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.

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.

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.

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.

Multiple Regulations of Parasitic Protozoan Viruses: A Double-Edged Sword for Protozoa.

Parasite infections affect human and animal health significantly and contribute to a major burden on the global economy. Parasitic protozoan viruses (PPVs) affect the protozoan parasites' morphology, phenotypes, pathogenicity, and growth rates. This discovery provides an opportunity to develop a novel preventive and therapeutic strategy for parasitic protozoan diseases (PPDs). Currently, there is greater awareness regarding PPVs; however, knowledge of viruses and their associations with host diseases remains limited. Parasite-host interactions become more complex owing to PPVs; however, few studies have investigated underlying viral regulatory mechanisms in parasites. In this study, we reviewed relevant studies to identify studies that investigated PPV development and life cycles, the triangular association between viruses, parasites, and hosts, and the effects of viruses on protozoan pathogenicity. This study highlights that viruses can alter parasite biology, and viral infection of parasites may exacerbate the adverse effects of virus-containing parasites on hosts or reduce parasite virulence. PPVs should be considered in the prevention of parasitic epidemics and outbreaks, although their effects on the host and the complexity of the triangular association between PPVs, protozoans, and hosts remain unclear. IMPORTANCE PPVs-based regulation of parasitic protozoa can provide a theoretical basis and direction for PPD prevention and control, although PPVs and PPV regulatory mechanisms remain unclear. In this review, we investigated the differences between PPVs and the unique properties of each virus regarding virus discovery, structures, and life cycles, focused on the Trichomonas vaginalis virus, Giardia lamblia virus, Leishmania RNA virus, and the Cryptosporidium parvum virus 1. The triangular association between PPVs, parasitic protozoa, and hosts reveals the "double-edged sword" property of PPVs, which maintains a balance between parasitic protozoa and hosts in both positive and negative respects. These studies discuss the complexity of parasitic protozoa and their co-existence with hosts and suggest novel pathways for using PPVs as tools to gain a deeper understanding of protozoal infection and treatment.

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.

Giardia duodenalis Induces Proinflammatory Cytokine Production in Mouse Macrophages via TLR9-Mediated p38 and ERK Signaling Pathways.

Giardia duodenalis, also known as Giardia lamblia or Giardia intestinalis, is an important opportunistic, pathogenic, zoonotic, protozoan parasite that infects the small intestines of humans and animals, causing giardiasis. Several studies have demonstrated that innate immunity-associated Toll-like receptors (TLRs) are critical for the elimination of G. duodenalis; however, whether TLR9 has a role in innate immune responses against Giardia infection remains unknown. In the present study, various methods, including reverse transcriptase-quantitative polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, immunofluorescence, inhibitor assays, and small-interfering RNA interference, were utilized to probe the role of TLR9 in mouse macrophage-mediated defenses against G. lamblia virus (GLV)-free or GLV-containing Giardia trophozoites. The results revealed that in G. duodenalis-stimulated mouse macrophages, the secretion of proinflammatory cytokines, including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-12 p40, was enhanced, concomitant with the significant activation of TLR9, whereas silencing TLR9 attenuated the host inflammatory response. Notably, the presence of GLV exacerbated the secretion of host proinflammatory cytokines. Moreover, G. duodenalis stimulation activated multiple signaling pathways, including the nuclear factor κB p65 (NF-κB p65), p38, ERK, and AKT pathways, the latter three in a TLR9-dependent manner. Additionally, inhibiting the p38 or ERK pathway downregulated the G. duodenalis-induced inflammatory response, whereas AKT inhibition aggravated this process. Taken together, these results indicated that G. duodenalis may induce the secretion of proinflammatory cytokines by activating the p38 and ERK signaling pathways in a TLR9-dependent manner in mouse macrophages. Our in vitro findings on the mechanism underlying the TLR9-mediated host inflammatory response may help establish the foundation for an in-depth investigation of the role of TLR9 in the pathogenicity of G. duodenalis.

