Spatio-temporal disparities of Clonorchis sinensis infection in animal hosts in China: a systematic review and meta-analysis.

BACKGROUND: Clonorchis sinensis, one of the most important food-borne zoonotic trematodes, remains prevalent in China. Understanding its infection status in animals is crucial for controlling human clonorchiasis. Here we conducted a systematic review and meta-analysis to focus on the spatio-temporal disparities of C. sinensis infection in animals in China. METHODS: Data on C. sinensis prevalence in snails, the second intermediate hosts, or animal reservoirs in China were extracted from electronic databases including PubMed, Embase, Web of Science, Chinese Wanfang database, CNKI, VIP, and China Biomedical Literature database. A random-effects meta-analysis model was utilized to estimate the pooled prevalence in each of the above animal hosts. Subgroup analysis and multivariable meta-regression were performed to explore potential sources of heterogeneity across studies and compare the temporal disparity of infection rates between high and low epidemic areas. Scatter plots were used to depict the biogeographical characteristics of regions reporting C. sinensis infection in animals. RESULTS: The overall pooled prevalence of C. sinensis was 0.9% (95% CI: 0.6-1.2%) in snails, 14.2% (12.7-15.7%) in the second intermediate host, and 14.3% (11.4-17.6%) in animal reservoirs. Prevalence in low epidemic areas (with human prevalence < 1%) decreased from 0.6% (0.2-1.2%) before 1990 to 0.0% (0.0-3.6%) after 2010 in snails (P = 0.0499), from 20.3% (15.6-25.3%) to 8.8% (5.6-12.6%) in the second intermediate hosts (P = 0.0002), and from 18.3% (12.7-24.7%) to 4.7% (1.0-10.4%) in animal reservoirs. However, no similar decrease in prevalence was observed in high epidemic areas (with human prevalence ≥ 1.0%). C. sinensis infections were predominantly reported in areas with altitudes below 2346 m and annual cumulative precipitation above 345 mm and were mostly concentrated in eastern China. CONCLUSIONS: There are spatio-temporal disparities in the animal infections of C. sinensis in different areas of China. Animal infections are primarily concentrated in regions with low altitude and high precipitation. The results suggest that implementing One Health-based comprehensive measures targeting both humans and animals, especially in high epidemic areas, is essential for successful eradication of C. sinensis in China.

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.

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.

Interspecies Phylogenetic Analysis of Clonorchis sinensis in High-incidence Areas of Hunan Province, China.

OBJECTIVE: This study aims to investigate the infection of Clonorchis sinensis ( C. sinensis) in high-incidence areas of Hunan Province, China. The phylogenetic analysis of the C. sinensis species in the highly infected areas was carried out. METHOD: Infection of the definitive human host and intermediate fish host by C. sinensis was investigated, and the mitochondrial genes cox1 and Nad1were used as genetic markers for phylogenetic analysis. RESULTS: In 2016-2020, the average population infection rate of Hunan was 1.38%, while in Tongdao County the rate was up to 26.90%, and the highest fish infection rate was detected in Qiyang County (99.44% in the dorsal fin of crucian carp). High genetic sequence similarity was observed in the samples from Qiyang and Lengshuitan which exhibited high homology with those from Guangdong and Gansu, whereas the parasitic species from Tongdao was highly homologous with those located in high-latitude areas. Moreover, no significant difference was found in the gene sequence of the parasitic species in definitive hosts dogs and cats. CONCLUSION: The systematically study of C. sinensis infection in the high-incidence areas will contribute greatly to the prevention and effectively controlling the spread of Clonorchis sinensis in Hunan Province The endemic of C. sinensis infection in Hunan Province is the result of co-action of local and foreign parasite species.

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.

Are Melanoides tuberculata and Tarebia granifera (Gastropoda, Thiaridae), suitable first intermediate hosts of Clonorchis sinensis in Vietnam?

