Clonorchis sinensis aggravated liver fibrosis by activating PARP-1 signaling to induce parthanatos via DNA damage.

Clonorchis sinensis is an important food-borne zoonotic parasite that is highly associated with liver fibrosis and cholangiocarcinoma. Further understanding of the pathogenesis of C. sinensis, especially liver fibrosis, could help us develop novel strategies for controlling clonorchiasis. Poly (ADP-ribose) polymerase-1 (PARP-1) can induce cellular parthanatos which is reported to be involved in liver fibrosis. Currently, whether C. sinensis could activate PARP-1 signaling to induce parthanatos or whether parthanatos play a role in C. sinensis-induced liver fibrosis is not clear. In the present study, the expression of PARP-1 and parthanatos indicators were detected in C. sinensis-infected mouse liver and in human intrahepatic biliary epithelial cells (HiBEpiCs) incubated with excretory/secretory products (ESPs) of C. sinensis. To explore the role of PARP-1 in C. sinensis infection, PARP-1 inhibitor NMS-P118 was used to block PARP-1 expression in vivo and vitro. The mortality rate, body weight, worm load, liver and bile duct lesions as well as PARP-1 and parthanatos indicators in C57BL/6 mice infected with C. sinensis, or in HiBEpiCs incubated with C. sinensis ESPs and NMS-P118 were analyzed and compared to the group without NMS-P118. The results showed that C. sinensis infection induced the activation of PARP-1 signaling as well as the translocation of AIF and MIF into the nucleus in mouse liver. ESPs of C. sinensis could induce PARP-1 up-regulation, ATP depletion and DNA damage in HiBEpiCs, indicating that C. sinensis could induce parthanatos. Inhibiting PARP-1 with NMS-P118 significantly reduced liver fibrosis and the number of larvae, increased the survival rate and body weight gain of the mice infected with C. sinensis. In addition, NMS-P118 decreased the expression of PARP-1 and alleviated ATP depletion as well as DNA damage in HiBEpiCs incubated with ESPs of C. sinensis. Our data indicated that C. sinensis and its ESPs could activate PARP-1 signaling to induce cellular parthanatos. NMS-P118 treatment alleviated liver fibrosis and promoted survival of the mice by inhibiting PARP-1, which suggested that PARP-1 could be used as a potential therapeutic target against clonorchiasis.

TLR3 activation by Clonorchis sinensis infection alleviates the fluke-induced liver fibrosis.

Clonorchis sinensis is a zoonotic parasite associated with liver fibrosis and cholangiocarcinoma development. The role of toll-like receptors (TLRs) in C. sinensis infection has not yet been fully elucidated. Here, the TLR3 signaling pathway, cytokine expression and liver fibrosis were examined in C. sinensis-infected wildtype (WT) and TLR3-/- mice. Polyinosinic-polycytidylic acid (Poly (I:C)) was used to treat C. sinensis infections. The results showed that TLR3 deficiency caused severe clonorchiasis with increased parasite burden, exacerbated proinflammatory cytokine expression and liver lesions, promoted the TGF-ß1/Smad2/3 pathway and myofibroblast activation, exacerbated liver fibrosis (compared to WT mice). Poly (I:C) intervention increased the body weight, decreased mouse mortality and parasite burden, reduced liver inflammation, and alleviated C. sinensis-induced liver fibrosis. Furthermore, C. sinensis extracellular vesicles (CsEVs) promote the production of IL-6, TNF in WT biliary epithelial cells (BECs) via p38/ERK pathway, compared with control group, while TLR3 deletion induced much higher levels of IL-6 and TNF in TLR3-/- BECs than that in WT BECs. Taken together, TLR3 inhibit IL-6 and TNF production via p38/ERK signaling pathway, a phenomenon that resulted in the alleviation of C. sinensis-induced liver fibrosis. Poly (I:C) is a potential treatment for clonorchiasis.

Clonorchis sinensis aggravates biliary fibrosis through promoting IL-6 production via toll-like receptor 2-mediated AKT and p38 signal pathways.

