Oral delivery of Eimeria acervulina transfected sequentially with two copies of the VP2 gene induces immunity against infectious bursal disease virus in chickens.

Chicken coccidiosis caused by Eimeria spp. can occur on almost all poultry farms, causing huge economic losses to the industry. Genetically manipulated Eimeria parasites as a vaccine vector to deliver viral antigens have been reported. In our preliminary study, transgenic E. acervulina expressing a VP2 gene (Ea-VP2) of the infectious bursal disease virus (IBDV) demonstrated partial protection against IBDV infection. To enhance immune responses, we aimed to increase the VP2 gene copy number in transgenic E. acervulina. In this study, we used a novel plasmid vector carrying a VP2 gene fused with three flag tags and a red fluorescent reporter gene (mCherry). The vector was introduced into Ea-VP2 sporozoites through nucleofection, leading to the generation of Ea-2VP2. Subsequent analysis revealed a notable escalation in the fluorescent rate, increasing from 0.11 to 95.1% following four consecutive passages facilitated by fluorescent-activated cell sorting. Verification via PCR, Western blot, and immunofluorescence confirmed the successful construction of the Ea-2VP2 population. Despite lower fecundity compared to wild-type E. acervulina, Ea-2VP2 maintained immunogenicity. Our research effectively created a transgenic E. acervulina strain transfected sequentially with two copies of the VP2 gene from IBDV. This modification resulted in an increased humoral immune response after primary immunization in chickens. Additionally, it demonstrated a degree of protection within the bursa against IBDV infection. Future studies will focus on further enhancing immune response levels.

Anti-inflammatory and immunoregulatory effects of colistin sulphate on human PBMCs.

In previous studies, CST has been identified as having an immunostimulatory effect on Caenorhabditis elegans and macrophage of rats. Here, we further investigated its immunomodulatory effects on human peripheral blood mononuclear cells (PBMCs). LPS-stimulated PBMCs inflammatory model was established. Flow cytometry was applied to measure phagocytosis of PBMCs. Cytokine mRNA and protein expression levels of LPS-stimulated PBMCs with or without CST were measured by qRT-PCR and ELISA. The transcriptomic profile of CST-treated PBMCs was investigated by RNA-sequencing. Gene Ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) were applied to find potential signalling pathways. PBMCs showed a significant increase in phagocytic activity at 6 h after being incubated with CST at the concentration of 10 µg/mL. In the presence of LPS, CST maintained and promoted the expression of TNF-α and chemokine CCL24. The content of pro-inflammatory cytokines, such as IL-1ß, IL-6 and IFN-γ, which were released from LPS-stimulated PBMCs, was reduced by CST at 6 h. Anti-inflammatory cytokines, such as IL-4, IL-13 and TGF-ß1, were significantly increased by CST at 24 h. A total of 277 differentially expressed immune-related genes (DEIRGs) were detected and cytokine-cytokine receptor interaction was highly enriched. CST presented obvious anti-inflammatory and immunoregulatory effects in LPS-induced PBMCs inflammatory model not only by improving the ability of PBMCs to clear pathogens but also by decreasing pro-inflammatory cytokines and increasing anti-inflammatory cytokines. And the mechanism may be related to cytokine-cytokine receptor interaction.

Developing efficient strategies for localizing the enhanced yellow fluorescent protein subcellularly in transgenic Eimeria parasites.

Eimeria species serve as promising eukaryotic vaccine vectors. And that the location of heterologous antigens in the subcellular components of genetically modified Eimeria may determine the magnitude and type of immune responses. Therefore, our study aimed to target a heterologous fluorescent protein to the cell surface or microneme, two locations where are more effective in inducing protective immunity, of Eimeria tenella and E. acervulina sporozoites. We used an enhanced yellow fluorescent protein (EYFP) as a tagging biomarker, fusing variously with some localization or whole sequences of compartmental proteins for targeting. After acquiring stable transgenic Eimeria populations, we observed EYFP expressing in expected locations with certain strategies. That is, EYFP successfully localized to the surface when it was fused between signal peptides and mature products of surface antigen 1 (SAG1). Furthermore, EYFP was efficiently targeted to the apical end, an optimal location for secretory organelle known as the microneme, when fused to the C terminus of microneme protein 2. Unexpectedly, EYFP exhibited dominantly in the apical end with only weak expression on the surface of the transgenic sporozoites when the parasites were transfected with plasmid with EYFP fused between signal peptides and mature products of E. tenella SAG 13. These strategies worked in both E. tenella and E. acervulina, laying a solid foundation for studying E. tenella and E. acervulina-based live vaccines that can be further tailored to the inclusion of cargo immunogens from other pathogens.

Microneme-located VP2 in Eimeria acervulina elicits effective protective immunity against infectious bursal disease virus.

Using transgenic Eimeria spp. to deliver exogenous antigens is a viable option for developing multivalent live vaccines. Previous research revealed that the location of antigen expression in recombinant Eimeria dictates the magnitude and type of immune responses. In this study, we constructed genetically modified Eimeria acervulina that expressed VP2 protein, a protective antigen from infectious bursal disease virus (IBDV), on the surface or in the microneme of sporozoites. After vaccination, VP2-specific antibody was readily detected in specific pathogen-free chickens receiving transgenic E. acervulina parasites expressing VP2 in microneme, but animals vaccinated with which expressing VP2 on surface failed to produce detectable antibody after two times immunizations. Moreover, the bursal lesion of microneme-located VP2 transgenic E. acervulina immunized chickens was less severe compared with un-immunized animals after IBDV challenge infection. Therefore, genetically modified E. acervulina that express IBDV-derived VP2 in micronemes are effective in inducing specific antibody responses against VP2, while parasites that have VP2 expression on cell surface are not suitable. Thus, the use of Eimeria parasites as vaccine vectors needs to consider the proper targeting of exogenous immunogens. Our results have implications for the design of other vector vaccines.

