Altered landscape of total RNA, tRNA and sncRNA modifications in the liver and spleen of mice infected by Toxoplasma gondii.

BACKGROUND: Pathogens can impact host RNA modification machinery to establish a favorable cellular environment for their replication. In the present study, we investigated the effect of Toxoplasma gondii infection on host RNA modification profiles and explored how these modifications may influence the host-parasite interaction. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the modification levels of ∼ 80 nt tRNA and 17-50 nt sncRNAs in mouse liver, spleen, and serum using liquid chromatography and tandem mass spectrometry analysis. The results revealed alterations in RNA modification profiles, particularly during acute infection. The liver exhibited more differentially abundant RNA modifications than the spleen. RNA modification levels in serum were mostly downregulated during acute infection compared to control mice. Correlations were detected between different RNA modifications in the liver and spleen during infection and between several RNA modifications and many cytokines. Alterations in RNA modifications affected tRNA stability and protein translation. CONCLUSIONS/SIGNIFICANCE: These findings provide new insight into the role of RNA modifications in mediating the murine host response to T. gondii infection.

Prevalence and Molecular Characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Cattle in Heilongjiang Province, Northeast China.

Crytosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are important diarrheal pathogens with a global distribution that threatens the health of humans and animals. Despite cattle being potential transmission hosts of these protozoans, the associated risks to public health have been neglected. In the present study, a total of 1155 cattle fecal samples were collected from 13 administrative regions of Heilongjiang Province. The prevalence of Cryptosporidium spp., G. duodenalis, and E. bieneusi were 5.5% (64/1155; 95% CI: 4.2-6.9), 3.8% (44/1155; 95% CI: 2.7-4.9), and 6.5% (75/1155; 95% CI: 5.1-7.9), respectively. Among these positive fecal samples, five Cryptosporidium species (C. andersoni, C. bovis, C. ryanae, C. parvum, and C. occultus), two G. duodenalis assemblages (E and A), and eight E. bieneusi genotypes (BEB4, BEB6, BEB8, J, I, CHS7, CHS8, and COS-I) were identified. Phylogenetic analysis showed that all eight genotypes of E. bieneusi identified in the present study belonged to group 2. It is worth noting that some species/genotypes of these intestinal protozoans are zoonotic, suggesting a risk of zoonotic disease transmission in endemic areas. The findings expanded our understanding of the genetic composition and zoonotic potential of Cryptosporidium spp., G. duodenalis, and E. bieneusi in cattle in Heilongjiang Province.

Survey of the Prosthogonimus spp. metacercariae infection in the second intermediate host dragonfly in Heilongjiang Province, China.

Prosthogonimiasis poses a threat to the reproductive system of poultry and wild birds, which are the definitive hosts of the parasite causing this disease. However, the parasite infection of the second intermediate host (dragonfly), the primary vector of this pathogen, is rarely reported. In this study, the prevalence of Prosthogonimus infection in dragonflies was investigated from June 2019 to October 2022 in Heilongjiang Province, northeast China. The species of metacercariae isolated from dragonfly were identified by morphological characteristics, molecular biology techniques, and animal infection experiments. The results showed that 11 species of dragonflies and one damselfly were identified and among six of the dragonflies infected by Prosthogonimus metacercariae, Sympetrum depressiusculum (28.53%) had the highest infection rate among all positive dragonflies, followed by Sympetrum vulgatum (27.86%) and Sympetrum frequens (20.99%), which are preferred hosts, and the total prevalence was 20.39% (2061/10,110) in Heilongjiang Province. Three species of Prosthogoniumus metacercariae were isolated, including Prosthogonimus cuneatus, Prosthogonimus pullucidus, and Prosthogonimus sp., among which P. cuneatus was the dominant species in dragonflies in Heilongjiang Province. This is the first report on the prevalence of Prosthogonimus in dragonflies in China, which provides baseline data for the control of prosthogonimiasis in Heilongjiang Province and a reference for the prevention of prosthogonimiasis in other areas of China.

The complete mitochondrial genome of Ogmocotyle ailuri: gene content, composition and rearrangement and phylogenetic implications.