Clonorchis sinensis is an important food-borne zoonotic parasite which has been linked to biliary fibrosis and cholangiocarcinoma. However, the details of the pathogenesis of C. sinensis were unclear. To explore the role and regulatory mechanism of toll-like receptor 2 (TLR2) in C. sinensis-induced biliary fibrosis, we established the C. sinensis-infected C57BL/6 mouse model with TLR2-/- and wild type (WT) mice. The mortality rate, liver lesions, TLR2 and TGF-ß1 expression, phosphorylation of Smad2/3, AKT, p38, ERK and p65, and cytokine productions were analyzed. Furthermore, similar parameters were examined in mouse biliary epithelial cells (BECs) co-cultured with C. sinensis excretory/secretory proteins (ESPs). The results showed that TLR2 expression was enhanced significantly in C. sinensis-infected WT mice and mouse BECs. C. sinensis-infected TLR2-/- mice exhibited an increased weight and a decreased mortality rate; significantly alleviated liver lesions and biliary fibrosis, reduced numbers of myofibroblasts; decreased expression of TGF-ß1 and phosphorylation level of AKT, p38 and Smad2/3; significantly decreased production of IL-6, TNF-α and IL-4, while increased production of IFN-γ compared with C. sinensis-infected WT mice. Furthermore, C. sinensis ESPs could activate TLR2-mediated AKT and p38 pathways to increase the production of IL-6 in mouse BECs. In conclusion, these data indicate that C. sinensis infection activated TGF-ß1-Smad2/3 through TLR2-mediated AKT and p38 pathways to promote IL-6 production, which resulted in myofibroblast activation and aggravating biliary fibrosis in mice.

Antitumor effect of invasive Lactobacillus plantarum delivering associated antigen gene sHSP between Trichinella spiralis and Lewis lung cancer cells.

Cancer is a frequent disease that seriously harms human health, but there are no ideal therapies for it. Currently, some food-grade microorganisms such as Lactobacillus plantarum have shown better anti-tumor effects. Here, recombinant Lactobacillus plantarum lives vector vaccine NC8-sHSP was generated by using the invasive Lactobacillus plantarum NC8 expressing FnBPA to deliver the associated antigen gene sHSP between trichinella spiralis and Lewis lung cancer cells (LLC) to host cells. NC8-sHSP colonized the mouse intestine to deliver plasmids to intestinal epithelial cells and controlled the growth of LLC by inducing humoral, cellular, and mucosal immunity. The tumor inhibition rates were 62.36% and 68.37% in the prophylactic assay and 40.76% and 44.22% in the treatment assay, respectively. Recombination of Lactobacillus plantarum did not cause significant damage. In conclusion, the recombinant invasive Lactobacillus plantarum constructed in this study has better anti-Lewis lung cancer effects in mice, which will provide new ideas for the application of food-grade microorganisms in anti-tumor and the development of oral tumor vaccines.

Extracellular vesicles of Clonorchis sinensis promote IL-6 and TNF-α secretion via the Toll-like receptor 9-mediated ERK pathway in biliary epithelial cells.

Clonorchis sinensis is closely associated with cholangitis, cholecystitis, biliary fibrosis and cholangiocarcinoma. The present study elucidated the role of extracellular vesicles of C. sinensis (CsEVs) in activating Toll-like receptor 9 (TLR9) and regulating inflammatory responses. The results showed that TLR9 expression was increased in the livers of C. sinensis-infected mice. CsEVs were cup-shaped or saucer-shaped and 80-120 nm in diameter. CsEVs activated TLR9 and promoted IL-6 and TNF-α expression in mouse biliary epithelial cells (BECs), and TLR9 siRNA interference reduced the secretion of the two cytokines. CsEV stimulation promoted the phosphorylation of ERK, p38, AKT, and p65, and TLR9 siRNA interference regulated the phosphorylated ERK, AKT and p65 levels. The ERK inhibitor decreased the CsEVs-induced IL-6 and TNF-α secretion. The present study elucidated for the first time that CsEVs induced IL-6 and TNF-α production in BECs via the TLR9-mediated ERK pathway.

Prevalence of fish-borne zoonotic trematode infection in Jilin Province, China.