BACKGROUND: Two thiarid snail species, Melanoides tuberculata and Tarebia granifera have been reported as first intermediate hosts of the liver fluke Clonorchis sinensis; however, their role as true first intermediate hosts has not been verified. Thus, the present study aimed to clarify the suitability of these two snail species as first intermediate hosts of C. sinensis. This was accomplished by collecting snails from a highly endemic area for C. sinensis in Vietnam, the Thac Ba reservoir, and identifying shed cercariae using molecular techniques. We also conducted experimental infections of five snail species including M. tuberculata and T. granifera with eggs of C. sinensis. METHODOLOGY/PRINCIPAL FINDINGS: A total of 11,985 snails, representing 10 species were sampled. Five snail species, M. tuberculata, T. granifera, Lymnaea swinhoei, Parafossarulus manchouricus, and Bithynia fuchsiana were found shedding cercariae with an overall prevalence of infection ranging from 0.7% to 11.5%. Seven cercarial types were recorded. Cercariae of C. sinensis were only found in Parafossarulus manchouricus. Using a multiplex PCR approach for detecting C. sinensis infection, the prevalence in P. manchouricus was 4.2%. Additionally, all five snail species were experimentally exposed to C. sinensis eggs, however only P. manchouricus was successfully infected with an infection rate of 7.87%. CONCLUSIONS/SIGNIFICANCE: We confirmed that in the Thac Ba reservoir, Vietnam, the two thiarids, M. tuberculata and T. granifera are not suitable first intermediate hosts of C. sinensis. Only P. manchouricus was found infected by C. sinensis in nature, and was the only species that became infected experimentally.

[Prevalence of Clonorchis sinensis infections in freshwater fish in mainland China: A meta-analysis].

OBJECTIVE: To understand the real prevalence of Clonorchis sinensis infections in the freshwater fish in mainland China, so as to provide insights into clonorchiasis control and detection of freshwater fish. METHODS: All literatures reporting the prevalence of C. sinensis infections in the freshwater fish, the second intermediate host of the parasite, were jointly retrieved in Chinese and English electronic databases from January 1, 2010 to December 31, 2020, including Wanfang Data, CNKI, PubMed, Web of Science, Embase and Cochrane Library. All studies were screened based on inclusion and exclusion criteria, and the quality of all enrolled literatures was evaluated. The pooled prevalence of C. sinensis infections in freshwater fish and its 95% confidence interval (CI) were estimated using the software Stata version 15.0, and subgroup analyses were performed to investigate the region-, season- and sample source-specific pooled prevalence of C. sinensis infections in freshwater fish. In addition, the sensitivity and publication bias of all included studies were analyzed. RESULTS: A total of 40 eligible literatures were included in this study, including 37 Chinese literatures and 3 English literatures, and there were 10 high-quality literatures, 27 moderate-quality literatures and 3 low-quality literatures. A total of 53 species containing 37 959 freshwater fish were reported in these 40 studies, and 73.58% (39/53) of freshwater fish species were identified with C. sinensis infections. Meta-analysis showed 23.5% [95% CI: (0.19, 0.28)] pooled prevalence of C. sinensis infections in freshwater fish in mainland China, and subgroup analyses higher prevalence of C. sinensis infections in freshwater fish in northeastern China [35.7%, 95% CI: (0.22, 0.50)] than in central [25.9%, 95% CI: (0.04, 0.48)] and southern China [20.6%, 95% CI: (0.09, 0.32)], higher prevalence of C. sinensis infections in freshwater fish sampled in spring [44.1%, 95% CI: (0.35, 0.53)] than in autumn [6.7%, 95% CI: (0.05, 0.08)] and summer [3.3%, 95% CI: (-0.01, 0.07)], and higher prevalence of C. sinensis infections in freshwater fish sampled from natural water [25.2%, 95% CI: (0.17, 0.33)] than from retail trades [22.2%, 95% CI: (0.17, 0.28)] and breeding chain [12.3%, 95% CI: (0.03, 0.22)]. However, all included studies had a publication bias with a low sensitivity. CONCLUSIONS: The prevalence of C. sinensis infections is high in freshwater fish in mainland China, and there are still challenges for clonorchiasis control. Reinforcement of health education, diagnostics development and food safety supervision is recommended in future clonorchiasis control programs.