Construction and evaluation of a fluorescence-based live attenuated Escherichia coli delivery system for generating oral vaccine candidate.

Enter toxigenic Escherichia coli (ETEC) is a major pathogen of swine industry that can have a substantial impact on morbidity and mortality. Therefore, it is necessary to develop effective vaccines for the prevention of ETEC infection. Live attenuated bacteria delivery system are effective tools for mucosal immunization. The purpose of this study was to construct a novel delivery system that can present the LTR192G-STb fusion protein as oral vaccine candidate. Firstly, the PRPL-mKate2 fluorescent cassette was inserted into the genome (yaiT pseudogene) of an attenuated E. coli by homologous recombination methods to construct the delivery system O142(yaiT::PRPL-mKate2). Secondly, the oral vaccine O142(yaiT:: LT192-STb) (ER-B) was derived for replacing the PRPL-mKate2 by LT192-STb fusion gene, and then it was tested for its feasibility as oral vaccine candidate. Subsequently, BALB/c mice were orogastrically immunized with ER-B. Results showed that mice orally immunized with ER-B produced high levels of specific IgA and IgG antibodies. The induced antibodies demonstrated neutralizing effects to enter toxins LT and STb. In addition, results of cellular immune responses showed that stimulation index values of immunized mice were significantly higher than the control group (P < 0.05) and with a marked shift towards Th 2 immunity. These data indicated that the recombinant E. coli ER-B could be a valuable candidate of future vaccines against ETEC infection.

Simultaneous oral immunization of mice with live attenuated Escherichia coli expressing LT192-STa 13 and LT 192-STb fusion immunogen, respectively, for polyvalent vaccine candidate.

Previous epidemiological study showed that most of the porcine enterotoxin Escherichia coli (ETEC) strains harbor multiple enterotoxins but lack any of the fimbriae or non-fimbrial adhesion genes. Therefore, effective ETEC vaccines need to aim directly at all the enterotoxin antigens. The objective of this study was to evaluate the simultaneous immune effect of two live attenuated E. coli strains expressing LTR192G-STaA13Q and LTR192G-STb fusion immunogen, respectively. The results showed that both local mucosal and systemic immune responses against LT, STa, STb, and F41 were induced in BALB/c mice immunized orally with the recombinant E. coli strains ER-A and ER-B simultaneously. In addition, results of cellular immune responses showed that stimulation index (SI) values of immunized mice were significantly higher than control mice (P < 0.05) and a marked shift toward type-2 helper T lymphocyte (Th 2) immunity. Moreover, the induced antibodies demonstrated neutralizing effects on LT, STa, and STb producing E. coli infection. These data indicated that the use of recombinant E. coli ER-A and ER-B could be a valuable strategy for future polyvalent vaccine development of ETEC.

Generation of an attenuated strain oral vaccine candidate using a novel double selection platform in Escherichia coli.

Live attenuated bacteria delivered orally are interesting tools for mucosal immunization. The objective of this study was to construct a novel counter-selection platform based on an attenuated wild-type Escherichia coli (E. coli) strain and to utilize it for the delivery of LTR192G-STaA13Q fusion protein as an oral vaccine. First, a counter-selectable marker, namely, PRPL-Kil, was inserted into an attenuated wild-type E. coli strain through the use of the red and G-DOC homologous recombination systems to construct the counter-selection platform, and PRPL-Kil was subsequently replaced by the LT192-STa13 fusion gene to construct the oral vaccine O142 (yaiT::LT192-STa13) (ER-A). Subsequently, BALB/c mice were orogastrically inoculated with ER-A. Our results showed that ER-A could induce the production of specific IgA and IgG against fimbriae (F41) and enterotoxins (LT and STa), with neutralizing activity in BALB/c mice. In addition, assays of cellular immune responses showed that the stimulation index (SI) values of immunized mice were significantly higher than those of control mice (P<0.05), and revealed a marked shift toward Th2-mediated immunity. These findings suggest that ER-A is a suitable candidate for an oral vaccine strain to protect animals from enter toxigenic Escherichia coli (ETEC) infection.