Fish-borne zoonotic trematodes (FZTs) are the most serious food-borne parasites in Asia and have become a burden to public health and a new challenge in food safety. In Jilin Province, China, the prevalence of FZTs in intermediate and definitive hosts has not been extensively explored. In the present study, we investigated the prevalence of FZTs in Jilin Province, China. From July to November 2020, a total of 132 freshwater snails (the first intermediate host of FZTs), 4122 wild freshwater fishes (the second intermediate host of FZTs) and 143 fecal samples from canines, ducks and swine (the definitive host of FZTs) were collected from the Yitong River basin of Jilin Province. FZT species were identified by morphological observation combined with internal transcribed spacer (ITS) sequence analysis. The prevalence of FZTs was then calculated. The results showed that the prevailing species of FZTs in Jilin Province, China, were Clonorchis sinensis, Metorchis orientalis and Echinochasmus japonicus. The total prevalence of FZTs was 29.74% (1226/4122) in fish, the total infection rates were 2.27% (3/132) in snails, 75.00% (21/28) in canines and 37.18% (29/78) in ducks. The coinfection rates of the two trematodes were 13.39% (552/4122) in fish, 35.71% (10/28) in canines and 7.69% (6/78) in ducks. The coinfection rate of the three flukes was 2.60% (107/4122) in fish. Nine of the 12 fish species examined were infected with FZT metacercariae.

First report of Eimeria and Entamoeba infection in alpacas (Vicugna pacos) in Shanxi Province, northern China.

Intestinal protozoa Eimeria and Entamoeba can infect many animal species including alpacas. However, data on the prevalence and pathogenicity of species of the two genera Eimeria and Entamoeba in alpacas in China is scarce. The current study was carried out to investigate the prevalence of Eimeria and Entamoeba in alpacas in two cities (Taiyuan and Xinzhou) in Shanxi Province, northern China, using PCR-based approaches. Eimeria spp. were only found in Taiyuan city, and the overall prevalence was 1.64%. All samples collected from male alpacas were PCR-negative for Eimeria. Four Eimeria-positive samples were tested positive as Eimeria lamae. The molecular prevalence of Entamoeba in alpacas was 18.03% (66/366), including 16.39% (50/305) in alpacas from Taiyuan city and 26.23% (16/61) from Xinzhou city, respectively. The Entamoeba prevalence in male alpacas (25.00%) was significantly higher than that in female alpacas (15.69%). Entamoeba bovis was the predominant species, and no Entamoeba histolytica infection was detected. Nine unique SSU rRNA gene sequences of Entamoeba were obtained which formed a new cluster. The results showed that sex and location might be the risk factors associated with prevalence of Eimeria spp., and sex might be the risk factor associated with prevalence of Entamoeba spp.. This is the first report of Entamoeba in alpacas worldwide. These findings expand our understanding of the prevalence and genetic diversity of Eimeria and Entamoeba in alpacas.

Serological evidence of Toxoplasma gondii and Chlamydia infection in alpacas (Vicugna pacos) in Shanxi Province, northern China.

Toxoplasma gondii, Neospora caninum, Chlamydia and bluetongue virus (BTV) are four important pathogens which can cause reproductive loss. The present study was conducted to estimate the seroprevalence of T. gondii, N. caninum, Chlamydia and BTV in alpacas in Shanxi Province, northern China. A total of 251 serum samples were collected from alpacas, and antibodies against T. gondii and Chlamydia were examined by the modified agglutination test (MAT) and indirect hemagglutination assay (IHA), respectively. Antibodies to N. caninum and BTV were determined by using the commercially available competitive enzyme-linked immunosorbent assay (cELISA) kits, respectively. The overall seroprevalence of T. gondii was 9.16% (95% CI 5.59-12.73) in the three sampled counties, of which, no T. gondii-seropositive samples were detected in alpacas in Fanshi County. Gender differences in the T. gondii seroprevalence were observed. The overall Chlamydia seroprevalence was 13.94% (95% CI: 9.66-18.22), and there was a statistically significant difference in Chlamydia seroprevalence in alpacas between the two counties, Jiexiu and Fanshi. All serum samples tested negative for N. caninum and BTV antibodies, respectively. To our knowledge, this is the first report of T. gondii and Chlamydia seroprevalence in alpacas in China, which provides baseline information for controlling T. gondii and Chlamydia infection in alpacas in China.

Prevalence and genotype distribution of Enterocytozoon bieneusi in farmed raccoon dogs (Nyctereutes procyonoides) in Shandong Province, eastern China.

