Rapid visual detection of Giardia duodenalis in faecal samples using an RPA-CRISPR/Cas12a system.

Giardiasis is a common intestinal infection caused by Giardia duodenalis, which is a major economic and health burden for humans and livestock. Currently, a convenient and effective detection method is urgently needed. CRISPR/Cas12a-based diagnostic methods have been widely used for nucleic acid-based detection of pathogens due to their high efficiency and sensitivity. In this study, a technique combining CRISPR/Cas12a and RPA was established that allows the detection of G. duodenalis in faecal samples by the naked eye with high sensitivity (10-1 copies/µL) and specificity (no cross-reactivity with nine common pathogens). In clinical evaluations, the RPA-CRISPR/Cas12a-based detection assay detected Giardia positivity in 2% (1/50) of human faecal samples and 47% (33/70) of cattle faecal samples, respectively, which was consistent with the results of nested PCR. Our study demonstrated that the RPA-CRISPR/Cas12a technique for G. duodenalis is stable, efficient, sensitive, specific and has low equipment requirements. This technique offers new opportunities for on-site detection in remote and poor areas.

Determination of cyclic adenosine phosphate and protein content in dormant chlamydospore and nondormant chlamydospore of Arthrobotrys flagrans.

Arthrobotrys flagrans, a nematode-eating fungus, is an effective component of animal parasitic nematode biocontrol agents. In the dried formulation, the majority of spores are in an endogenous dormant state. This study focuses on dormant chlamydospore and nondormant chlamydospore of A. flagrans to investigate the differences in cyclic adenosine monophosphate (cAMP) and protein content between the two types of spores. cAMP and soluble proteins were extracted from the nondormant chlamydospore and dormant chlamydospore of two isolates of A. flagrans. The cAMP Direct Immunoassay Kit and Bradford protein concentration assay kit (Coomassie brilliant blue method) were used to detect the cAMP and protein content in two types of spores. Results showed that the content of cAMP in dormant spores of both isolates was significantly higher than that in nondormant spores (p < 0.05). The protein content of dormant spores in DH055 bacteria was significantly higher than that of nondormant spores (p < 0.05). In addition, the protein content of dormant spores of the SDH035 strain was slightly higher than that of nondormant spores, but the difference was not significant (p > 0.05). The results obtained in this study provide evidence for the biochemical mechanism of chlamydospore dormancy or the germination of the nematophagous fungus A. flagrans.

The Detection of Circulating Antigen Glutathione S-Transferase in Sheep Infected with Fasciola hepatica with Double-Antibody Sandwich Signal Amplification Enzyme-Linked Immunosorbent Assay.

Fasciolosis is a global zoonotic parasitic disease caused by F. hepatica infection that is particularly harmful to cattle and sheep. A biotin-streptavidin signal amplification ELISA (streptavidin-ELISA/SA-ELISA) based on circulating antigens can allow for the early detection of F. hepatica-infected animals and is suitable for batch detection. It is considered to be a better means of detecting F. hepatica infection than traditional detection methods. In this study, using the serum of sheep artificially infected with F. hepatica, the cDNA expression library of F. hepatica was screened, 17 immunodominant antigen genes of F. hepatica were obtained, and glutathione s-transferase (GST) was selected as the candidate detection antigen. Firstly, the GST cDNA sequence was amplified from F. hepatica, followed by the preparation of recombinant protein GST (rFhGST). Then, monoclonal and polyclonal antibodies against rFhGST were prepared using the GST protein. Afterward, the immunolocalization of the target protein in the worm was observed via confocal microscopy, and it was found that the GST protein was localized in the uterus, intestinal tract, and body surface of F. hepatica. Finally, a double-antibody sandwich SA-ELISA based on the detection of circulating antigens was established. There was no cross-reaction with positive sera infected with Dicrocoelium lanceatum (D. lanceatum), Haemonchus contortus (H. contortus), Neospora caninum (N. caninum), or Schistosoma japonicum (S. japonicum). Forty serum and fecal samples from the same batch of sheep in Nong'an County, Changchun City, Jilin Province, China were analyzed using the established detection method and fecal detection method. The positive rate of the SA-ELISA was 17.5%, and the positive rate of the fecal detection method was 15%. The detection results of this method were 100% consistent with commercial ELISA kits. A total of 152 sheep serum samples were tested in Nong'an County, Changchun City, Jilin Province, and the positive rate was 5.92%. This study laid the foundation for the development of serological detection preparations for F. hepatica infection based on the detection of circulating antigens.

