Clonorchiasis and opisthorchiasis: epidemiology, transmission, clinical features, morbidity, diagnosis, treatment, and control.

Clonorchis sinensis, Opisthorchis viverrini, and Opisthorchis felineus are important liver flukes that cause a considerable public health burden in eastern Asia, southeastern Asia, and eastern Europe, respectively. The life cycles are complex, involving humans, animal reservoirs, and two kinds of intermediate hosts. An interplay of biological, cultural, ecological, economic, and social factors drives transmission. Chronic infections are associated with liver and biliary complications, most importantly cholangiocarcinoma. With regard to diagnosis, stool microscopy is widely used in epidemiologic surveys and for individual diagnosis. Immunologic techniques are employed for screening purposes, and molecular techniques facilitate species differentiation in reference laboratories. The mainstay of control is preventive chemotherapy with praziquantel, usually combined with behavioral change through information, education and communication, and environmental control. Tribendimidine, a drug registered in the People's Republic of China for soil-transmitted helminth infections, shows potential against both C. sinensis and O. viverrini and, hence, warrants further clinical development. Novel control approaches include fish vaccine and biological control. Considerable advances have been made using multi-omics which may trigger the development of new interventions. Pressing research needs include mapping the current distribution, disentangling the transmission, accurately estimating the disease burden, and developing new diagnostic and treatment tools, which would aid to optimize control and elimination measures.

Effectiveness of health education in improving knowledge, attitude and practice related to foodborne zoonotic trematodes in Vietnam, with a particular focus on Clonorchis sinensis.

OBJECTIVE: Foodborne zoonotic trematodes (FZT), which infect the liver, lungs and intestines of humans, are an emerging public health concern in tropical countries including Vietnam. In northern Vietnam, Clonorchis sinensis is recognised as the most important species of the FZT. Up to now, small-scale studies conducted in the country have indicated that health education could improve participants' knowledge and practices related to clonorchiasis, however strong evidence is still lacking. We conducted an intervention trial between 2020 and 2021 in four communes in Yen Bai and Thanh Hoa province, aiming to evaluate the impact of an extended educational package on knowledge, attitude and practices related to clonorchiasis, as well as on knowledge on the large liver fluke and minute intestinal flukes. METHODS: To this end, baseline and post-intervention questionnaires were conducted. Generalised estimating equations models were run to analyse the impact of the intervention on knowledge, attitudes and practices over time in the intervention versus control group. Finally, 172 people in the intervention group and 162 in the control group were included for analysis. RESULTS: Results demonstrated that the intervention significantly improved clonorchiasis knowledge, attitudes and practices, with higher odds for a correct knowledge response (odds ratio (OR) = 2.80, 95% confidence interval (CI) = 1.84-4.27, p-value < 0.001), increased average mean attitude score (mean = 0.363, 95%CI = 0.182-0.544, p-value < 0.001), and a reduced odds of consuming raw fish (OR = 0.15, 95%CI = 0.06-0.40, p = 0.002) in the intervention group compared to the control group post-intervention versus baseline. Additionally, participants in the intervention group exhibited enhanced basic knowledge of the large liver fluke and minute intestinal flukes post-intervention. CONCLUSIONS: These findings demonstrate the potential of health education programs in preventing clonorchiasis in endemic areas, emphasising the importance of continued health education as a critical component of integrated control programs for clonorchiasis.

Effects of co-infection with Clonorchis sinensis on T cell exhaustion levels in patients with chronic hepatitis B.

To investigate the effects of co-infection with Clonorchis sinensis (C. sinensis) on T cell exhaustion levels in patients with chronic hepatitis B, we enrolled clinical cases in this study, including the patients with concomitant C. sinensis and HBV infection. In this study, we detected inhibitory receptors and cytokine expression in circulating CD4+ and CD8+ T cells by flow cytometry. PD-1 and TIM-3 expression levels were significantly higher on CD4+ T and CD8+ T cells from co-infected patients than on those from the HBV patients. In addition, CD4+ T cells and CD8+ T cells function were significantly inhibited by C. sinensis and HBV co-infection compared with HBV single infection, secreting lower levels of Interferon gamma (IFN-γ), Interleukin-2 (IL-2), and TNF-α. Our current results suggested that C. sinensis co-infection could exacerbate T cell exhaustion in patients with chronic hepatitis B. PD-1 and TIM-3 could be novel biomarkers for T cell exhaustion in patients with Clonorchis sinensis and chronic hepatitis B co-infection. Furthermore, it may be one possible reason for the weaker response to antiviral therapies and the chronicity of HBV infection in co-infected patients. We must realize the importance of C. sinensis treatment for HBV-infected patients. It might provide useful information for clinical doctors to choose the right treatment plans.

