectively, as determined by routine meat inspection (Trieu, Reference Tang, Wong and Lai2012), while the seroprevalence of porcine cysticercosis in Daklak province, in the central highlands, was estimated at 0.94% using the lentil lectin-purified glycoprotein enzyme-linked immunoelectrotransfer blot (LLGP-EITB) assay (Ng-Nguyen et al., Reference Ng-Nguyen, Stevenson and Traub2018). Until now, T. asiatica cysticercosis has not been confirmed in pigs in Vietnam, although the adult intestinal parasite was found in humans (Somers et al., 2007).
Pigs also serve as intermediate hosts of Taenia hydatigena, a non-zoonotic tapeworm species, which uses dogs as their definite hosts. This parasite was reported to be highly prevalent in pigs in Asia (Nguyen et al., Reference Ng-Nguyen, Noh, Breen, Stevenson, Handali and Traub2016). In Vietnam, the prevalence of T. hydatigena was estimated at 31.8% in southern provinces and 25.1% in northern Vietnam (Huan, Reference Huan1994; Lan et al., Reference Lan, Quyen and Hoat2011). This parasite is reported to cause cross-reactions in serological tests used for diagnosis of T. solium cysticercosis in pigs (Dorny et al., Reference Dorny, Phiri, Vercruysse, Gabriel, Willingham, Brandt, Victor, Speybroeck and Berkvens2004). In regions where the prevalence of T. hydatigena in pigs is high, serological tests that are prone to cross-reactions should not be used to diagnose zoonotic cysticercosis. The diagnostic performance of serological tests to detect T. solium cysticercosis in pigs has not yet been assessed in Vietnam.
Therefore, we performed a cross-sectional survey in pig abattoirs in Phu Tho province, northern Vietnam, aiming to determine the prevalence of Taenia species and to assess the diagnostic performance of serological methods for T. solium cysticercosis in pigs.
Materials and methods
Study area
Our study was conducted in Tan Son and Yen Lap districts of Phu Tho province (fig. 1). This province is located in the northern mountainous region of Vietnam and shares a border with Hanoi in the west. About 1.4 million people belonging to 34 ethnic groups are dwelling over an area of 3528.1 km2 (GSO, 2019). In Phu Tho province, the pig husbandry system is representative for the midland area, including both traditional smallholder farms and commercial farms. The pig population is estimated to be around 800,000; in 2017, 128.2 thousand tons of pork were produced for local consumption or export (SDAH, Reference Rodriguez-Hidalgo, Benitez-Ortiz, Praet, Saa, Vercruysse, Brandt and Dorny2018), ranking seventh out of 24 northern provinces (Vietnam Livestock Production, Reference Vien, Thach, Thuan, Dung, Tan, Duc and Tuyet2018). Furthermore, this province was reported to have a high prevalence of human taeniasis (8.68% by the Kato–Katz method) (Vien et al., Reference Tsang, Brand and Boyer2013). The habit of eating undercooked pork and liver is common, especially ‘thit lon chua’ (fermented pork), ‘gan tai’ (rare liver) and ‘gan nuong’ (grilled liver) (Vien et al., Reference Tsang, Brand and Boyer2013). At the clinic of the National Institute of Malariology, Parasitology and Entomology (NIMPE) in Hanoi, NCC patients frequently originate from Phu Tho province.
Fig. 1. Map of Vietnam showing Phu Tho province and the location of the study sites.
In Tan Son, one of our study districts, a preliminary study on Taenia spp. infection in humans estimated that 7.3% and 1.2% of the population had antibodies and circulating antigens, respectively (NIMPE, unpublished results). However, no data on Taenia spp. in pigs are available. Yen Lap is a neighbouring district of Tan Son in the high mountain region of Phu Tho province. In both districts, ethnic minorities account for more than 70% of the population and pork is mainly sold on wet markets.
Table 1. Number of pigs harbouring cysticerci and suspected lesions in different organs based on meat inspection and dissection.
a White solid spots in the liver of 1 mm or less.