Establishment of an ultrasensitive and visual detection platform for Neospora caninum based-on the RPA-CRISPR/Cas12a system.

Neospora caninum is a protozoan parasite that causes neosporosis in cattle, and leads to a high rate of abortion and severe financial losses. Rapid and accurate detection is particularly important for preventing and controlling neosporosis. In our research, a highly effective diagnostic technique based on the RPA-CRISPR/Cas system was created to successfully identify N. caninum against the Nc5 gene, fluorescent reporter system and the lateral flow strip (LFS) biosensor were exploited to display results. The specificity and sensitivity of the PRA-CRISPR/Cas12a assay were evaluated. We discovered that it was highly specific and did not react with any other pathogens. The limit of detection (LOD) for this technology was as low as one parasite per milliliter when employing the fluorescent reporter system, and was approximately ten parasites per milliliter based on the LFS biosensor and under blue or UV light. Meanwhile, the placental tissue samples were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (59.4 %, 19/32). The canine feces were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (8.6 %, 6/70). The RPA-CRISPR/Cas12a detection procedure was successfully finished in within 90 min and offers advantages of high sensitivity and specificity, speed and low cost. The technique was better suitable for extensive neosporosis screening in non-laboratory and resource-constrained locations. This study provided a new strategy for more rapid and portable identification of N. caninum.

Microstructure characterization of different types of chlamydospores in Arthrobotrys flagrans.

The morphological and structural differences of different types of chlamydospore of Arthrobotrys flagrans, a nematophagous fungus, were studied under light microscope and electron microscope to provide a reference for the biological control of parasitic nematodiasis. In this study, A. flagrans isolate F088 dormant chlamydospore and nondormant chlamydospore were selected as the research objects. The structural differences of these spores were observed by optical microscopy through lactol cotton blue, Trypan blue, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining. FunXite -1, 4',6-diamidino-2-phenylindole, and calcofluor white staining were used to observe the metabolic activity, cell wall, and nucleus differences of the two types of spores under fluorescence microscope. Ultrastructure of the two kinds of spores was observed using scanning electron microscope (SEM) and transmission electron microscope (TEM). Since lacto phenol cotton blue, trypan blue staining cannot distinguish dormant spores from dead spores, MTT assay was performed. Fluorescence microscopy observation showed that the cytoplasmic metabolic activity of nondormant spores was stronger than that of dormant spores. The nucleus of dormant spores was bright blue, and their fluorescence was stronger than that of nondormant spores. The cell wall of nondormant spores produced stronger yellow-green fluorescence than that of dormant spores. Ultrastructural observation showed that there were globular protuberances on the surface of the two types of spores but with no significant difference between them. The inner wall of dormant spore possesses a thick zona pellucida with high electron density which was significantly thicker than that of nondormant spores, and their cytoplasm is also changed. In this study, the microstructure characteristics of dormant and nondormant chlamydospores of A. flagrans fungi were preliminarily clarified, suggesting that the state of cell wall and intracellular materials were changed after spores entered to dormancy.

Development of an LFD-RPA Assay for Rapid Detection of Pentatrichomonas hominis Infection in Dogs.