Trematodes of the genus Ogmocotyle are intestinal flukes that can infect a variety of definitive hosts, resulting in significant economic losses worldwide. However, there are few studies on molecular data of these trematodes. In this study, the mitochondrial (mt) genome of Ogmocotyle ailuri isolated from red panda (Ailurus fulgens) was determined and compared with those from Pronocephalata to investigate the mt genome content, genetic distance, gene rearrangements and phylogeny. The complete mt genome of O. ailuri is a typical closed circular molecule of 14 642 base pairs, comprising 12 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions. All genes are transcribed in the same direction. In addition, 23 intergenic spacers and 2 locations with gene overlaps were determined. Sequence identities and sliding window analysis indicated that cox1 is the most conserved gene among 12 PCGs in O. ailuri mt genome. The sequenced mt genomes of the 48 Plagiorchiida trematodes showed 5 types of gene arrangement based on all mt genome genes, with the gene arrangement of O. ailuri being type I. Phylogenetic analysis using concatenated amino acid sequences of 12 PCGs revealed that O. ailuri was closer to Ogmocotyle sikae than to Notocotylus intestinalis. These data enhance the Ogmocotyle mt genome database and provide molecular resources for further studies of Pronocephalata taxonomy, population genetics and systematics.

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.

First description of the mitogenome and phylogeny:Aedes vexansand Ochlerotatus caspius of the Tribe Aedini (Diptera: Culicidae).

Culicidae, the mosquito family, includes more than 3600 species subdivided into the subfamilies Anophelinae and Culicinae. One-third of mosquitoes belong to the Aedini tribe, which is subordinate to the subfamily Culicinae, which comprises common vectors of viral zoonoses. The tribe of Aedini is extremely diverse in morphology and geographical distribution and has high ecological and medical significance. However, knowledge about the systematics of the Aedini tribe is still limited owing to its large population and the similar morphological characteristics of its species. This study provides the first description of the complete mitochondrial (mt) genome sequence of Aedes vexans and Ochlerotatus caspius belonging to the Aedini tribe. The mt genomes of A. vexans and O. caspius are circular molecules that are 15,861 bp and 15,954 bp in size, with AT contents of 78.54% and 79.36%, respectively. Both the circular mt genomes comprise 37 functional subunits, including 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes (tRNAs), and a control region (also known as the AT-rich region). The most common start codons are ATT/ATG, apart from cox1 (TCG) and nad5 (GTG), while TAA is the termination codon for all PCGs. All tRNAs have a typical clover leaf structure, except tRNA Ser1. Phylogenetic analysis of the concatenated, aligned amino acid sequences of the 13 PCGs showed that A. vexans gathered with Aedes sp. in a sister taxon, and O. caspius gathered with Ochlerotatus sp. in a sister taxon. The findings from the present study support the concept of monophyly of all groups, ratify the current taxonomic classification, and provide vital molecular marker resources for further studies of the taxonomy, population genetics, and systematics of the Aedini tribe.

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.

Characterization of the mitochondrial genome of Tetrameres grusi and insights into the phylogeny of Spirurina.

Tetrameres grusi is a significant parasitic nematode of cranes that is classified into suborder Spirurina. However, for more than a century, this classification has been controversial. Mitochondrial genomes are valuable resources for parasite taxonomy, population genetics and systematics studies. Here, the mitochondrial genome of T. grusi was determined and subsequently compared with those from Spirurina species using concatenated datasets of amino acid sequences predicted from mitochondrial protein-coding genes. The complete mitochondrial genome of T. grusi is circular with 13,709 bp, and it contains 12 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one non-coding region. All of the protein-coding genes are transcribed in the same direction. There were 18 intergenic spacers of 1-44 bp, and six locations with gene overlaps, ranging from 1 bp to 28 bp, in the mitochondrial genome of T. grusi. The AT content of this mitochondrial genome was 71.56%. This was similar to mitochondrial genomes of other Spirurina species, which also exhibited strong AT content bias, not only in the nucleotide composition but also in codon usage. The sequenced mitogenomes of the 25 Spirurina nematodes showed three classes of gene arrangements based on the 12 protein-coding genes, and the gene arrangement of the T. grusi mitochondrial genome belonged to the Class I. Phylogenetic analyses using mitochondrial genomes of 25 Spirurina nematodes revealed that T. grusi (Habronematoidea) was closer to Gongylonema pulchrum (Spiruroidea) than Spirocerca lupi (Thelazioidea). The availability of the complete mitochondrial genome sequence of T. grusi provides new and useful genetic markers for further studies on Spirurina nematodes.

Prevalence of Theileria in cattle in China: A systematic review and meta-analysis.