Study design
We conducted a cross-sectional survey between July and August 2018. Three study sites per district were chosen based on convenience of sample collection (e.g. being able to purchase the entire visceral organs). The three communes, Thu Ngac, My Thuan, Tan Phu were selected in Tan Son, while in Yen Lap, two communes, Xuan Vien, Xuan Thuy, and one town, Yen Lap town, were chosen.
A list of pig abattoirs in the six sites was requested from the District Veterinary Station of the Sub-Department of Animal Health in Phu Tho. The owners of all 76 abattoirs were informed about the study objectives, and invited to participate in the study. All abattoirs were small-scale enterprises processing few pigs per day. A total of 51 out of 76 abattoir owners agreed to take part in the study. Once approval was granted, the slaughterhouse was visited, and the process of the study was explained to the owners before sampling. Next, blood samples were taken immediately after slaughter of the pigs. The slaughterers performed a routine check of the abdominal cavity and meat cutting as ‘informal inspection’; no official meat inspection is performed in abattoirs in this region. All visible cysts and suspected lesions were collected and transferred to vials containing ethanol 70%. The liver, heart, tongue, masseter muscles and diaphragm were collected from the same pigs. Blood and tissues were transported to the lab in cool boxes. Blood was allowed to clot, serum was separated and stored at –20°C until further analysis.
Laboratory analysis
Dissection
The collected tissues were cut into slices of less than 0.5 cm and examined for cysticerci. Cysts detected on incisional and intact surfaces were visually classified as one of the Taenia species being T. solium, T. hydatigena and T. asiatica (Dorny et al., Reference Dorny, Phiri, Vercruysse, Gabriel, Willingham, Brandt, Victor, Speybroeck and Berkvens2004). Detected cysts were collected in separate tubes for each tissue and stored in 70% ethanol. Suspected lesions presenting as small white spots in the liver were also collected and stored in 70% ethanol.
Molecular techniques
Genomic DNA was extracted from all collected cysts and suspected lesions using the DNeasyBlood and Tissue Extraction Kit according to the manufacturers’ instructions (QIAGEN, Hilden, Germany). Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) targeting a mitochondrial 12 s ribosomal DNA fragment was used to differentiate the Taenia spp. using procedures described by Rodriguez-Hidalgo et al. (2002) and Devleesschauwer et al. (Reference Devleesschauwer, Aryal, Tharmalingam, Joshi, Rijal, Speybroeck, Gabriël, Victor and Dorny2013). Species confirmation was done by sequencing of the PCR products at the VIB Genetic Service Facility (University of Antwerp, Belgium). The sequences were edited and aligned using BioEdit (Hall, Reference Hall1999), BLAST was performed on National Center for Biotechnology Information and the sequences were submitted to GenBank.
Serological tests
Serum samples were examined for circulating Taenia spp. antigens using a commercial B158/B60 Ag-ELISA kit (apDia Turnhout, Belgium), following the manufacturer's instructions. Detection of specific antibodies to T. solium was performed using the LLGP-EITB, using native antigen extracts, and a EITB using a recombinant T24H antigen (rT24H-EITB) at the Centers for Disease Control and Prevention, Atlanta, USA (Tsang et al., Reference Trung, Praet, Cam, Lam, Manh, Gabriãl and Dorny1989; Noh et al., Reference Nguyen, Gabriël, Abatih and Dorny2014).
Sample size
We assumed a large population size with unknown prevalence of Taenia spp. cysticercosis in pigs (due to the absence of data on the prevalence of T. asiatica cysticercosis in pigs), a required confidence level of 95% and a desired precision of 5% (Martin et al., Reference Le, Nga, van De, Cong and Doanh1987). The planned sample size for the study was 384. Ultimately, 399 pigs were sampled from 51 abattoirs in Phu Tho.
Statistical analysis
All collected data were entered in Excel (Microsoft Office 2016, Microsoft Corporation, Washington, USA). A descriptive analysis of the occurrence of the different Taenia spp. was conducted. The prevalence of Taenia spp. and the associated 95% Wilson score confidence intervals (CIs) were calculated. In case of low cell counts, the Clopper–Pearson exact CIs were calculated. All statistical analyses were conducted using R software (R Core Team, Reference Polley and Mostert2020).