Pentatrichomonas hominis is a trichomonad protozoan that infects the cecum and colon of humans and other mammals. It is a zoonotic pathogen that causes diarrhea in both animals and humans. As companion animals, dogs infected with P. hominis pose a risk of transmitting it to humans. Current methods, such as direct smears and polymerase chain reaction (PCR), used for P. hominis detection have limitations, including low detection rates and the need for specialized equipment. Therefore, there is an urgent need to develop rapid, sensitive, and simple detection methods for clinical application. Recombinase polymerase amplification (RPA) has emerged as a technology for rapid pathogen detection. In this study, we developed a lateral flow dipstick (LFD)-RPA method based on the highly conserved SPO11-1 gene for detecting P. hominis infection by optimizing the primers, probes, and reaction conditions, and evaluating cross-reactivity with genomes of Giardia duodenalis and other parasites. The LFD-RPA method was then used to test 128 dog fecal samples collected from Changchun. The results confirmed the high specificity of the method with no cross-reactivity with the five other parasites. The lowest detection limit of the method was 102 copies/µL, and its sensitivity was 100 times higher than that of the conventional PCR method. Consistent with the positivity rate observed using nested PCR, 12 samples (out of 128) tested positive using this method (positivity rate, 9.38%). In conclusion, the LFD-RPA method developed in this study represents a simple and sensitive assay that allows for the rapid detection of P. hominis infection in dogs, especially in this field.

PET imaging with [68Ga]-labeled TGFß-targeting peptide in a mouse PANC-1 tumor model.

Purpose: Transforming growth factor ß (TGFß) is upregulated in many types of tumors and plays important roles in tumor microenvironment construction, immune escape, invasion, and metastasis. The therapeutic effect of antibodies and nuclide-conjugated drugs targeting TGFß has not been ideal. Targeting TGFß with small-molecule or peptide carriers labeled with diagnostic/therapeutic nuclides is a new development direction. This study aimed to explore and confirm the imaging diagnostic efficiency of TGFß-targeting peptide P144 coupled with [68Ga] in a PANC-1 tumor model. Procedures: TGFß-targeting inhibitory peptide P144 with stable activity was prepared through peptide synthesis and screening, and P144 was coupled with biological chelator DOTA and labeled with radionuclide [68Ga] to achieve a stable TGFß-targeting tracer [68Ga]Ga-P144. This tracer was first used for positron emission tomography (PET) molecular imaging study of pancreatic cancer in a mouse PANC-1 tumor model. Results: [68Ga]Ga-P144 had a high targeted uptake and relatively long uptake retention time in tumors and lower uptakes in non-target organs and backgrounds. Target pre-blocking experiment with the cold drug P144-DOTA demonstrated that the radioactive uptake with [68Ga]Ga-P144 PET in vivo, especially in tumor tissue, had a high TGFß-targeting specificity. [68Ga]Ga-P144 PET had ideal imaging efficiency in PANC-1 tumor-bearing mice, with high specificity in vivo and good tumor-targeting effect. Conclusion: [68Ga]Ga-P144 has relatively high specificity and tumor-targeted uptake and may be developed as a promising diagnostic tool for TGFß-positive malignancies.

Development of Nested Polymerase Chain Reaction with Novel Specific Primers for Detection of Tritrichomonas muris Infection in Laboratory Mice.

A variety of rodent ceca are parasitized by Tritrichomonas muris (T. muris), a flagellated protozoan. To date, there are no ideal methods for the detection of T. muris infections in laboratory mice; thus, new molecular methodologies for its specific detection need to be developed. In this study, using staining and SEM, it was observed that T. muris has a pear-shaped body and contains three anterior flagella. A nested PCR system with novel specific primers was designed based on the conserved regions of the SSU rRNA gene of T. muris. The nested PCR system for T. muris showed good specificity and high sensitivity for at least 100 T. muris trophozoites/mL and 0.1 ng/µL of fecal genomic DNA, which means that 176 trophozoites per gram of mouse feces could be detected. When using this nested PCR system, the detection rate was 18.96% (58/306), which was higher than the detection rate of 14.05% (43/306) detected via smear microscopy in fecal samples from five mouse strains. The sensitivity and specificity of nested PCR in detecting T. muris was found to be 100%, and it demonstrated a 26% increase in diagnostic sensitivity compared to the smear microscopy method in the present study. In conclusion, the nested PCR developed with novel primers based on the SSU rRNA gene of T. muris has good accuracy, specificity, and sensitivity for the detection of T. muris infections in laboratory mice.