Enterocytozoon bieneusi is a common microsporidian species, which can infect humans and various species of animals. However, little is known about E. bieneusi prevalence and genotypes in farmed raccoon dogs (Nyctereutes procyonoides) in Shandong Province, China. In this study, a total of 356 fecal samples were collected from farmed raccoon dogs in Weihai, Weifang, and Yantai cities in Shandong Province, China. A total of 23 (6.5%) samples were E. bieneusi-positive by nested PCR amplification of the internal transcribed spacer (ITS) region of ribosomal DNA. Statistical analysis showed that E. bieneusi prevalence in male raccoon dogs was higher than that in female raccoon dogs, and the highest E. bieneusi prevalence was detected in adult raccoon dogs. Sequence analysis revealed four known E. bieneusi genotypes (D, type IV, CHG1, and Peru8), and type IV (11/23) was the predominant genotype. The genotypes type IV, Peru8, and CHG1 were reported in raccoon dogs for the first time in China. Phylogenetic analysis showed that three human-pathogenic genotypes (D, type IV, and Peru8) were clustered into group 1, and the CHG1 belonged to group 2. These findings expand the current understanding of E. bieneusi prevalence and genotype distribution in raccoon dogs in China. Our study also shows that raccoon dogs are hosts for E. bieneusi belonging to several genotypes, including zoonotic ones, highlighting the possibility of transmission of this pathogen between raccoon dogs and humans.

Prevalence and Subtypes of Blastocystis in Alpacas, Vicugna pacos in Shanxi Province, China.

Blastocystis, an enteric protist, has been reported to be an important cause of protozoal gastrointestinal manifestations in humans and animals worldwide. Animals harboring certain Blastocystis subtypes (STs) may serve as a potential source of human infection. However, information about the prevalence and genetic diversity of Blastocystis in alpacas is limited. In the present study, a total of 366 fecal samples from alpacas in Shanxi Province, northern China, were examined for Blastocystis by PCR amplification of the small subunit rRNA gene, followed by sequencing and phylogenetic analysis. The prevalence of Blastocystis in alpacas was 23.8%, and gender difference in the prevalence of Blastocystiswas observed. The most predominant Blastocystis ST was ST10, followed by ST14 and ST5. The detection of ST5, a potentially zoonotic genotype, indicates that alpacas harboring ST5 could be a potential source of human infection with Blastocystis. These data provide new insight into the prevalence and genetic diversity of Blastocystis in alpacas.

Application of Toxoplasma gondii GRA15 peptides in diagnosis and serotyping.

Previous studies showed that three clonal strain types (I, II, and III) of Toxoplasma gondii can be distinguished using serotyping based on a series of polymorphic proteins. However, to establish a systematic serotyping method with higher resolution even being equal to that of genotyping, more specific peptide markers are needed. The objective of the present study was to determine the possibility of the polymorphic dense granule protein 15 (GRA15) for diagnosis and serotyping of T. gondii infection. Three different T. gondii GT1 strain GRA15 gene fragments encoding a 584-residue peptide, a 199-residue peptide and a 84-residue peptide were amplified, expressed and purified, respectively. Anti-T. gondii GT1 strain antibodies, anti-T. gondii RH strain antibodies and anti-T. gondii PRU strain antibodies were used for immunoblotting analysis of the three peptides. Western blotting analysis showed that the 584-residue peptide of GT1 strain GRA15 was a potential candidate for serological diagnosis of T. gondii infection. RH strain from GT1 strain could be distinguished by serotyping based on the GRA15199 or GRA1584, and T. gondii GT1 strain could be distinguished from PRU strain by using serotyping based on the GRA1584. These findings reveal, for the first time, a novel potential role of GRA15-derived peptides in diagnosis and serological differentiation of T. gondii infection.

Molecular characterization of Eimeria spp. and Blastocystis in rabbits in Shandong Province, China.

Eimeria spp. and Blastocystis are the common parasites that parasitize the intestinal tract of rabbits, which can seriously threaten the health of rabbits and lead to economic losses to the rabbit industry. However, information about the prevalence and transmission of these two parasites in rabbits is limited in China. The objective of this study was to survey the prevalence of Eimeria spp. and Blastocystis in rabbits in Shandong Province. A total of 616 rabbit fecal samples were collected from two cities (Rizhao and Weihai) in Shandong Province, eastern China, and Eimeria spp. and Blastocystis were identified by polymerase chain reaction based on species-specific markers. The prevalence of Eimeria spp. was 20% (123/616) and the Blastocystis prevalence was 0.97% (6/616). Five different Eimeria species (Eimeria intestinalis, E. perforans, E. magna, E. media, and E. irresidua) and the ST4 subtype of Blastocystis were identified in rabbits by sequence analysis. This is the first report of Blastocystis prevalence and subtype ST4 in rabbits in Shandong Province. The findings provide baseline data for the prevention and control of Eimeria spp. and Blastocystis in rabbits in Shandong Province, China.