Introduction.

Cholangiocarcinoma (CCA) is a lethal cancer arising in the bile ducts within and just outside the liver. It occurs worldwide and falls into two etiologically defined groups, one related to chronic liver fluke infection and the other not. Liver fluke-related CCA is found in continental Southeast Asia (caused by Opisthorchis viverrini with infection leading to opisthorchiasis), East Asia (Clonorchis sinensis), and Eastern Europe and Russia (Opisthorchis felineus). Both O. viverrini and C. sinensis are classified as group one carcinogens, while recent data from O. felineus suggest the same. In Southeast Asia, an estimated 67.3 million people are at risk of O. viverrini infection and subsequently developing CCA. When the three liver fluke species are considered, an estimated 700 million people are at risk of infection and developing CCA globally. The northeast of Thailand (Isan) is the world's hot spot of liver fluke infection and CCA. Early detection, diagnosis, and surgical intervention/curative treatment of CCA are critical to increase life expectancy and quality of life of people in the region and globally. Despite concentrated recent efforts focusing on a multidisciplinary approach to understand the ecology, epidemiology, biology, public health, and social significance of infection by cancer causing liver flukes, it remains an underestimated and under-resourced public health problem. In addition, it is still believed to be a regional problem without global significance-this is not the case. This book focuses on O. viverrini as the main causative agent of CCA in Southeast Asia, but many aspects detailed in the following chapters also relate to the two other liver fluke species. Our aim is to produce a holistic framework including the basic biology of O. viverrini and its relation to the epidemiology of the disease through diagnosis to treatment, including palliative methods, pathology, and control.

Clonorchis sinensis Reinfection Rate and Reinfection Determinants: A Prospective Cohort Study in Hengxian County, Guangxi, China.

BACKGROUND: To understand Clonorchis sinensis reinfection and the determinants of reinfection in endemic areas is important in establishment of control measures. METHODS: A prospective cohort study was implemented in Hengxian County, Guangxi, China. Individuals with C. sinensis infection were completely treated, and those cured were enrolled as study subjects and followed up for 3, 6, and 12 months. The reinfection frequency and incidence were calculated, and a multivariable Cox proportional hazard model was constructed to capture reinfection determinants. RESULTS: Among 635 enrolled subjects, 436 (68.7%) completed follow-up. Of these, 177 (40.6%) were reinfected; 133 (75.1%) were reinfected once, 41 (23.2%) twice, and 3 (1.7%) three times. The incidence of reinfection was 64.0 per 100 person-years. Men (adjusted hazard ratio [aHR], 1.67; 95% confidence interval [CI], 1.14-2.44), those with underlying diseases (aHR, 1.41; 95% CI, 1.02-1.95), and those with moderate- or heavy-intensity infections (aHR, 1.45; 95% CI, 1.14-1.85) had increasing reinfection probabilities. CONCLUSIONS: C. sinensis reinfection is high in endemic areas. Men and high-intensity infection are important determinants of reinfection. Repeated chemotherapy is necessary to control reinfection and its associated morbidities, especially in high-risk individuals. In addition, behavioral education is advised to decrease overall reinfection in endemic areas.

Epidemiological characterization of Clonorchis sinensis infection in humans and freshwater fish in Guangxi, China.