Interspecies Phylogenetic Analysis of / 生物医学与环境科学（英文）

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Prevalence of Clonorchis sinensis infection in fish in South-East Asia: A systematic review and meta-analysis.

Clonorchis sinensis, an important fish-borne zoonotic trematode, is widely distributed in South-East Asia, especially in China. Infections from human and animal reservoir hosts occur due to the consumption of raw or undercooked fish with C. sinensis metacercariae. This study aimed to evaluate the prevalence of C. sinensis metacercariae in fish in South-East Asia via systematic review and meta-analysis. We searched PubMed, ScienceDirect, China National Knowledge Infrastructure, Wanfang and Chongqing VIP databases for studies published between 1976 and 2020 that are related to the prevalence of C. sinensis metacercariae in fish. Studies were screened with keywords based on inclusion and exclusion criteria. Seventy-one eligible articles were identified, covering three countries: China, Korea and Vietnam. The pooled prevalence of C. sinensis metacercariae in fish from South-East Asia was 30.5%, with 35.1% in China, 29.7% in Korea and 8.4% in Vietnam. In subgroup analyses of climate, season, water source and publication date, the highest prevalence was identified in the Dwb climate type (43.3%), summer (70.2%), river (34.5%) and pre-2001 publications (38.9%), respectively. In comparison, the lowest prevalence was found in the Dfa climate type (14.5%), winter (19.5%), lake (8.0%) and post-2001 publications (23.8%). Meta-regression results indicated that country (p = .009), the published time (p = .035) and water source subgroups (p = .003) may be the source of heterogeneity. Overall, our study indicates that a high prevalence of C. sinensis infections occurs in fish in China, Korea and Vietnam, illuminating a significant public health concern in these countries.

Oral delivery of Bacillus subtilis spores expressing Clonorchis sinensis paramyosin protects grass carp from cercaria infection.

Clonorchis sinensis (C. sinensis), an important fishborne zoonotic parasite threatening public health, is of major socioeconomic importance in epidemic areas. Effective strategies are still urgently expected to prevent against C. sinensis infection. In the present study, paramyosin of C. sinensis (CsPmy) was stably and abundantly expressed on the surface of Bacillus subtilis spores. The recombinant spores (B.s-CotC-CsPmy) were incorporated in the basal pellets diet in three different dosages (1 × 105, 1 × 108, 1 × 1011 CFU/g pellets) and orally administrated to grass carp (Ctenopharyngodon idella). The immune responses and intestinal microbiota in the treated grass carp were investigated. Results showed that specific anti-CsPmy IgM levels in sera, skin mucus, bile, and intestinal mucus, as well as mRNA levels of IgM and IgZ in the spleen and head kidney, were significantly increased in B.s-CotC-CsPmy-1011 group. Besides, transcripts levels of IL-8 and TNF-αin the spleen and head kidney were also significantly elevated than the control groups. Moreover, mRNA levels of tight junction proteins in the intestines of B.s-CotC-CsPmy-1011 group increased. Potential pathogenetic bacteria with lower abundance and higher abundances of candidate probiotics and bacteria associated with digestion in 1 × 1011 CFU/g B.s-CotC-CsPmy spores administrated fishes could be detected compared with control group. The amount of metacercaria in per gram fish flesh was statistically decreased in 1 × 1011 CFU/g B.s-CotC-CsPmy spores orally immunized group. Our work demonstrated that B. subtilis spores presenting CsPmy on the surface could be a promising effective, safe, and needle-free candidate vaccine against C. sinensis infection for grass carp.

[Investigation on Clonorchis sinensis infections in marketed cats in Nanning City].