Giardia VSPAS7 protein attenuates Giardia intestinalis-induced host macrophage pyroptosis.

BACKGROUND: The unicellular protozoan parasite Giardia intestinalis, which primarily infects humans and animals such as cattle and sheep, is having a major negative impact on public health. Giardia is able to evade the recognition and elimination of the host immune system because of the trophozoite surface and extracellular vesicles (EVs) covered by variant-specific surface proteins (VSPs). As key proteins for immune evasion, whether VSPs can regulate Giardia-induced pyroptosis and promote Giardia evasion of host immune responses has not been reported. METHODS: To examine the role of Giardia VSPAS7 on Giardia-induced activation of the signaling pathway, secretion of pro-inflammatory cytokines, pyroptosis and the mechanism involved, we constructed the pcDNA3.1-vspas7 expression plasmid and transfected this plasmid into mouse macrophages. Key proteins for pyroptosis, IL-1ß secretion and LDH release were detected in pcDNA3.1-vspas7-transfected wild-type (WT) cells and NLRP3-deficient cells by western blot, ELISA and LDH assays, respectively. The interactions of Giardia VSPAS7 and mouse NLRP3 were examined using immunofluorescence assays (IFA), co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays. RESULTS: VSPAS7 could decrease the levels of phosphorylated-p65 (P-p65), P-IκBα and P-ERK caused by Giardia and reduce the production levels of Giardia-induced pro-inflammatory cytokine IL-6, IL-12 p40 and TNF-α. The results showed that VSPAS7 inhibited Giardia-mediated activation of NF-κB, ERK/MAPK signaling and secretion of pro-inflammatory cytokines. Furthermore, VSPAS7 suppressed Giardia-induced macrophage pyroptosis by reducing GSDMD cleavage, caspase-1 activation, IL-1ß secretion and LDH release. We further found that VSPAS7 could interact with mouse NLRP3 directly, and in NLRP3-deficient cells the suppression of Giardia-induced macrophage pyroptosis by VSPAS7 was significantly attenuated. CONCLUSIONS: Overall, VSPAS7 could inhibit Giardia-induced activation of signaling pathways and pyroptosis in host macrophages, allowing Giardia evasion of host immune responses. Studies on Giardia VSP-mediated immune evasion provide an important theoretical basis for in-depth studies on Giardia pathogenicity.

Pathological characteristics and congenital immunological responses of pigeons-infected with Neospora caninum.

Pigeons are natural intermediate host of Neospora caninum (N. caninum). In comparison to ruminants, N. caninum causes milder clinical symptoms and less financial loss to pigeons. Natural infectious rates and high prevalence of N. caninum in pigeons, and death cases of N. caninum-infected pigeons under experimental conditions have been reported, but the detailed pathological characteristics and congenital immunological responses of pigeons-infected with N. caninum remain not well described. In this study, pigeons were infected intraperitoneally with 107 N. caninum tachyzoites. N. caninum in tissues was detected by qPCR. Pathological changes of tissues were examined by hematoxylin-eosin staining. Blood smears were prepared for counting eosinophils changes in blood. Heterophil extracellular traps (HETs) in vivo and in vitro were quantified by Pico Green. N. caninum-induced HETs structures were observed by immunofluorescence staining. The model of pigeons-infected with N. caninum was successfully established. Lung and duodenum were the main target organs of pigeons-infected with N. caninum. N. caninum caused hemorrhage, edema and inflammatory cell infiltration in liver, pulmonary congestion and hemorrhage, organizational destruction in lung, and shorter villi or even disappear in duodenum. N. caninum also increased the number of eosinophils in blood of pigeons. Moreover, N. caninum-induced HETs release in the congenital immunological system of pigeons were first demonstrated, and the HETs structures were consisted of DNA as the skeleton and modified with citH3 and elastase. N. caninum-induced HETs release was related with NADPH oxidase, TLR 2 and 4, ERK1/2 and p38 MAPK signaling pathways, and glycolysis. In summary, it is the first report on the detailed pathological characteristics and congenital immunological responses of pigeons-infected with N. caninum, which may provide theoretical basis for the prevention and control of Neosporosis in pigeons.