BACKGROUND: Clonorchiasis is a widespread yet neglected foodborne disease with over 85% of all cases found in China. Guangxi province, located in southeastern China, ranks among the highest endemic provinces. We explore the epidemiological status and determinants of Clonorchis sinensis (C. sinensis) infection in humans and freshwater fish in Guangxi, China. METHODS: Data on C. sinensis infection in humans from January 2008 to December 2017were extracted from the China Information System for Disease Control and Prevention. An active surveillance of C. sinensis infection in fish was conducted in 2016-2017. County level data including potential environmental, social-economical and behavioral determinants was also collected. Univariate and multivariate logistic regression models were used to explore the determinants of C. sinensis infection in humans and fish. Simple and multiple zero-inflated Poisson regression models were fit to assess the associated factors of clonorchiasis in humans at the county level. RESULTS: Totally, 4526 C. sinensis cases were reported between 2008 and 2017, with an annual prevalencerate of 0.96/100,000 persons. Of 101 counties in Guangxi, 97 reported at least 1 case. Among 2,098 fish samples, 203 (9.7%) from 70 counties contained C. sinensis. The rate was higher in small fish including Pseudorasbora parva (45.3%), Misgurnus anguillicaudatus (41.2%), Hemicculter leuciclus (34.5%), unclassified small fishes (30.9%), Cyprinidae (20.0%), Cirrhinus molitorella (16.4%), Carassius auratus (13.6%) and Cyprinus carpio (13.3%), while it was lower in fish species that are usually used in preparing raw fish dishes including Ctenopharyngodon idellus (3.6%), Spinibarbus denticulatus (3.7%), Monopterus albus (6.4%), Cyprinus carpio (4.4%), Oreochromis mossambicus (3.3%) and Spualiobarbus Curriculus (6.6%). The C. sinensis infection in fish was only associated with fish species. The estimated human clonorchiasis prevalence at the county level was positively associated with raw fish consumption habits and certain rivers. CONCLUSIONS: Clonorchiasis is highly prevalent in both humans and freshwater fish in Guangxi. Environmental, social-economic and behavioral determinants contribute to the high prevalence as well as the significant differential distribution by county. Regular surveillance should be implemented for clonorchiasis to demonstrate the change in epidemiology and burden, which will benefit the design of interventions.

Current status of Clonorchis sinensis and clonorchiasis in Korea: epidemiological perspectives integrating the data from human and intermediate hosts.

Clonorchis sinensis is a carcinogenic liver fluke that causes clonorchiasis in humans. Clonorchiasis is prevalent in East Asian countries, and approximately 15­20 million individuals are estimated to be infected with this fluke globally. This review highlights the current status of C. sinensis and clonorchiasis in Korea from the epidemiological perspective involving the analysis of humans and intermediate hosts. Despite the recent decline in C. sinensis infection rate in Korea, C. sinensis infections remain endemic in 5 major river basins (Han-gang, Geum-gang, Seomjin-gang, Yeongsan-gang and Nakdong-gang; gang means river) with a high incidence of cholangiocarcinoma. A noticeable pattern involves increasing mild infections among patients diagnosed positive for C. sinensis eggs. The infection rate of C. sinensis metacercariae in the second intermediate host, freshwater fish, is also maintained at a substantial level. Thus, the One Health approach integrating different sectors and disciplines is recommended to accelerate and sustain control of C. sinensis, thereby leading to successful eradication. Health promotion via information dissemination and health education should be extended to prevent the consumption of raw freshwater fish by residents living in high-risk areas.

Similarities and differences among the Opisthorchiidae liver flukes: insights from Opisthorchis felineus.

The foodborne liver trematode Opisthorchis felineus (Rivolta, 1884) is a member of the triad of phylogenetically related epidemiologically important Opisthorchiidae trematodes, which also includes O. viverrini (Poirier, 1886) and Clonorchis sinensis (Loos, 1907). Despite similarity in the life cycle, Opisthorchiidae liver flukes also have marked differences. Two species (O. viverrini and C. sinensis) are recognized as Group 1A biological carcinogens, whereas O. felineus belongs to Group 3A. In this review, we focus on these questions: Are there actual differences in carcinogenicity among these 3 liver fluke species? Is there an explanation for these differences? We provide a recent update of our knowledge on the liver fluke O. felineus and highlight its differences from O. viverrini and C. sinensis. In particular, we concentrate on differences in the climate of endemic areas, characteristics of the life cycle, the range of intermediate hosts, genomic and transcriptomic features of the pathogens, and clinical symptoms and morbidity of the infections in humans. The discussion of these questions can stimulate new developments in comparative studies on the pathogenicity of liver flukes and should help to identify species-specific features of opisthorchiasis and clonorchiasis pathogenesis.

