An investigation of the prevalence and diversity of Anisakis in China: marine food safety implications.

Anisakis can cause Anisakiasis in humans if raw or undercooked fish is consumed. Symptoms of infection may include vomiting, acute abdominal symptoms, or allergies. In this study, we collected 187 commercially available marine fish from the Yellow Sea, East China Sea, and South China Sea. Among them, 79 were found positive containing 520 Anisakis worms. The average prevalence rate was found 42% in this investigation. Ninety-two worms from different sea areas were selected and analyzed for identification, revealing the presence of five different species, which are Anisakis pegreffii, Hysterothylacium aduncum, Hysterothylacium zhoushanense, Hysterothylacium amoyense, and Hysterothylacium sp. In the meta-analysis, three databases: PubMed, CNKI, and BaiduXueshu were searched for surveys on the prevalence of Anisakis in Chinese waters from January 2000 to December 2023. A total of 26 studies were included in this analysis of which 25 publications were retrieved from different databases and one being the present study. The pooled prevalence of Anisakis was 45% among commercially available marine fish. Variances in the prevalence of Anisakis were noted among the four seas, with the highest rates in the East China Sea and the Bohai Sea, reaching 53% [0.38; 0.68] and 49% [0.36; 0.62], respectively. The Prevalence of Anisakis infection was significantly higher in astern parts such as Liaoning, Shanghai, and Zhejiang. Analysis of the host fish subgroups revealed that the orders of Anguilliformes, Scombriformes, and Gadiformes had high rates of infection. These findings suggest a significant prevalence of Anisakis, posing an increasing risk of infection for individuals. This study provides impactful information for implementing preventative measures against Anisakis.

Characterization of the complete mitochondrial genomes of Diplodiscus japonicus and Diplodiscus mehari (Trematoda: Diplodiscidae): Comparison with the members of the superfamily Paramphistomoidea and phylogenetic implication.

Diplodiscus japonicus and Diplodiscus mehari (Trematoda: Diplodiscidae) are two important parasites in wood frogs, which have large infection rates and essential importance of ecology, economy and society. In this study, the complete mitochondrial (mt) genomes of D. japonicus and D. mehari were sequenced, then compared with other related trematodes in the superfamily Paramphistomoidea. The complete circular mt sequence of D. japonicus and D. mehari were 14,210 bp and 14,179 bp in length, respectively. Both mt genomes comprised 36 functional subunits, consisting of 12 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and one non-coding region. The mt genes of D. japonicus and D. mehari were transcribed in the same direction, and the gene arrangements were identical to those of Paramphistomoidea trematodes. In the 12 PCGs, GTG was the most common initiation codon, whereas TAG was the most common termination codon. All tRNAs had a typical cloverleaf structure except tRNA Ser1. A comparison with related Paramphistomoidea trematode mt genomes suggested that the cox1 gene of D. mehari was the longest in these trematodes. Phylogenetic analyses revealed that Paramphistomoidea trematodes formed a monophyletic branch, Paramphistomidae and Gastrothylacidae were more closely related than Diplodiscidae. And the further analysis with Pronocephalata branch found that the flukes parasitic in amphibians (frogs) formed one group, and the flukes from ruminants (cattle, sheep, ect) formed another group. Our study demonstrated the importance of sequencing mt genomes of D. japonicus and D. mehari, which will provide significant molecular resources for further studies of Paramphistomoidea taxonomy, population genetics and systematics.

Multiple biochemical indices and metabolomics of Clonorchis sinensis provide a novel interpretation of biomarkers.