OBJECTIVE: To understand Clonorchis sinensis infections in cats in Nanning City, so as to provide evidence for the control of the reservoir host of C. sinensis. METHODS: The cat livers were purchased from cat slaughterhouses in Nanning City. The cat gallbladder and liver were dissected, and liver flukes were collected and counted. Then, the worms were subjected to morphological observation, amplification of the ITS2 gene and sequencing. The species of the worms were identified using BLAST. RESULTS: A total of 105 cat livers were collected from two cat slaughterhouses, and 68 were detected with C. sinensis infections, with an infection rate of 64.76%. The highest burden was 980 worms in a single liver, and the mean burden was 72 worms in a liver. There were 3 types of liver flukes with various size and morphology, and all were identified as C. sinensis by means of morphological observation, ITS2 gene amplification, sequencing and sequence alignment. CONCLUSIONS: There is a high infection rate of C. sinensi in marketed cats in Nanning City, and it is therefore suggested that targeted interventions should be intensified for the management of C. sinensis infections in cats.

A Clonorchiasis Case of a Leopard Cat, Prionailurus bengalensis euptilurus, Diagnosed by Ultrasonography and Egg Detection in Republic of Korea.

A clonorchiasis case in a captive leopard cat, Prionailurus bengalensis euptilurus, was confirmed by ultrasonographic findings and egg morphologies found in the bile juice sample in the Korea. The leopard cat was introduced from the wild habitat of Gyeongsangnam-do, to Cheongju Zoo in Cheongju-si, Chungcheongbuk-do, Korea in August 2014. Physical examinations were basically performed for quarantine and check-up health. The cat was comparatively good in health except anorexia. The cyst-like bile duct dilation and the increased echogenicity of gall bladder wall and hepatic parenchyma were observed by ultrasonography. Ultrasound-guided needle biopsy was conducted for collecting bile juice and the specimens were observed under light microscope. The numerous small trematode eggs were detected in the bile juice sample of the light microscopy. The eggs were 25-33 (28±3) µm by 18-22 (20±1) µm in size and showed typical characteristics of Clonorchis sinensis egg, i.e., a dominantly developed operculum, shoulder rim and dust-like wrinkles in surface. To treat the liver fluke infection, 20 mg/kg of praziquantel was orally administered only once to the case. Follow-up studies including fecal examinations were conducted during 2 years after treatment. But no more eggs were detected from the case. In the present study, we described the first clonorchiasis case of leopard cat, which was confirmed by ultrasonographic findings and egg morphologies from the bile juice sample in Korea.

Infection Status with Clonorchis sinensis Metacercariae in Fish from Yangcheon (Stream) in Sancheong-gun, Gyeongsangnam-do, Korea.

The infection status with Clonorchis sinensis metacercariae (CsMc) was examined in freshwater fishes from Yangcheon (a branch of Gyeongho-gang), which is located in Sancheong-gun, Gyeongsangnam-do, the Republic of Korea. Total 2,201 fishes in 26 species were examined by the artificial digestion method through 7 years. CsMc were detected in 1,171 (53.2%) fishes in 21 spp. (80.8%) and their density was 85 per fish infected. Total 532 (99.6%) out of 534 Pungtungia herzi (index fish) examined were infected with 147 CsMc per fish infected. Metacercarial densities in this fish were highest in 2015 (179 CsMc), followed by 2012 (168), 2013 (152), 2016 (145), 2014 (114), and 2017 (89) respectively. In the gobioninid fish group, i.e., P. herzi, Sarcocheilichthys spp., Squalidus spp., Pseudogobio esocinus, Hemibarbus longirostris, and Hemibarbus labeo, 841 (92.7%) fishes were infected with 117 CsMc per fish infected. Total 250 (54.7%) acheilognathinid fish (bitterlings), Acheilognathus spp. and Acanthorhodeus spp. were infected with 5.8 CsMc. In the rasborinid fish (chubs) group, i.e., Zacco platypus, Zacco temminckii, and Zacco koreanus, 77 (13.7%) out of 563 fish examined were infected with 2.4 CsMc in average. The susceptibility indices of CsMc were 49.09 in the overall positive fish group, 104.15 in the gobioninid group, 3.17 in the acheilognathinid group and 0.35 in the rasborinid fish group respectively. Only 1 CsMc was detected in 3 fish species, Coreoperca herzi, Channa argus, and Lepomis macrochirus, respectively. Conclusively, it was confirmed that CsMc are moderately prevalent in fishes from Yangcheon in Sancheon-gun, Gyeongsangnam-do, Korea.