Expression and immunoreactivity of a recombinant multi-epitope antigen designed based on four major structural proteins of avian infectious bronchitis virus.

The development of rapid, simple, and sensitive diagnostic methods for identification of avian infectious bronchitis virus (IBV) is crucial for the effective control of avian infectious bronchitis. In the present study, a tandemly arranged multiepitope peptide (named SEMN) was designed with four antigenic regions derived from four major structural proteins of IBV. Then, we performed codon optimization of SEMN gene by changing the codon-adaptation index from 0.45 to 0.94 and expressed the optimized gene in codon bias-adjusted Escherichia coli Rosetta (DE3), followed by determination of the immunoreactivity of the purified protein. Bioinformatics analysis of SEMN showed a high antigenicity, surface probability and hydrophilicity. The recombinant protein rSEMN was expressed both in soluble forms and as inclusion bodies, and the molecular weight of rSEMN was about 39 kDa. The preliminary diagnostic performance of rSEMN was confirmed by Western blotting analysis using chicken anti-IBV polyclonal antibodies. Further studies are needed to evaluate the immunogenicity in animal models and to give a final assessment of the diagnostic utility of this recombinant multi-epitope antigen.

Molecular detection and genotype distribution of Enterocytozoon bieneusi in farmed silver foxes (Vulpes vulpes) and arctic foxes (Vulpes lagopus) in Shandong Province, eastern China.

Enterocytozoon bieneusi is an opportunistic enteric pathogen which can infect a wide range of animal species and humans. It is the most diagnosed species of Microsporidia in humans and has an impact on public health. Many infected animals including foxes may be a potential source for transmitting E. bieneusi to humans. However, limited information is available on the E. bieneusi prevalence and genotypes in farmed foxes in China. Therefore, in the present study, 344 fresh fecal samples were collected from farmed foxes (Vulpes vulpes and Vulpes lagopus) in Shandong Province, and the prevalence and genotypes of E. bieneusi were examined based on sequence analysis of the ribosomal internal transcribed spacer (ITS) region. The overall E. bieneusi prevalence was 9% (31/344); of them, 6.5% (9/138) in farmed silver foxes (V. vulpes) and 10.7% (22/206) in farmed arctic foxes (V. lagopus). Moreover, four known (Hum-q1, NCF2, HND-1, and Type IV) and two novel E. bieneusi genotypes (SDF1 and SDF2) were identified in farmed foxes in the present study. All of the E. bieneusi genotypes belonged to the zoonotic group based on phylogenetic analysis. In addition, 2, 4, 0, and 11 samples were successfully amplified at MS1, MS3, MS4, and MS7 loci, respectively. The present study reveals E. bieneusi prevalence and genotype distribution in farmed foxes in Shandong Province and enlarged the host and geographic information of E. bieneusi in China.

Prevalence and multilocus genotypes of Enterocytozoon bieneusi in alpacas (Vicugna pacos) in Shanxi Province, northern China.

Enterocytozoon bieneusi is a single-celled obligate pathogen that seriously threatens animal and public health. However, information on the prevalence and genotypes of E. bieneusi in alpacas in China is limited. In the present study, 366 fresh fecal samples from alpacas in Shanxi Province, northern China, were collected to detect E. bieneusi by nested PCR amplification of the internal transcribed spacer (ITS) of nuclear ribosomal DNA (rDNA). The overall prevalence of E. bieneusi in alpacas was 4.4% (16/366), including 3.9% (12/305) in Yangqu County and 6.6% (4/61) in Dai county, respectively. Four known genotypes were identified, namely ALP1, ALP3, P, and SH11, all of which belong to the zoonotic group 1 by phylogenetic analysis. Moreover, ITS-positive samples were further characterized by PCR amplification of other four targets, including three microsatellites (MS1, MS3, and MS7) and one minisatellite (MS4). Multilocus sequence typing (MLST) showed that 5, 2, 3, and 3 types were identified at MS1, MS3, MS7, and MS4 loci, respectively, representing eight multilocus genotypes (MLGs). These findings contribute to the improved understanding of the prevalence and genotypes of E. bieneusi in alpacas in China and have important implications for controlling E. bieneusi infections in animals and humans.