Results
Informal inspection results
A total of 399 pigs from 51 abattoirs in Yen Lap and Tan Son districts, Phu Tho province were examined for cysticerci by slaughterers (table 1). Forty-one pigs (41/399, 10.8%) carried cysts consistent with the morphology of T. hydatigena cysticerci (i.e. large, visceral, fluid-filled cysts with a single white scolex) in the abdominal cavity and/or on the surface of abdominal organs. Among them, 15 pigs had T. hydatigena cysticerci on the liver surface only (fig. 2a).
Fig. 2. (a) Taenia hydatigena cysticerci on the liver surface; (b) white spot in the liver; (c) and T. hydatigena cyst under the liver surface.
Dissection results
Tongue, heart, liver, diaphragm and masseter muscles of all 399 pigs were dissected (table 1). No cysticerci of T. solium were detected. In 74 pigs, lesions without a clear diagnosis (small white solid spots, diameter smaller than or equal to 1 mm) were detected in the liver (fig. 2b). In one pig, a cyst (visceral, fluid-filled cyst of 1 cm in diameter with a single white scolex), perceived to be T. hydatigena due to its large size, was found inside the liver close to the surface (fig. 2c); and in the same pig, cysticerci consistent with the morphology of T. hydatigena were detected during inspection by slaughterers in the abdominal cavity.
Molecular results
Suspected lesions from 74 pigs and T. hydatigena cysticerci from 41 pigs were further analysed by PCR followed by RFLP. Cysticerci consistent with the morphology of T. hydatigena detected in 41 pigs were all confirmed to be T. hydatigena. Suspected lesions detected in 31 pig livers were also identified as T. hydatigena. Thus, the total number of pigs infected with T. hydatigena was 72 and the prevalence was estimated at 18.0% (95% Wilson score CI: 14.6–22.1%). Suspected lesions in two pigs showed four bands (100, 131, 190, 309 bp) in RFLP, consistent with T. asiatica (fig. 3), which were confirmed by sequencing (GenBank accession numbers MT448955.1 and MT463535.1). The prevalence of T. asiatica was estimated at 0.50% (95% exact CI: 0.06–1.80%). One suspected lesion collected from a pig liver revealed four bands (309, 190, 131, 100 bp) in RFLP and was identified to be Spirometra erinaceieuropaei by sequencing (GenBank accession number MT465993.1). No T. solium cysticerci were detected by morphological nor molecular identification in the 399 sampled pigs.
Fig. 3. PCR-RFLP results of suspected lesions in pigs sampled in Phu Tho, Vietnam. Lane 1: DNA ladder; lane 12: Taenia hydatigena – positive control; lanes 2–5, 7–10 (385, 289, 114 bp): T. hydatigena; lane 11 (393, 295, 218, 169, 130, 116 bp): T. asiatica; lane 6 (309, 190, 131, 100 bp): Spirometra erinaceieuropaei.
Serological test results
The Ag-ELISA identified 94/399 (23.6%) of pigs as positive for circulating cysticercus antigens, while 6/399 (1.5%) of the results were doubtful (grey zone) and 299/399 (74.9%) were negative (fig. 4). Cysticerci of T. hydatigena or T. asiatica were identified in 51/94 of sero-positive pigs (54.3%), with T. hydatigena being identified in 50/94 (53.2%). Conversely, the Ag-ELISA was negative in 22/74 (29.7%) of pigs found to harbour T. hydatigena or T. asiatica cysts. One pig with a doubtful Ag-ELISA result (grey zone) was found to harbour T. hydatigena cysts. The serum of the S. erinaceieuropaei-positive pig was negative in the Ag-ELISA. Overall, T. hydatigena confirmed cases were Ag-ELISA positive in 50 out of 72 (69.4%) pigs and serum of one out of two (50.0%) pigs infected with T. asiatica was positive.
Fig. 4. The results of Ag-ELISA and molecular analysis of cysts detected in pig samples in Phu Tho, Vietnam.