Clonorchis sinensis infection contributes to hepatocellular carcinoma progression in rat.

Clonorchis sinensis (C. sinensis) infection is a risk factor for cholangiocarcinoma. Whether it also contributes to the development of hepatocellular carcinoma (HCC) is still unclear. This study explored the potential relationship between C. sinensis infection and HCC. A total of 110 Sprague-Dawley rats were divided into four treatment groups, the negative control group (NC) received intragastric (i.g.) administration of saline, while the clonorchiasis group (CS) received i.g. administration of 150 C. sinensis metacercariae. The diethylnitrosamine-induced group (DEN) received intraperitoneal (i.p.) administration of DEN. The clonorchiasis DEN-induced group (CSDEN) received i.g. administration of 150 C. sinensis metacercariae followed by i.p. administration of DEN. Hematoxylin and eosin staining, immunohistochemistry, and Masson's trichrome staining were performed for histopathological analysis of the isolated tissues. RNA-seq technology and RT-PCR were employed for gene expression. In the DEN group, 15 rats survived, of which 9 developed liver cirrhosis and 7 developed HCC. In the CSDEN group, all of the 17 surviving rats developed cirrhosis, and 15 showed development of HCC. The incidence of liver cirrhosis and HCC was significantly higher in the CSDEN group than in the DEN group. KEGG pathway analysis of the differentially expressed genes suggested significant upregulation in inflammation-associated pathways. Immunohistochemistry and RT-PCR results showed significant upregulation of hepatic progenitor cell markers (CK19, SOX9, EpCAM) in the CS group compared to the NC group, as well as in the CSDEN group compared to the DEN group. Our study suggests that C. sinensis infection increases risk of HCC in a rat model by stimulating proliferation of hepatic progenitor cells.

High-quality reference genome for Clonorchis sinensis.

The Chinese liver fluke, Clonorchis sinensis, causes the disease clonorchiasis, affecting ~35 million people in regions of China, Vietnam, Korea and the Russian Far East. Chronic clonorchiasis causes cholangitis and can induce a malignant cancer, called cholangiocarcinoma, in the biliary system. Control in endemic regions is challenging, and often relies largely on chemotherapy with one anthelmintic, called praziquantel. Routine treatment carries a significant risk of inducing resistance to this anthelmintic in the fluke, such that the discovery of new interventions is considered important. It is hoped that the use of molecular technologies will assist this endeavour by enabling the identification of drug or vaccine targets involved in crucial biological processes and/or pathways in the parasite. Although draft genomes of C. sinensis have been published, their assemblies are fragmented. In the present study, we tackle this genome fragmentation issue by utilising, in an integrated way, advanced (second- and third-generation) DNA sequencing and informatic approaches to build a high-quality reference genome for C. sinensis, with chromosome-level contiguity and curated gene models. This substantially-enhanced genome provides a resource that could accelerate fundamental and applied molecular investigations of C. sinensis, clonorchiasis and/or cholangiocarcinoma, and assist in the discovery of new interventions against what is a highly significant, but neglected disease-complex.

Infection Characteristics of Clonorchis sinensis Metacercariae in Fish from Republic of Korea.

The infection nature of Clonorchis sinensis metacercariae (CsMc) in freshwater fish hosts is closely related to the transmission of human clonorchiasis. This article reviewed the infection characteristics of CsMc in freshwater fish in the Republic of Korea (Korea). The status of CsMc infection was examined in a total of 17,792 cyprinid fish of 49 species in 9 water systems, which included Hantan-/Imjin-gang, Han-gang, Geum-gang, Mangyeong-gang, Yeongsan-gang, Tamjin-gang, Seomjin-gang, Nakdong-gang, and streams in the east coastal areas from 2010 to 2020. The infection status of CsMc was examined according to cyprinid fish species and water systems, after which analyzed by endemicity and susceptibility index. The high endemicity was shown in the cyprinid fish from 3 regions (6.1%) in the upper reaches of Nakdong-gang, such as Banbyeon-cheon (stream), Yongjeon-cheon, and Wi-cheon. The moderate levels were observed in fishes from 8 regions (16.3%), and low endemicity was shown in fishes from 20 regions (40.8%). No CsMc were detected in fish from 18 regions (36.7%). The susceptibility of CsMc in index fish, Puntungia herzi, was found to be a reliable index without examination of other fish species. CsMc infection rates were closely related to subfamily groups in the cyprinid fish hosts in a highly endemic area. In Korea, a total of 58 fish species in 10 families has been listed as the second intermediate hosts for C. sinensis. This review provides several novel features of CsMc infection and clarifies the species of second intermediate freshwater fish host in Korea.