BACKGROUND: Clonorchiasis, an infectious disease caused by the liver fluke Clonorchis sinensis, may lead to the development of liver and gallbladder diseases, and even cholangiocarcinoma (CCA). However, the pathogenesis, host-pathogen interaction, and diagnostic markers for clonorchiasis remain unclear. METHODS: Eighteen rabbits were randomly divided into control group (n = 9) and C. sinensis-infected group (n = 9), and their plasma samples were collected at 7, 14, 28, and 63 days post-infection (dpi). Biochemical indices and metabolites in different infection periods were detected. A non-targeted ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach was employed to investigate the metabolic profiles of plasma in rabbits, and related metabolic pathways of differential metabolites and correlation between candidate biochemical indices and differential metabolites were analyzed. Finally, the candidate biomarkers were verified with human samples using a targeted metabolomics method. RESULTS: The result of biochemical indices indicated C. sinensis infection would affect the liver function biochemical indices, especially alanine aminotransferase, aspartate transaminase (AST), glutamyl transpeptidase (GGT), total bile acid, high-density lipoprotein, and cholinesterase. The metabonomic results showed that 58, 212, 23, and 21 differential metabolites were identified in different phases of the infection. Multivariate statistical analysis of differential metabolites revealed distinct metabolic signatures during different phases of infection, with most of these signatures being observed at 14 dpi, which mainly influences the amino acid metabolisms. For metabolites and biochemical indices, AST, GGT, hypoxanthine, L-pipecolic acid, and D-glucuronate represented potential noninvasive biomarkers for the diagnosis of C. sinensis (P < 0.05 and AUC > 0.8). Furthermore, GGT and D-glucuronate levels were positively correlated with the infection (r(28) = 0.98, P < 0.0001) and showed excellent diagnostic performance (AUC = 0.972; 95% confidence interval, 0.921 to 1.000). CONCLUSIONS: The present results provide new insights into plasma metabolic changes in rabbits during C. sinensis infection, and the potential biomarker may be used for developing an effective method to diagnose clonorchiasis in the future.

The complete mitochondrial genome of Prosthogonimus cuneatus and Prosthogonimus pellucidus (Trematoda: Prosthogonimidae), their features and phylogenetic relationships in the superfamily Microphalloidea.

Prosthogonimus cuneatus and Prosthogonimus pellucidus (Trematoda: Prosthogonimidae) are common flukes of poultry and other birds which can cause severe impacts on animal health and losses to the poultry industry. However, there are limited studies on the molecular epidemiology, population genetics, and systematics of Prosthogonimus species. In the present study, the complete mitochondrial (mt) genomes of P. cuneatus and P. pellucidus were determined to be 14,829 bp and 15,013 bp in length, respectively. Both mt genomes contain 12 protein-coding genes (PCGs) (cox1-3, nad1-6, nad4L, cytb, and atp6), 22 transfer RNA genes, two ribosomal RNA genes, and one non-coding region. Our comparative analysis shows that the atp6 genes of P. cuneatus and P. pellucidus are longer than any previously published atp6 genes of other trematodes. The lengths of the atp6 genes of P. cuneatus and P. pellucidus in this study seem unusual, and should therefore be studied further. The mt genes of P. cuneatus and P. pellucidus are transcribed in the same direction, and the gene arrangements are identical to those of Plagiorchis maculosus, Tamerlania zarudnyi, and Tanaisia sp., but different from those of Eurytrema pancreaticum, Dicrocoelium chinensis, and Brachycladium goliath. The mt genome A + T contents of P. cuneatus and P. pellucidus are 64.47% and 65.34%, respectively. In the 12 PCGs, ATG is the most common initiation codon, whereas TAG is the most common termination codon. The sequence identity of the same 12 PCGs among the eight trematodes (P. cuneatus, P. pellucidus, Pl. maculosus, D. chinensis, E. pancreaticum, B. goliath, T. zarudnyi, Tanaisia sp.) of Xiphidiata are 55.5%-81.7% at the nucleotide level and 43.9%-82.5% at the amino acid level. The nucleotide similarities among the complete mt genomes of the eight trematodes range from 54.1%-81.5%. Phylogenetic analysis based on the aligned concatenated amino acid sequences of the 12 PCGs shows that P. cuneatus and P. pellucidus cluster together and are sister to T. zarudnyi and Tanaisia sp., and this clade is more closely related to E. pancreaticum, Dicrocoelium spp. and Lyperosomum longicauda in the family Dicrocoeliidae, than it is to species in the families Plagiorchiidae and Brachycladiidae. These are the first reported complete mt genomes of Prosthogonimidae, and these data will provide additional molecular resources for further studies of Prosthogonimidae taxonomy, population genetics, and systematics.

Integrative Transcriptomics and Proteomics Analyses to Reveal the Developmental Regulation of Metorchis orientalis: A Neglected Trematode With Potential Carcinogenic Implications.