Rapid, sensitive, and visual detection of Clonorchis sinensis with an RPA-CRISPR/Cas12a-based dual readout portable platform.

Clonorchis sinensis is a food-borne parasite that parasitizes the liver and bile ducts of humans and many animals. This parasite exerts a high burden due to diverse hepatobiliary morbidities (e.g., cholangitis, cholecystitis, cholelithiasis, and cholangiocarcinoma), and an effective detection strategy is urgently needed. CRISPR/Cas12a exhibits nonspecific trans-cleavage activity upon binding to its specific target and has been widely used for nucleic acid detection. In this study, an RPA-CRISPR/Cas12a-based dual readout portable detection platform was established, which shows high sensitivity (one copy/µl) and specificity (no cross-reactivity with common pathogens) by rapid preamplification and combines lateral flow strips and visual fluorescence for visualization of results by the naked eye within 1 h. Moreover, 50 human fecal swabs and 50 fish flesh samples were detected by this platform and nested PCR. The CRISPR/Cas12a-based dual readout portable platform showed 10.0 % (5/50) C. sinensis-positive samples in human fecal swabs and 28.0 % (14/50) in fish flesh, which was consistent with the results of nested PCR. The results demonstrate that our portable platform has the advantages of stability, sensitivity, accuracy, and low equipment requirements. Furthermore, we provide novel point-of-care testing (POCT) for clinical use in remote rural and resource-constrained areas.

Unfolded protein response is involved in resistance to Neospora caninum infection via IRE1α-XBP1s-NOD2 Axis.

Neospora caninum, an intracellular protozoan parasite, causes neosporosis resulting in major losses in the livestock industry worldwide. However, no effective drugs or vaccines have been developed to control neosporosis. An in-depth study on the immune response against N. caninum could help to search for effective approaches to prevent and treat neosporosis. The host unfolded protein response (UPR) functions as a double-edged sword in several protozoan parasite infections, either to initiate immune responses or to help parasite survival. In this study, the roles of the UPR in N. caninum infection in vitro and in vivo were explored, and the mechanism of the UPR in resistance to N. caninum infection was analyzed. The results revealed that N. caninum triggered the UPR in mouse macrophages, such as the activation of the IRE1 and PERK branches, but not the ATF6 branch. Inhibition of the IRE1α-XBP1s branch increased the N. caninum number both in vitro and in vivo, while inhibition of the PERK branch did not affect the parasite number. Furthermore, inhibition of the IRE1α-XBP1s branch reduced the production of cytokines by inhibiting NOD2 signalling and its downstream NF-κB and MAPK pathways. Taken together, the results of this study suggest that the UPR is involved in the resistance of N. caninum infection via the IRE1α-XBP1s branch by regulating NOD2 and its downstream NF-κB and MAPK pathways to induce the production of inflammatory cytokines, which provides a new perspective for the research and development of anti-N. caninum drugs.

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.

A potential role for Giardia chaperone protein GdDnaJ in regulating Giardia proliferation and Giardiavirus replication.