Relationship between Clonorchis sinensis Infection and Cholangiocarcinoma in Korea.

This study provides an overview of the current status of clonorchiasis and cholangiocarcinoma (CCA), and their relationship in Korea during 2012-2020. Data were obtained from the Health Insurance Review & Assessment Service of Korea. Cluster, trend, and correlation analyses were performed. Gyeongsangnam-do and Seoul had the highest average number of cases (1,026 and 4,208) and adjusted rate (306 and 424) for clonorchiasis and CCA, respectively. The most likely clusters (MLC) for clonorchiasis and CCA were Busan/Gyeongsangnam-do/Ulsan/Daegu/Gyeongsangbuk-do (Relative Risk; RR = 4.55, Likelihood Ratio; LLR = 9,131.115) joint cluster and Seoul (RR = 2.29, LLR = 7,602.472), respectively. The MLC for clonorchiasis was in the southeastern part of Korea, while that for CCA was in the southern part. Clonorchiasis showed a decreasing trend in the southeastern districts, while increased in the southwestern districts. Cities in the central region had a decreasing trend, while the western districts had an increasing trend. In most adults (30-59), infection rate of clonorchiasis showed a significant decrease until 2018, while thereafter increased, although not significant. CCA showed a sharply decreasing tendency. The incidence of clonorchiasis and CCA were positively correlated. In general, the correlation was weak (r = 0.39, P < 0.001), but it was strongly positive around the 4 river basins (r = 0.74, P < 0.001). This study might provide an analytic basis for developing an effective system against clonorchiasis and CCA.

Status of Helminthic Infections in Residents around River Basins in the Republic of Korea for 10 Years (2011-2020).

The positive rate of Clonorchis sinensis is the highest among intestinal parasites in the Republic of Korea (Korea). More than 1.2 million people were at risk of C. sinensis infection in Korea in 2012. An intensive control program is being implemented for residents of the 5 major river basins to reduce helminthic infections, including C. sinensis infection. This study evaluated the continuous intensive control program for parasitic diseases including clonorchiasis in areas near the 5 major river basins in Korea over the past 10 years (2011-2020). A total of 335,020 fecal samples (one sample per resident) prepared by the modified sedimentation technic were microscopically examined. Those who expelled helminth eggs were treated with anthelmintics through local health centers and re-examined 3 months later. The overall positive rate of helminths egg was 7.1%. The annual positive rates were dramatically decreased from 14.4% (2011) to 5.9% (2020). The egg positive rate was highest in C. sinensis (5.3%), followed by heterophyid flukes (1.5%) and Trichuris trichiura (0.2%). The prevalence of C. sinensis was significantly higher in males (7.6%) than in females (3.7%), and the highest in the 50-59 years (7.0%) age group. Our results are beneficial to establish prevention and control policies against helminthiases including clonorchiasis in endemic areas in this country.

Clonorchis sinensis excretory-secretory products increase malignant characteristics of cholangiocarcinoma cells in three-dimensional co-culture with biliary ductal plates.

Clonorchis sinensis is a carcinogenic human liver fluke, prolonged infection which provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma (CCA). These effects are driven by direct physical damage caused by the worms, as well as chemical irritation from their excretory-secretory products (ESPs) in the bile duct and surrounding liver tissues. We investigated the C. sinensis ESP-mediated malignant features of CCA cells (HuCCT1) in a three-dimensional microfluidic culture model that mimics an in vitro tumor microenvironment. This system consisted of a type I collagen extracellular matrix, applied ESPs, GFP-labeled HuCCT1 cells and quiescent biliary ductal plates formed by normal cholangiocytes (H69 cells). HuCCT1 cells were attracted by a gradient of ESPs in a concentration-dependent manner and migrated in the direction of the ESPs. Meanwhile, single cell invasion by HuCCT1 cells increased independently of the direction of the ESP gradient. ESP treatment resulted in elevated secretion of interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-ß1) by H69 cells and a cadherin switch (decrease in E-cadherin/increase in N-cadherin expression) in HuCCT1 cells, indicating an increase in epithelial-mesenchymal transition-like changes by HuCCT1 cells. Our findings suggest that C. sinensis ESPs promote the progression of CCA in a tumor microenvironment via the interaction between normal cholangiocytes and CCA cells. These observations broaden our understanding of the progression of CCA caused by liver fluke infection and suggest a new approach for the development of chemotherapeutic for this infectious cancer.