Metorchis orientalis is a neglected zoonotic parasite of the gallbladder and bile duct of poultry, mammals, and humans. It has been widely reported in Asian, including China, Japanese, and Korea, where it is a potential threat to public health. Despite its significance as an animal and human pathogen, there are few published transcriptomic and proteomics data available. Transcriptome Illumina RNA sequencing and label-free protein quantification were performed to compare the gene and protein expression of adult and metacercariae-stage M. orientalis, resulting in 100,234 unigenes and 3,530 proteins. Of these, 13,823 differentially expressed genes and 1,445 differentially expressed proteins were identified in adult versus metacercariae. In total, 570 genes were differentially expressed consistent with the mRNA and protein level in the adult versus metacercariae stage. Differential gene transcription analyses revealed 34,228 genes to be expressed in both stages, whereas 66,006 genes showed stage-specific expression. Compared with adults, the metacercariae stage was highly transcriptional. GO and KEGG analyses based on transcriptome and proteome revealed numerous up-regulated genes in adult M. orientalis related to microtubule-based processes, microtubule motor activity, and nucleocytoplasmic transport. The up-regulated genes in metacercariae M. orientalis were mainly related to transmembrane receptor protein serine/threonine kinase activity, transmembrane receptor protein serine/threonine kinase signaling pathway. Transcriptome and proteome comparative analyses showed numerous up-regulated genes in adult stage were mainly enriched in actin filament capping, spectrin, and glucose metabolic process, while up-regulated genes in metacercariae stage were mainly related to cilium assembly, cilium movement, and motile cilium. These results highlight changes in protein and gene functions during the development of metacercariae into adults, and provided evidence for the mechanisms involved in morphological and metabolic changes at both the protein and gene levels. Interestingly, many genes had been proved associated with liver fibrosis and carcinogenic factors were identified highly expressed in adult M. orientalis, which suggests that M. orientalis is a neglected trematode with potential carcinogenic implications. These data provide attractive targets for the development of therapeutic or diagnostic interventions for controlling M. orientalis.

Identification of Myoferlin, a Potential Serodiagnostic Antigen of Clonorchiasis, via Immunoproteomic Analysis of Sera From Different Infection Periods and Excretory-Secretory Products of Clonorchis sinensis.

Clonorchiasis, which is caused by Clonorchis sinensis, is an important foodborne disease worldwide. The excretory-secretory products (ESPs) of C. sinensis play important roles in host-parasite interactions by acting as causative agents. In the present study, the ESPs and sera positive for C. sinensis were collected to identify proteins specific to the sera of C. sinensis (i.e., proteins that do not cross-react with Fasciola hepatica and Schistosoma japonicum) at different infection periods. Briefly, white Japanese rabbits were artificially infected with C. sinensis, and their sera were collected at 7 days post-infection (dpi), 14 dpi, 35 dpi, and 77 dpi. To identify the specific proteins in C. sinensis, a co-immunoprecipitation (Co-IP) assay was conducted using shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) to pull down the sera roots of C. sinensis, F. hepatica, and S. japonicum. For the annotated proteins, 32, 18, 39, and 35 proteins specific to C. sinensis were pulled down by the infected sera at 7, 14, 35, and 77 dpi, respectively. Three proteins, Dynein light chain-1, Dynein light chain-2 and Myoferlin were detected in all infection periods. Of these proteins, myoferlin is known to be overexpressed in several human cancers and could be a promising biomarker and therapeutic target for cancer cases. Accordingly, this protein was selected for further studies. To achieve a better expression, myoferlin was truncated into two parts, Myof1 and Myof2 (1,500 bp and 810 bp), based on the antigenic epitopes provided by bioinformatics. The estimated molecular weight of the recombinant proteins was 57.3 ku (Myof1) and 31.3 ku (Myof2). Further, both Myof1 and Myof2 could be probed by the sera from rabbits infected with C. sinensis. No cross-reaction occurred with the positive sera of S. japonica, F. hepatica, and negative controls. Such findings indicate that myoferlin may be an important diagnostic antigen present in the ESPs. Overall, the present study provides new insights into proteomic changes between ESPs and hosts in different infection periods by LC-MS/MS. Moreover, myoferlin, as a biomarker, may be used to develop an objective method for future diagnosis of clonorchiasis.