Modeling the Transmission Dynamics of Clonorchiasis in Foshan, China.

Clonorchiasis, known as the Chinese liver fluke disease, is caused by Clonorchis sinensis infection with food-borne liver fluke, which is transmitted via snails to freshwater fish and then to human beings or other piscivorous mammals. Clonorchis sinensis infection is mainly related to liver and biliary disorders, especially cholangiocarcinoma, and has an increased human-health impact due to the greater consumption of raw freshwater fish. In this article, we propose a deterministic model to describe the spread of clonorchiasis among human-snail-fish populations and use the model to simulate the data on the numbers of inspected and infected individuals of Foshan City, located in Guangdong Province in the southeast of P.R China, from 1980-2010. Mathematical and numerical analyses of the model are carried out to understand the transmission dynamics of clonorchiasis and explore effective control measures for the local outbreaks of the disease. We find that (i) the transmission of clonorchiasis from cercariae to fish plays a more important role than that from eggs to snails and from fish to humans; (ii) As the cycle of infection-treatment-reinfection continues, it is unlikely that treatment with drugs alone can control and eventually eradicate clonorchiasis. These strongly suggest that a more comprehensive approach needs to include environmental modification in order to break the cercariae-fish transmission cycle, to enhance awareness about the disease, and to improve prevention measures.

A duplex PCR for the simultaneous detection of Fasciola hepatica and Clonorchis sinensis.

Both Fasciola hepatica and Clonorchis sinensis are endemic in China, South Korea, Japan and other Southeast Asian countries. Reliable and sensitive diagnostic methods are needed for detecting their infections in humans and animals. Differential simplex and duplex polymerase chain reaction (PCR) methods were developed. The PCRs targeted the second internal transcribed spacer (its2) (408 bp) of F. hepatica, and NADH dehydrogenase subunit 2 gene (nad2) (527 bp) of C. sinensis. Both simplex PCRs detected as little as 2 pg genomic DNA in one microliter in a 25 µL PCR reaction system. The duplex PCR had similar detection limit as well, and detected as low as one egg in 200 mg feces. These methods were analytical specific with no amplification being observed from the gemonic DNA of Fasciolopsis buski, Haemonchus contortus, Ascaris ovis or Eimeri ahsata. Of 158 sheep fecal samples collected from various farms, four and one samples were PCR-positive for F. hepatica and C. sinensis, respectively. The duplex PCR method described here is time-saving and convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of F. hepatica and C. sinensis in endemic area.

Infections by Hepatic Trematodes in Cats from Slaughterhouses in Vietnam.

Domestic cats are a final host for some hepatic trematodes, and cat meat is used for human consumption in several parts of northern Vietnam. Since there is no tradition for commercial cat rearing in the area, cats are purchased alive from other areas as well, i.e., southern Vietnam and neighboring countries, and brought to slaughterhouses. This uncontrolled trade in live cats could pose a risk for spread of various parasites, including hepatic trematodes. Hence, in this study, we investigated the infections by hepatic trematodes in cats from the slaughterhouses. Cat livers were sampled from 12 slaughterhouses. Clonorchis sinensis and Platynosomum fastosum were found in 14 of 78 necropsied cats of local origin, while more than half of the non-local cats (170 of 318) were infected by C. sinensis, P. fastosum, or Opisthorchis viverrini. The non-local cats had higher prevalence (odds ratio = 6.61, P < 0.01, 95% CI: 2.34-19.41) and intensity of infection (count ratio = 6.47, P < 0.01, 95% CI: 1.77-23.59) by C. sinensis than local cats. Prevalence of P. fastosum infection did not differ significantly between the 2 groups of cats. Opisthorchis viverrini was found at low prevalence (2.5%) in non-local cats. The presence of O. viverrini in cats sampled in northern Vietnam and the high prevalence and intensity of infection in cats of non-local origin suggest that cats are transported over great distances, and this poses a risk of spreading these trematodes.