DX5+ NKT cells' increase was correlated with liver damage in FVB mice not in BALB/c mice infected by Clonorchis sinensis.

AIMS: DX5+ NKT cells' distribution and population change in BALB/c and FVB mice infected by C sinensis and their function in liver damage were investigated. METHODS AND RESULTS: Mice were infected by Clonorchis sinensis metacercariae, and lymphocytes were isolated from the livers, spleens and peripheral blood. NK, DX5+ NKT, INF-γ+ DX5+ NKT cells and liver fibrosis were analysed. The DX5+ NKT cells displayed the largest amount in normal BALB/c mice liver followed by peripheral blood and spleen. Although the hepatic DX5+ NKT cells of BALB/c mice were more than that of FVB mice, they did not show significant percentage change after C sinensis infection. The hepatic DX5+ NKT cells of FVB mice increased remarkably after infection accompanied with heavier liver injury and fibrosis than the BALB/c mice. And hydroxyproline content was also positively correlated with DX5+ NKT cells only in FVB mice. However, the increase of IFN-γ producing DX5+ NKT cells was lower in FVB mice than in BALB/c mice which showed sharp increase with mild liver damage after infection. The frequencies of anti-fibrotic NK cells were similar in both of the two mouse strains. CONCLUSIONS: C sinensis could induce different DX5+ NKT cells responses in different mouse strains which may play roles in liver injury and fibrosis in FVB mice.

Soil-transmitted helminths, intestinal protozoa and Clonorchis sinensis infections in southeast China.

BACKGROUND: Extensive parasitic diseases epidemiology in Zhejiang province has not been carried out since the second national survey in 2004. Therefore, dynamics in prevalence and infection pattern of the major intestinal parasites should be explored. METHODS: The distribution of three parasites including soil-transmitted helminths (STH), intestinal protozoa and C. sinensis in Zhejiang from 2014 to 2015 were explored. Kato-Katz technique was used for STH and C. sinensis detection, whereas transparent adhesive paper anal swab was used for pinworm detection, and iodine smear was used for protozoa detection. A questionnaire survey on alimentary habits and sanitary behaviors was conducted in half of the studied counties. RESULTS: This study recruited 23,552 participants: 19,935 from rural and 3617 from urban area. Overall prevalence of intestinal helminth infections was 1.80%. In this study, seven helminth species were identified including A. duodenale, N. americanus, Trichuris trichiura, Ascaris lumbricoides, C. sinensis, Fasciolopsis buski and pinworm. The average prevalence of STH infection was 1.71%: 1.94% in rural and 0.44% in urban area. Hookworm was the most prevalent infection at 1.58%: 1.79% in rural and 0.44% in urban area. Prevalence varied considerably in the studied counties. Prevalence was highest in Yongkang county at 10.25%. Only 2.79% of children from rural area were infected with pinworm. A proportion of 0.40% of rural participants were infected with protozoa, whereas Endolimax nana was the most prevalent at 0.23%. C. sinensis showed infection only in one man. Awareness on C. sinensis was 24.47% in rural and 45.96% in urban area, respectively. CONCLUSIONS: Prevalence of STH and protozoa infections declined considerably whereas C. sinensis infections remained few in Zhejiang province compared with the prevalence reported in previous large scale surveys (19.56% for national STH infection in 2004, 18.66% and 4.57% for provincial STH and protozoa infection, respectively in 1999). The findings of this study showed that hookworm, mainly N. americanus remained a parasitic threat to population health, mainly in the central and western Zhejiang. Therefore, more health education regarding fertilization and farming habits is necessary in rural areas. The awareness concerning hookworm infection should be reinforced.