Characterization of the complete mitochondrial genomes of Coronocyclus labiatus and Cylicodontophorus bicoronatus: Comparison with Strongylidae species and phylogenetic implication.

Coronocyclus labiatus and Cylicodontophorus bicoronatus are two significant horse parasitic nematodes which are classified into subfamily Cyathostominae, family Strongylidae, however, the classification of these nematodes has been controversial for more than a century. Mitochondrial (mt) genomes are considered valuable sources for parasite taxonomy, population genetics, and systematics studies. In the present study, the mt genomes of Co. labiatus and Cd. bicoronatus (type species) were determined and subsequently compared with those from closely related species by phylogenetic analysis based on concatenated datasets of amino acid sequences predicted from mt protein-coding genes. The complete mt genomes of Co. labiatus and Cd. bicoronatus were circular with 13,827 bp and 13,753 bp in size, respectively. Both mt genomes consisted of a total of 12 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and two non-coding regions. All protein coding genes were transcribed in the same direction, and the gene order in both mt genomes belonged to the gene arrangement type 3 (GA3). There were 19 intergenic spacers with 1 bp to 35 bp and one overlap with 4 bp in mt genome of Co. labiatus, and 22 intergenic spacers with 1-29 bp in size but no overlap in the mt genome of Cd. bicoronatus. The A + T content of Co. labiatus and Cd. bicoronatus mt genomes were 75.87 % and 75.16 %, respectively. Similar to mt genones of other Strongylidae species published in GenBank, they also exhibited a strong A + T bias not only in the nucleotide composition but also in codon usage. Comparative analyses of mt genomes nucleotide sequence showed that mt genomes of Co. labiatus and Cd. bicoronatus had higher identities to that of Cylicostephanus goldi (90.3 % and 86.9 %, respectively), followed by those of two Cyathostomum species (89.9∼90.0 %; 86.4 %), respectively. Phylogenetic analyses using mt genomes of 26 Strongyloidea nematodes revealed that Co. labiatus was closely related to Cyathostomum species, and Cd. bicoronatus formed a distinct branch with Cyathostominae species, which was closer to Triodontophorus than Poteriostomum imparidentatum. We concluded Coronocyclus might be closely related with Cyathostomum but represent a distinct genus based on comparative mt genome sequences and phylogenetic analyses. The availability of complete mt genome sequences of Co. labiatus and Cd. bicoronatus provides new and useful genetic markers for further studies on Strongylidae nematodes.

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.

Comparative analysis of mitochondrial DNA datasets indicates that Cylicostephanus minutus represents a species complex.

Cyathostomins are one kind of the most important parasites in equids. Cylicostephanus minutus is a member of the subfamily Cyathostominae. In the present study, we determined the complete mitochondrial (mt) genomes from four Cs. minutus isolates and reconstructed the phylogenetic relationship of Strongylidae to test the hypothesis that Cs. minutus represents a species complex. The complete mt genome sequences of Cs. minutus were 13,772-13,822 bp in length, and contained 36 genes (12 protein coding genes, 22 tRNA genes, two rRNA genes), and two non-coding regions (NCRs). The intraspecific identity of nucleotide sequences and amino acid sequences in Cs. minutus (1-4) were 89.3-97.9% and 97.0-98.8%, respectively. Two operational taxonomic units (OTUs) were determined based on the mt genome sequences, OTU 2 (Csm 1 and Csm 2) and OTU 3 (Csm 3 and Csm 4). Sequence analysis showed the divergence between OTU 2 and OTU 3 was 8.9-10.7%. Pairwise comparisons of 12 protein coding genes between OTU 2 and OTU 3 showed a difference of 3.0-13.3% at the nucleotide level and 0-6.7% at the amino acid level. Phylogenetic analysis showed the separation of Cs. minutus isolates from the same host into different distinct clades based on mt genomes. Comparisons of partial mt cox1, nad5, and cytb and ITS2 sequences from 20 Cs. minutus isolates from the same host and the same geographical location with other Cs. minutus sequences available in GenBank revealed significant nucleotide differences. Phylogenetic analysis showed a separation of Cs. minutus into three distinct clades. Thus, the comparative and phylogenetic analyses of mtDNA datasets indicated that Cs. minutus represents a complex of at least three species. Our results have further confirmed the existence of a cryptic Cs. minutus species, and provides a reference for the taxonomical, population genetics, and systematics studies of other cyathostomin species.