Oral delivery of Bacillus subtilis spores expressing cysteine protease of Clonorchis sinensis to grass carp (Ctenopharyngodon idellus): Induces immune responses and has no damage on liver and intestine function.

Clonorchis sinensis (C. sinensis) is a fish-borne trematode. Human can be infected by ingestion of C. sinensis metacercariae parasitized in grass carp (Ctenopharyngodon idella). For induction of effective oral immune responses, spores of Bacillus subtilis (B. subtilis) WB600 were utilized as vehicle to delivery CsCP (cysteine protease of C. sinensis) cooperated with CotC (B.s-CotC-CP), one of coat proteins, to the gastrointestinal tract. After routine culture of 8-12 h in LB medium, B. subtilis containing CotC-CsCP was transferred into the sporulation culture medium. SDS-PAGE, western blotting and the growth curve indicated that the best sporulation time of recombinant WB600 was 24-30 h at 37 °C with continuous shaking (250 rpm). Grass carp were fed with three levels of B.s-CotC-CP (1 × 106, 1 × 107, and 1 × 108 CFU g-1) incorporated in the basal pellets diet. The commercial pellets or supplemented with spores just expressing CotC (1 × 107 CFU g-1) were served as control diet. Our results showed that grass carp orally immunized with the feed-based B.s-CotC-CP developed a strong specific immune response with significantly (P < 0.05) higher levels of IgM in samples of serum, bile, mucus of surface and intestinal compared to the control groups. Abundant colonization spores expressing CsCP were found in hindgut that is conducive to absorption and presentation of antigen. Moreover, B. subtilis spores appeared to show no sign of toxicity or damage in grass carp. Our cercariae challenge experiments suggested that oral administration of spores expressing CsCP could develop an effective protection against C. sinensis in fish body. Therefore, this study demonstrated that the feed-based recombinant spores could trigger high levels of mucosal and humoral immunity, and would be a promising candidate vaccine against C. sinensis metacercariae formation in freshwater fish.

Immune response induced by oral delivery of Bacillus subtilis spores expressing enolase of Clonorchis sinensis in grass carps (Ctenopharyngodon idellus).

Clonorchiasis, caused by the consumption of raw or undercooked freshwater fish containing infective metacercariae of Clonorchis sinensisis (C.sinensis), remains a common public health problem. New effective prevention strategies are still urgent to control this food-borne infectious disease. The previous studies suggested Bacillus subtilis (B. subtilis) spores was an ideal vaccines delivery system, and the C.sinensis enolase (CsENO) was a potential vaccine candidate against clonorchiasis. In the current study, we detected CsENO-specific IgM levels by ELISA in sera, intestinal mucus and skin mucus in grass carps (Ctenopharyngodon idella) through oral administration with B. subtilis spores surface expressing CsENO. In addition, immune-related genes expression was also measured by qRT-PCR. Grass carps orally treated with B. subtilis spores or normal forages were used as controls. The results of ELISA manifested that specific IgM levels of grass carps in CsENO group in sera, intestine mucus and skin mucus almost significantly increased from week 4 post the first oral administration when compared to the two control groups. The levels of specific IgM reached its peak in intestine mucus firstly, then in sera, and last in skin mucus. qRT-PCR results showed that 5 immune-related genes expression had different degree of rising trend in CsENO group when compared to the two control groups. Our study demonstrated that orally administrated with B. subtilis spores expressing CsENO induced innate and adaptive immunity, systemic and local mucosal immunity, and humoral and cellular immunity. Our work may pave the way to clarify the exact mechanisms of protective efficacy elicited by B. subtilis spores expressing CsENO and provide new ideas for vaccine development against C. sinensis infection.