BACKGROUND: Giardia duodenalis (referred to as Giardia) is a flagellated binucleate protozoan parasite, which causes one of the most common diarrheal diseases, giardiasis, worldwide. Giardia can be infected by Giardiavirus (GLV), a small endosymbiotic dsRNA virus belongs to the Totiviridae family. However, the regulation of GLV and a positive correlation between GLV and Giardia virulence is yet to be elucidated. METHODS: To identify potential regulators of GLV, we performed a yeast two-hybrid (Y2H) screen to search for interacting proteins of RdRp. GST pull-down, co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) assay were used to verify the direct physical interaction between GLV RdRp and its new binding partner. In addition, their in vivo interaction and colocalization in Giardia trophozoites were examined by using Duolink proximal ligation assay (Duolink PLA). RESULTS: From Y2H screen, the Giardia chaperone protein, Giardia DnaJ (GdDnaJ), was identified as a new binding partner for GLV RdRp. The direct interaction between GdDnaJ and GLV RdRp was verified via GST pull-down, co-immunoprecipitation and BiFC. In addition, colocalization and in vivo interaction between GdDnaJ and RdRp in Giardia trophozoites were confirmed by Duolink PLA. Further analysis revealed that KNK437, the inhibitor of GdDnaJ, can significantly reduce the replication of GLVs and the proliferation of Giardia. CONCLUSION: Taken together, our results suggested a potential role of GdDnaJ in regulating Giardia proliferation and GLV replication through interaction with GLV RdRp.

Immunogenicity and efficacy of serogroup A and D bacterins against Pasteurella multocida in mice.

Introduction: Pasteurella multocida is a widespread respiratory pathogen in pigs, causing swine pneumonia and atrophic rhinitis, and the capsular serogroups A and D are the main epidemic serogroups in infected animals. This study investigated the protective effects of serogroup A and D bacterins against current circulating P. multocida strains, to better understand the immunity generated by bacterins. Method: 13 serogroup A (seven A: L3 and six A: L6 strains) and 13 serogroup D (all D: L6 strains) P. multocida strains were isolated, and used as inactivated whole cell antigen to prepare P. multocida bacterins. Mice were immunized with these bacterins at 21-day interval and intraperitoneally challenged with the homologous and heterologous P. multocida strains, respectively. The antibody titer levels and immunization protective efficacy of vaccines were evaluated. Results: All of the bacterins tested induced high titer levels of immunoglobulin G antibodies against the parental bacterial antigen in mice. Vaccination with the six A: L6 bacterins provided no protection against the parent strain, but some strains did provide heterologous protection against A: L3 strains. Vaccination with the seven A: L3 bacterins provided 50%-100% protection against the parent strain, but none gave heterologous protection against the A:L6 strains. Immunization with the thirteen D: L6 bacterins offered 60%-100% protection against the parent strain, and almost all D: L6 strains gave cross-protection. Discussion: This study found that the cross-protectivity of serogroup A strains was poor, while serogroup D strains was effective, which provided some insights for P. multocida vaccine development.

The NLRP3 inflammasome recognizes alpha-2 and alpha-7.3 giardins and decreases the pathogenicity of Giardia duodenalis in mice.