The serum of 1/399 (0.3%) pig, positive in Ag-ELISA, was also positive in LLGP-EITB, with bands at GP50 and GP42, and in the rT24H-EITB test. This serum originated from a pig with many T. hydatigena cysts on the liver. All other serum samples were negative in both LLGP-EITB and rT24H-EITB tests.
Discussion
Our study is the first to confirm the presence of T. asiatica cysticerci in pigs in Vietnam. This finding completes the life cycle of this parasite in the country. Previously, only intestinal T. asiatica tapeworms were found in humans (Le et al., Reference Le, Do, Nguyen, Nguyen and Do2002). However, the typical cysticerci of T. asiatica, cystic or oval-shaped, with a length of 1.9–3.0 mm and a thin and transparent cyst wall, as described by Eom et al. (Reference Eom, Jeon and Kong2002, Reference Eom, Rim and Jeon2020), were not observed in this study. The two molecularly confirmed cases of T. asiatica presented as small, white, round spots in the liver. Fan et al. (Reference Fan, Chung, Lin and Wu1990, Reference Fan, Chung, Lin and Pawlowski1992) stated that T. asiatica cysticerci start calcification 45 days post inoculation and present as calcified spots in the liver only six months post inoculation. This type of hepatic lesion can easily be missed during meat inspection or confused with other parasites such as T. hydatigena, or lesions caused by migrating Ascaris suum larvae (so-called ‘milk spots’) (Polley & Mostert, Reference Noh, Rodriguez, Lee, Handali, Gonzalez, Gilman, Tsang, Garcia and Wilkins1980; Blazek et al., Reference Blazek, Schramlova and Hulinska1985). Because of the difficulty to detect T. asiatica during meat inspection, infected livers are likely to enter the food chain and infect humans, especially considering the local culinary habit to consume undercooked liver (Vien et al., Reference Tsang, Brand and Boyer2013).
On the other hand, we were not able to detect cysticerci of T. solium in any of the investigated tissues of 399 pigs raised in traditional farms. Interestingly, T. solium antibodies were detected in one serum sample, as determined by both LLGP-EITB and rT24H-EITB. This pig harboured many T. hydatigena cysticerci but, conversely, no T. solium cysticerci were detected. This result may be explained by the study procedures – that is, dissecting only the predilection organs, while cysticerci may have been located somewhere else in the carcass. Alternatively, the pig may have been exposed to T. solium eggs without establishing cysticerci (Conlan et al., Reference Conlan, Vongxay, Khamlome, Dorny, Sripa, Elliot, Blacksell, Fenwick and Thompson2012; Devleesschauwer et al., Reference Devleesschauwer, Aryal, Tharmalingam, Joshi, Rijal, Speybroeck, Gabriël, Victor and Dorny2013). The possibility of cross-reactions with T. hydatigena in the LLGP-EITB and rT24H-EITB tests is less plausible in this case, as the serum reacted to the GP42 and GP50 bands in the LLGP-EITB while only the GP50 band has been found to be less specific (Gomez-Puerta et al., Reference Gomez-Puerta, Vargas-Calla, Castillo, Lopez-Urbina, Dorny, Garcia, Gonzalez and O'Neal2019); and the GP50 is not included in the rT24-EITB.
This study revealed a high prevalence of T. hydatigena cysticercosis in pigs (18.0%, 95% CI: 14.5–22.1%), although lower than found in previous studies (25.1–31.8%) in pigs in Vietnam (Nguyen et al., Reference Ng-Nguyen, Noh, Breen, Stevenson, Handali and Traub2016). Similarly, in Laos, the occurrence of T. hydatigena (22.4%) is much more common than T. solium (0.8%) in pigs (Conlan et al., Reference Conlan, Vongxay, Khamlome, Dorny, Sripa, Elliot, Blacksell, Fenwick and Thompson2012). The low prevalence of zoonotic cysticercosis might be the consequence of the improved general living conditions, hygiene and sanitation of the local populations. In contrast, conditions allowing the completion of the T. hydatigena life cycle persist in the region: the traditional backyard farming type is still prominent (Lapar et al., Reference Lapar, Toan, Staal, Minot, Tisdell, Que and Tuan2012), allowing pigs to have access to faeces of dogs that are free-roaming and are often fed condemned organs, which may contain T. hydatigena cysticerci.