The importance of wild fish in the epidemiology of Clonorchis sinensis in Vietnam.

Preliminary findings of a high prevalence of Clonorchis sinensis in wild-caught fish in a North Vietnam reservoir (Thac Ba reservoir, Yen Bai Province) prompted a longitudinal epidemiological study of fish infections. Monthly collections of fish from September 2014 to August 2015 were processed for recovery of metacercariae; 1219 fish, representing 22 species, were examined. Seven species were infected with C. sinensis metacercariae. Four species, Toxabramis houdemeri, Hemiculter leucisculus, Cultrichthys erythropterus, and Culter recurvirostris, had high prevalence (31.1 to 76.7 %); metacercarial intensities ranged from 3.9 to 65.7 metacercariae/fish. A seasonal variation of C. sinensis prevalence was observed in T. houdemeri. Variation in intensity of infection occurred in C. erythropterus and H. leucisculus. Intensity and prevalence of C. sinensis in the most highly infected species, T. houdemeri, varied by fish size; prevalence was higher in fish weighing more than 3 g, and intensity was higher in fish weighing more than 5 g. The distribution of metacercariae in the body region of T. houdemeri was significantly higher in the caudal fin (14.7 metacercariae/g), compared to the body and head regions (0.7 and 1.4 metacercariae/g, respectively). Further epidemiological investigations on C. sinensis in this reservoir region should include assessing the relative risk of the different fish species for humans based on the latter's food preferences, and the prevalence of C. sinensis in the community. The snail intermediate host(s) in the reservoir should also be identified along with the ecological factors influencing its exposure to C. sinensis eggs and its subsequent transmission of cercariae to fish. Also needed are investigations on the relative importance of wild and domestic reservoir hosts as sources of egg contamination of the reservoir.

Hemozoin is a product of heme detoxification in the gut of the most medically important species of the family Opisthorchiidae.

Many species of trematodes such as Schistosoma spp., Fasciola hepatica and Echinostoma trivolvis are blood-feeding parasites. Nevertheless, there is no consensus on the feeding habits of the family Opisthorchiidae (Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis). Previously, histological studies of O. felineus and C. sinensis revealed some dark stained material in their gut lumen. In this study we conducted a comprehensive analysis of the gut contents of three members of the family Opisthorchiidae (O. felineus, O. viverrini and C. sinensis). Using transmission electron microscopy, we demonstrated for the first known time the presence of disintegrating blood cells in the gut of O. felineus as well as electron-dense crystals in the gut of O. felineus and C. sinensis. Electron energy loss spectroscopy revealed iron atoms in these crystals, and mass spectrometry of the purified pigment demonstrated the presence of heme. Fourier-transform infrared spectroscopy identified the signature peaks of the common iron-carboxylate bond characteristic in crystals isolated from O. felineus and C. sinensis. Scanning electron microscopy showed layered ovoid crystals of various sizes from 50 nm to 2 µm. Morphological, chemical and paramagnetic properties of these crystals were similar to those of hemozoin from Schistosoma mansoni. Crystal formation occurs on the surface of lipid droplets in O. felineus and C. sinensis guts. Our results suggest that the diet of O. felineus and C. sinensis includes blood. Detoxification of the free heme produced during the digestion proceeds via formation of insoluble crystals that contain iron and heme dimers, i.e. crystals of hemozoin. Furthermore, we believe that biocrystallisation of hemozoin takes place on the surface of the lipid droplets, similar to S. mansoni. Hemozoin was not detected in the closely related species O. viverrini.