BACKGROUND: Giardia duodenalis is a parasitic organism that can cause giardiasis, an intestinal infection, particularly prevalent in young children, with clinical symptoms of diarrhea. We previously reported that extracellular G. duodenalis triggers intracellular nucleotide-binding oligomerization-like receptor 3 (NLRP3) inflammasome activation and regulates the host inflammatory response by secreting extracellular vesicles (EVs). However, the exact pathogen-associated molecular patterns in G. duodenalis EVs (GEVs) involved in this process and the role of the NLRP3 inflammasome in giardiasis remain to be elucidated. METHODS: Recombinant eukaryotic expression plasmids of pcDNA3.1(+)-alpha-2 and alpha-7.3 giardins in GEVs were constructed, transfected into primary mouse peritoneal macrophages and screened by measuring the expression levels of the inflammasome target molecule caspase-1 p20. The preliminary identification of G. duodenalis alpha-2 and alpha-7.3 giardins was further verified by measuring the protein expression levels of key molecules of the NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1ß], pro-caspase-1, and caspase-1 p20), the secretion levels of IL-1ß, the level of apoptosis speck-like protein (ASC) oligomerization and the immunofluorescence localization of NLRP3 and ASC. The roles of the NLRP3 inflammasome in G. duodenalis pathogenicity were then evaluated using mice in which NLRP3 activation was blocked (NLRP3-blocked mice), and body weight, parasite burden in the duodenum and histopathological changes in the duodenum were monitored. In addition, we explored whether alpha-2 and alpha-7.3 giardins triggered IL-1ß secretion in vivo through the NLRP3 inflammasome and determined the roles of these molecules in G. duodenalis pathogenicity in mice. RESULTS: Alpha-2 and alpha-7.3 giardins triggered NLRP3 inflammasome activation in vitro. This led to caspase-1 p20 activation, upregulation of the protein expression levels of NLRP3, pro-IL-1ß and pro-caspase-1, significant enhancement of IL-1ß secretion, ASC speck formation in the cytoplasm and also induction of ASC oligomerization. Deletion of the NLRP3 inflammasome aggravated G. duodenalis pathogenicity in mice. Compared to wild-type mice gavaged with cysts, mice gavaged with cysts in NLRP3-blocked mice displayed increased trophozoite loads and severe duodenal villus damage, characterized by necrotic crypts with atrophy and branching. In vivo assays revealed that alpha-2 and alpha-7.3 giardins could induce IL-1ß secretion through the NLRP3 inflammasome and that immunization with alpha-2 and alpha-7.3 giardins decreased G. duodenalis pathogenicity in mice. CONCLUSIONS: Overall, the results of the present study revealed that alpha-2 and alpha-7.3 giardins trigger host NLRP3 inflammasome activation and decrease G. duodenalis infection ability in mice, which are promising targets for the prevention of giardiasis.

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.

Cryptosporidium parvum maintains intracellular survival by activating the host cellular EGFR-PI3K/Akt signaling pathway.

Autophagy is a critical cellular mechanism in helping infected cells remove intracellular pathogens and is countered by pathogens maintaining intracellular survival by regulating autophagy through the manipulation of the host cellular signal transduction pathway. Cryptosporidium parvum is a zoonotic intracellular but extracytoplasmic protozoon that causes diarrhea in infants and young children worldwide. However, it is still unclear how Cryptosporidium adapts to intracellular survival. In the present study, we demonstrated that C. parvum could activate the EGFR-PI3K/Akt signaling pathway to promote intracellular survival in HCT-8 cells. The western blot results showed that C. parvum induced EGFR and Akt phosphorylation in HCT-8 cells. The EGFR inhibitor AG1478 decreased EGFR and Akt phosphorylation, and the PI3K inhibitor LY294002 impaired Akt phosphorylation induced by C. parvum in HCT-8 cells. Inhibition of EGFR or Akt decreased the number of intracellular parasites. Second, low-dose infection of C. parvum triggered complete autophagy and enhanced autophagic flux in HCT-8 cells. The expressions of mTOR and p62 were decreased, and the expressions of LC3 and Beclin1 were increased in C. parvum-infected HCT-8 cells. Transfection with siBeclin1 or siATG7 reduced LC3 accumulation, while lysosome inhibitor E64d+pepA increased LC3 accumulation induced by C. parvum in HCT-8 cells. Intracellular parasite proliferation was decreased when treated with autophagy inducer rapamycin, whereas autophagy inhibitor 3-MA, E64d+pep A, siBeclin1 or siATG7 increased intracellular parasites. Third, C. parvum inhibited autophagy killing to promote its own intracellular survival by activating EGFR-Akt signaling pathway. The EGFR inhibitor AG1478 enhanced autophagic flux, and Akt inhibitor IV increased LC3 accumulation and inhibited C. parvum proliferation in HCT-8 cells. Akt inhibitor IV-inhibited C. parvum proliferation was attenuated by E64d+pepA. In summary, C. parvum could maintain intracellular survival by inhibiting autophagy via EGFR-PI3K/Akt pathway. These results revealed a new mechanism for the interaction of C. parvum with host cells.