The storage stability of Bacillus subtilis spore displaying cysteine protease of Clonorchis sinensis and its effect on improving the gut microbiota of mice.

Bacillus subtilis (B. subtilis) spore can serve as an ideal vehicle for expressing heterologous antigens, and elicit specific immune responses by oral administration. In previous studies, we successfully constructed the recombinant B. subtilis spores expressing cysteine protease of Clonorchis sinensis (C. sinensis, B.s-CsCP), and confirmed that oral administration of B.s-CsCP could elicit good protective immune responses in mice. In this study, Gram staining was used to observe the morphology of B.s-CsCP in different form, and the storage of liquid spores and lyophilized spores at different temperatures was compared. The mice were orally immunized with three different doses of spores (2×108, 1×109, and 5×109 CFU/day) for three times in total at biweekly interval. Then, antibody levels of mice were measured, the safety of spores was evaluated, and the changes of gut microbiota after oral gavage of spores (1×109 dose) were investigated. Results showed that B. subtilis was a typical Gram-positive bacterium, and its spore had good resistance to chemical dye. Liquid B. subtilis spores resuspended in sterile water could be stored for a long time at 4 °C or below, while lyophilized spores could be well stored even at RT and better at lower temperatures. Oral administration of B. subtilis spores to mice could stimulate both local mucosal and systemic immune responses in a dose-dependent manner without toxic side effects. Besides, beneficial bacteria producing butyrate such as Odoribacter were increased, while potential pathogens such as Escherichia-Shigella were decreased in mice intestine. Therefore, our work further confirmed that B. subtilis spores expressing CsCP could be a promising oral vaccine against C. sinensis with the advantages of stability, safety, easy storage, and promotion of intestinal health.Key Points• Recombinant CsCP B. subtilis spores could be easily preserved in either liquid or freeze-dried state.• Oral immunization of recombinant spores in mice could increase both local and system immune levels in a dose-dependent manner.• Oral administration of recombinant spores increased the number of beneficial bacteria and reduced the number of harmful bacteria in the intestinal tract of mice.

Prospects for liver fluke vaccines.

Fasciola spp., Opisthorchis spp. and Clonorchis sinensis are common liver flukes that can cause a variety of diseases, mainly cholangiocarcinoma induced by clonorchiasis and liver damage and associated pathology induced by fascioliasis. Because these trematodes are parasites of humans and domestic animals, they have greatly affected the economy of agricultural industries and public health worldwide. Due to the emergence of drug resistance and the living habits of flukes, among other reasons, a possibility of reinfection remains even when antiparasitic drugs are used. Therefore, developing a safe, efficient and cost-effective vaccine against trematodes is an important goal. Here, we briefly describe the progress in the development of vaccines against liver flukes. Related innovations may provide effective protection against these helminths and the diseases that they cause.

High Endemicity with Clonorchis sinensis Metacercariae in Fish from Yongjeon-cheon (Stream) in Cheongsong-gun, Gyeongsangbuk-do, Korea.

The infection status with Clonorchis sinensis metacercariae (CsMc) was examined in freshwater fishes from Yongjeon-cheon (a branch of Nakdong-gang) located in Cheongsong-gun, Gyeongsangbuk-do, the Republic of Korea (Korea). A total of 750 fishes in 19 species were examined by the artificial digestion method for 2 years (2019 and 2020). CsMc were detected in 378 (51.4%) out of 735 fishes in 14 species (73.7%), and the infection intensity was 666 per fish infected. In 2019, CsMc were found in 172 (68.0%) out of 253 fishes in 10 species, and the infection intensity was 565 per fish infected. In 2020, CsMc were detected in 206 (62.2%) out of 331 fishes in 10 species, and the infection intensity was 751 per fish infected. The other zoonotic trematode, ie. Metagonimus spp., Centrocestus armatus, Echinostoma spp. and Clinostomum complanatum, metacercariae were also detected in fishes from the survey streams, but their endemicities were relatively low. Conclusively, it was first confirmed that CsMc are highly endemic in fishes from Yongjeon-cheon in Cheongsong-gun, Gyeongsangbuk-do, Korea.