Theileria, one of the causative agents of blood protozoan, has brought a huge economic loss to the cattle industry worldwide. However, the epidemiology of Theileria in Chinese cattle has not been systematically investigated. This comprehensive review aimed at investigating the prevalence of Theileria infection in cattle in China. A total of 48 published papers on Theileria infection in cattle in China (including data from 21,366 animals) from inception to October 8, 2021 met the inclusion standard after searching in five databases (Technology Periodical Database, Wan Fang Database, China National Knowledge Infrastructure, PubMed, and ScienceDirect). The pooled prevalence of Theileria in cattle in China was 32.4% identified by using a random effects model. The prevalence in Northeastern China (45.3%) was higher than that in other regions. In the sex subgroup, the prevalence of Theileria was higher in females (48.9%) than that in males (45.8%). The prevalence of Theileria was higher in cattle of free range (34.4%) compared with that of intensive farming (22.3%). The prevalence prior to 2013 (36.1%) was higher than that after 2013 (33.6%). Among three cattle species, dairy cows had the lowest prevalence (21.5%). The prevalence of Theileria (T.) annulata (22.2%) and T. sergenti (26.2%) was higher than other species of Theileria (T. buffeli: 17.5%, T. luwenshuni: 0.9%, T. orientalis: 15.5%, T. ovis: 0.21%, T. sinensis: 20.2%, T. uilenbergi: 6.2%, Others: 0.9%). We also analyzed the impact of different geographic factor subgroups (longitude, latitude, precipitation, temperature, humidity, and altitude) on the prevalence of Theileria in cattle. Among them, climatic factors of longitude, latitude, precipitation, humidity, temperature were associated with the prevalence of Theileria. These analyses suggested that Theileria was common in cattle in China. Targeted prevention programs based on geographic and climatic conditions in different areas may play an important role in reducing Theileria infection among cattle.

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.

Proteomic Analysis of Fasciola hepatica Excretory and Secretory Products Co-Immunoprecipitated Using Time Course Infection Sera.

Fasciola hepatica is a widespread pathogen that is known for its harmful effects on the health and productivity of ruminant animals. To identify the proteins present in all periods of infection with F. hepatica but not in those with Fasciola gigantica by shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS), we collected the ESPs and sera of F. hepatica and F. gigantica. In this study, the sheep were artificially infected with F. hepatica and the sera were collected at five different periods: 3 days post-infection (dpi), 7 dpi, 21 dpi, 63 dpi, and 112 dpi. The interacting proteins were pulled down from the sheep sera of all five periods and the sera with F. gigantica by co-immunoprecipitation (Co-IP) assay, before being identified by LC-MS/MS analysis. Thirty, twenty-two, twenty-three, twenty-seven, and twenty-two proteins were pulled down by the infected sera at 3 dpi, 7 dpi, 21 dpi, 63 dpi, and 112 dpi, respectively. Among them, 12 proteins existed in all periods, while six proteins could be detected in all periods in F. hepatica but not in F. gigantica. Protein relative pathway analysis revealed that these proteins mainly refer to the metabolism, regulation of genetic activity, and signal transduction of F. hepatica. In conclusion, this study provides meaningful data for the diagnosis of fasciolosis and to understand the interactions between F. hepatica and the host.

The prevalence of Toxoplasma gondii in sheep in China: A systematic review and meta-analysis.

Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii, which is widespread in warm-blooded animals and humans. Currently, many studies regarding T. gondii infection in sheep have been published worldwide. However, there is no meta-analysis of sheep infection in China. In this study, five databases were used to retrieve articles related to T. gondii in China. A total of 59 studies from 1987 to June 30, 2020, have been included. We estimated that the combined prevalence of T. gondii in the selected period was 8.5% (3197/28,099). In the analysis of publication year, the lowest positive rate after 2010 was 7.4% (1883/17,313). Geographically, the highest prevalence of T. gondii in sheep was recorded in Southwest China 19.2% (490/2080). Analysis according to age showed that the prevalence of infection in sheep older than 12 months was significantly (P < 0.05) higher than that in younger animals. The prevalence of T. gondii in sheep farmed by farmers was 7.2% (566/6336), which was higher than 5.3% (660/3121) of large-scale farming, suggesting a role for the feeding and husbandry. We also analyzed the impact of different geographic and climatic factors on the prevalence of T. gondii infection in sheep. The results showed that the prevalence was higher in low altitude, warm, humid, and high rainfall areas. We suggest that appropriate control programs should be formulated according to the differences in reproduction patterns and geographical conditions in different regions to reduce the prevalence of T. gondii among sheep in China.

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 and Risk Factors of Toxoplasma gondii Infection Among Five Wild Rodent Species from Five Provinces of China.