Comparative analyses of complete ribosomal DNA sequences of Clonorchis sinensis and Metorchis orientalis: IGS sequences may provide a novel genetic marker for intraspecific variation.

Both Clonorchis sinensis and Metorchis orientalis are the fish-borne zoonotic trematodes, and have a wide distribution of southeastern Asia, especially in China. Due to the similar morphology, life cycle, and parasitic positions are difficult to differentiate between both metacercariae. In the present study, the complete rDNA sequences of five C. sinensis and five M. orientalis were obtained and compared for the first time. And the IGS rDNA sequences were tested as a genetic marker. The results showed complete rDNA lengths of C. sinensis were range from 8049 bp to 8391 bp, including 1991 bp, 1116 bp, 3854 bp, and 1088-1430 bp belonging to 18S, ITS, 28S and IGS, respectively. And the complete rDNA lengths of M. orientalis were range from 7881 bp to 9355 bp, including 1991 bp, 1077 bp, 3856 bp, and 957-2431 bp belonging to 18S, ITS, 28S and IGS, respectively. Comparative analyses reveal length difference main in IGS, which has higher intraspecific and interspecific variations than other ribosomal regions. Forty four repeat (forward and inverted) sequences were found in the complete rDNAs of C. sinensis and M. orientalis. The phylogenetic analyses showed that the sequences of ITS1, ITS2, 18S and 28S could be used as different level genetic markers. In IGS phylogenetic tree, Opisthorchiidae, Paramphistomidae, Dicrocoeliidae, and Schistosomatidae formed monophyletic groups, and the same length sequences were clustered together in the same species. These findings of the present study provide the new molecular data for studying the complete rDNA of C. sinensis and M. orientalis, and indicate IGS sequences may used as a novel genetic marker for studying intraspecific variation in trematodes.

The complete mitochondrial genome of Echinostoma miyagawai: Comparisons with closely related species and phylogenetic implications.

Echinostoma miyagawai (Trematoda: Echinostomatidae) is a common parasite of poultry that also infects humans. Es. miyagawai belongs to the "37 collar-spined" or "revolutum" group, which is very difficult to identify and classify based only on morphological characters. Molecular techniques can resolve this problem. The present study, for the first time, determined, and presented the complete Es. miyagawai mitochondrial genome. A comparative analysis of closely related species, and a reconstruction of Echinostomatidae phylogeny among the trematodes, is also presented. The Es. miyagawai mitochondrial genome is 14,416 bp in size, and contains 12 protein-coding genes (cox1-3, nad1-6, nad4L, cytb, and atp6), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and one non-coding region (NCR). All Es. miyagawai genes are transcribed in the same direction, and gene arrangement in Es. miyagawai is identical to six other Echinostomatidae and Echinochasmidae species. The complete Es. miyagawai mitochondrial genome A + T content is 65.3%, and full-length, pair-wise nucleotide sequence identity between the six species within the two families range from 64.2-84.6%. The Es. miyagawai sequences is most similar to Echinostoma caproni. Sequence difference are 15.0-33.5% at the nucleotide level, and 8.6-44.2% at the amino acid level, among the six species, for the 12 protein-coding genes. ATG and TAG are the most common initiation and termination codons, respectively. Twenty of the Es. miyagawai transfer RNA genes transcribe products of the conventional cloverleaf structure, while two of the transfer RNA genes, namely trnS1(AGC) and trnS2(UGA), have unpaired D-arms. Phylogenetic analyses using our mitochondrial data indicate that Es. miyagawai is closely related to other Echinostomatidae species, except for Echinostoma hortense, which forms a distinct paraphyletic branch, and Echinochasmus japonicus, which is outside the clade containing all other Echinostomatidae species. These phylogenetic results support the elevation of subfamily Echinostomatidae. Our dataset also provides a significant resource of molecular markers to study the taxonomy, population genetics, and systematics of the echinostomatids.