The study is also the first to report the presence of S. erinaceieuropaei in a pig in Vietnam. The literature on this parasite in pigs is limited; it was reported in pork from feral hogs in the US, but not in the liver (Bengtson & Rogers, Reference Bengtson and Rogers2001). Spirometra erinaceieuropaei is a zoonotic parasite causing sparganosis in humans. It has been identified in humans in Vietnam and other countries in Asia, such as Korea, China and Thailand (Anantaphruti et al., Reference Anantaphruti, Nawa and Vanvanitchai2011; Le et al., Reference Le, Nga, van De, Cong and Doanh2017; Tang et al., Reference Somers, Dorny, Geysen, Nguyen, Thach, Vercruysse and Nguyen2017; Kim et al., Reference Kim, Ahn, Sohn, Nawa and Kong2018; Muigg et al., Reference Michelet and Dauga2019). This larva found in the pig was very small and, thus, could easily have been ignored during meat inspection. Humans or dogs and cats in the area may get infected by eating undercooked infected livers.
In this study, we used the B158/B60 Ag-ELISA on the collected pig serum samples. This test detects infection with viable Taenia cysticerci, and is genus- (not species) specific (Dorny et al., Reference Dorny, Phiri, Vercruysse, Gabriel, Willingham, Brandt, Victor, Speybroeck and Berkvens2004; Rodriguez-Hidalgo et al., Reference Rodriguez-Hidalgo, Geysen, Benítez-Ortiz, Geerts and Brandt2006). Consequently, pigs with either T. hydatigena or T. asiatica infection were found positive. The high prevalence of T. hydatigena and the low prevalence of zoonotic cysticercosis in pigs in this study suggest that the Ag-ELISA is not suitable to diagnose zoonotic cysticercosis in Vietnam. This situation is different from some African countries where the specificity of Ag-ELISA test for T. solium was reported to be as high as 94.7% (Dorny et al., Reference Dorny, Phiri, Vercruysse, Gabriel, Willingham, Brandt, Victor, Speybroeck and Berkvens2004), because of the lower prevalence of T. hydatigena (Nguyen et al., Reference Ng-Nguyen, Noh, Breen, Stevenson, Handali and Traub2016), the absence of T. asiatica (Eom et al., Reference Eom, Jeon and Rim2009) and the high to very high prevalence of T. solium in those regions (Dorny et al., Reference Dorny, Phiri, Vercruysse, Gabriel, Willingham, Brandt, Victor, Speybroeck and Berkvens2004).
In conclusion, T. solium cysticercosis was not found in pigs slaughtered in low-throughput abattoirs in this area by slicing predilection sites. Furthermore, T. asiatica was detected at a low prevalence in pig livers and was confirmed by molecular techniques. The prevalence of non-zoonotic T. hydatigena was high. Finally, results of serological techniques, especially the Ag-ELISA, to study the prevalence of T. solium in the area, should be interpreted with caution because of their low specificity.
Acknowledgements
The authors thank the District Station of Animal Health in Tan Son and Yen Lap, Phu Tho, a student team (Nguyen Van Toan, Hoang Thanh Truc, Can Quoc Hung) from the Faculty of Veterinary Medicine in National University of Agriculture and Dinh The Dung from the Vietnam-Russia Tropical Center for supporting the field work. The authors also thank their colleagues in the National Center for Veterinary Diagnosis, Hanoi, Vietnam.
Financial support
This work was supported by the Directorate General for Development, Belgium, through the individual PhD program (Man T.T. Nguyen) of the Institute of Tropical Medicine Antwerp, Belgium.
Conflicts of interest
The authors declare that there is no conflict of interest.
Ethical standards
The authors declare compliance with animal welfare and ethical guidelines of the investigators for all animal manipulations in this study.