Toxoplasma gondii, an intracellular zoonotic parasite, can infect humans and various animals worldwide. Wild rodents plan an important role as intermediate hosts of T. gondii. Some studies on T. gondii from wild rodents have been published, but the investigation data of T. gondii in wild rodents in China are limited. Therefore, brain tissue samples from 382 wild rodents in four provinces and one autonomous region of China were screened by PCR amplification of T. gondii B1 gene. Furthermore, the wild rodents were identified as five species based on their morphological characteristics, including Citellus dauricus (n = 35 from Heilongjiang), Lasiopodomys brandti (n = 81 from Inner Mongolia), Apodemus agrarius (n = 12 from Heilongjiang), Rattus norvegicus (n = 2 from Heilongjiang; n = 99 from Zhejiang; n = 54 from Shanxi), and Mus musculus (n = 99 from Guangxi). The overall prevalence of T. gondii in these wild rodents was 5.24% (20/382) in this study. At different regions, the highest prevalence of T. gondii was in Guangxi (12.12%) compared with other regions (0% in Heilongjiang; 2.47% in Inner Mongolia; 2.02% in Zhejiang; 7.41% in Shanxi). At different climates, the higher prevalence was found in temperate/mesothermal climates (7.07%) compared with continental/microthermal climates (3.26%). Also, the results showed that M. musculus had the highest prevalence of T. gondii infection (12.12%) among the rodent species sampled. Moreover, sampling year was significantly related to the prevalence of T. gondii in investigated wild rodents (p = 0.0117). This is the first report of T. gondii in wild rodents from Zhejiang, Guangxi, Shanxi, Heilongjiang provinces and Inner Mongolia autonomous region in China, providing the fundamental information for further prevention and control of toxoplasmosis in China.

Molecular detection of Cryptosporidium spp. and Enterocytozoon bieneusi in Longjiang Wagyu cattle in Northeastern China.

Cryptosporidium spp. and Enterocytozoon bieneusi are two important zoonotic pathogens that can cause diarrhea and other gastrointestinal illnesses in humans and animals. However, the prevalence and genotype of the parasites in Longjiang Wagyu cattle in Heilongjiang Province, Northeast China have not been reported. In the present study, a total of 423 fecal samples of Longjiang Wagyu cattle collected from different farms in Heilongjiang Province, Northeast China, were examined for Cryptosporidium spp. and E. bieneusi using nested PCR. The overall infection rates for Cryptosporidium spp. and E. bieneusi were 6.38% (n = 27) and 7.09% (n = 30), respectively. The prevalence in different age groups ranged from 3.80% (95% confidence interval (CI) 1.01-6.59) to 8.36% (95% CI 4.83-11.90) for Cryptosporidium spp. and 5.97% (95% CI 2.52-9.43) to 7.94% (95% CI 4.49-11.40) for E. bieneusi. By analyzing the DNA sequences of the small subunit (SSU) rRNA gene, two Cryptosporidium species were detected in this study, namely C. parvum (n = 25) and C. ryanae (n = 2). The IIdA20G1 subtype was further identified by using the 60-kDa glycoprotein (gp60) gene of C. parvum. E. bieneusi was identified using three known sequences through the analysis of internal transcribed spacer (ITS) sequences: J (n = 23), I (n = 5), and BEB4 (n = 2), and all belonged to group 2. The results indicated that some of the Cryptosporidium species and E. bieneusi genotypes identified in Longjiang Wagyu cattle in the study areas might have zoonotic potential.

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.

Toxoplasma gondii Seropositivity and Associated Risk Factors in Cats (Felis catus) in Three Provinces in Northeastern China from 2013 to 2019.

Toxoplasma gondii is an important protozoan parasite that can infect a range of animals. Cats are the most important definitive hosts for T. gondii. Therefore, an assessment of the level of T. gondii infection in cats is of public health importance. As limited information about seroprevalence of T. gondii infection in cats in northeastern China was available, 854 serum samples, including 235 cats in Jilin province, 267 cats in Liaoning province, and 352 cats in Heilongjiang province, were examined by an indirect hemagglutination assay (IHA) test, between September 2013 and July 2019. The overall seroprevalence of T. gondii was 19.09% (163/854) at the cutoff 1:64, with 19.60% (69/352), 18.29% (43/235), and 19.10% (51/267) in Heilongjiang, Jilin and Liaoning, respectively. Logistic regression analysis showed that mode of life was highly related to T. gondii infection in cats at the investigation areas. These results could provide foundation data for prevention and control of T. gondii prevalence in these regions. Moreover, the results also suggested that the effective control strategies should continue to be performed.