Extended theory of planned behavior to explain the influence mechanism of low-speed driving behavior.

Low-speed driving is an underestimated dangerous behavior that may cause safety issues, such as speed dispersion and traffic flow bottlenecks. To investigate the influence mechanism of low-speed driving behavior, this study constructed the low-speed specific model (LSSM) by extending theory of planned behavior (TPB). The LSSM incorporated two factors, namely, risk perception and behavior habit, into the standard TPB components (attitude, subjective norm, perceived behavioral control, and behavior intention). Web-based questionnaires were used to collect data from a valid sample of 374, of which males accounted for 50%. The participants were aged from 18 to 65 years (M = 35.40, SD = 0.88). The structural equation model was applied to calculate and validate the interrelationships among the components of LSSM. Results showed that the LSSM could explain the variance in low-speed driving behavior and behavior intention by 46% and 76%, respectively. Meanwhile, attitude (ß = 0.52, p < 0.001) and behavior habit (ß = 0.48, p < 0.001) had the strongest positive influence and prediction power over low-speed driving behavior, respectively, whereas subjective norm (ß = 0.05, p > 0.01) and perceived behavioral control (ß = -0.12, p > 0.01) showed few significant in influencing the intention. LSSM also showed that people who were sensitive to driving risk perception would avoid low-speed driving behaviors and attitudes. Our findings may provide theoretical support for interventions on low-speed driving behavior.

Distinct non-synonymous mutations in cytochrome b highly correlate with decoquinate resistance in apicomplexan parasite Eimeria tenella.

BACKGROUND: Protozoan parasites of the genus Eimeria are the causative agents of chicken coccidiosis. Parasite resistance to most anticoccidial drugs is one of the major challenges to controlling this disease. There is an urgent need for a molecular marker to monitor the emergence of resistance against anticoccidial drugs, such as decoquinate. METHODS: We developed decoquinate-resistant strains by successively exposing the Houghton (H) and Xinjiang (XJ) strains of E. tenella to incremental concentrations of this drug in chickens. Additionally, we isolated a decoquinate-resistant strain from the field. The resistance of these three strains was tested using the criteria of weight gain, relative oocyst production and reduction of lesion scores. Whole-genome sequencing was used to identify the non-synonymous mutations in coding genes that were highly associated with the decoquinate-resistant phenotype in the two laboratory-induced strains. Subsequently, we scrutinized the missense mutation in a field-resistant strain for verification. We also employed the AlphaFold and PyMOL systems to model the alterations in the binding affinity of the mutants toward the drug molecule. RESULTS: We obtained two decoquinate-resistant (DecR) strains, DecR_H and XJ, originating from the original H and XJ strains, respectively, as well as a decoquinate-resistant E. tenella strain from the field (DecR_SC). These three strains displayed resistance to 120 mg/kg decoquinate administered through feed. Through whole-genome sequencing analysis, we identified the cytochrome b gene (cyt b; ETH2_MIT00100) as the sole mutated gene shared between the DecR_H and XJ strains and also detected this gene in the DecR_SC strain. Distinct non-synonymous mutations, namely Gln131Lys in DecR_H, Phe263Leu in DecR_XJ, and Phe283Leu in DecR_SC were observed in the three resistant strains. Notably, these mutations were located in the extracellular segments of cyt b, in close proximity to the ubiquinol oxidation site Qo. Drug molecular docking studies revealed that cyt b harboring these mutants exhibited varying degrees of reduced binding ability to decoquinate. CONCLUSIONS: Our findings emphasize the critical role of cyt b mutations in the development of decoquinate resistance in E. tenella. The strong correlation observed between cyt b mutant alleles and resistance indicates their potential as valuable molecular markers for the rapid detection of decoquinate resistance.

Live attenuated anticoccidial vaccines for chickens.

Chicken coccidiosis, caused by infection with single or multiple Eimeria species, results in significant economic losses to the global poultry industry. Over the past decades, considerable efforts have been made to generate attenuated Eimeria strains, and the use of live attenuated anticoccidial vaccines for disease prevention has achieved tremendous success. In this review, we evaluate the advantages and limitations of the methods of attenuation as well as attenuated Eimeria strains in a historical perspective. Also, we summarize the recent exciting research advances in transient/stable transfection systems and clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing developed for Eimeria parasites, and discuss trends and challenges of developing live attenuated anticoccidial vaccines based on transgenesis and genome editing.

Comparative transcriptome profiling of Eimeria tenella in various developmental stages and functional analysis of an ApiAP2 transcription factor exclusively expressed during sporogony.

BACKGROUND: The apicomplexan parasites Eimeria spp. are the causative agents of coccidiosis, a disease with a significant global impact on the poultry industry. The complex life cycle of Eimeria spp. involves exogenous (sporogony) and endogenous (schizogony and gametogony) stages. Unfortunately, the genetic regulation of these highly dynamic processes, particularly for genes involved in specific developmental phases, is not well understood. METHODS: In this study, we used RNA sequencing (RNA-Seq) analysis to identify expressed genes and differentially expressed genes (DEGs) at seven time points representing different developmental stages of Eimeria tenella. We then performed K-means clustering along with co-expression analysis to identify functionally enriched gene clusters. Additionally, we predicted apicomplexan AP2 transcription factors in E. tenella using bioinformatics methods. Finally, we generated overexpression and knockout strains of ETH2_0411800 to observe its impact on E. tenella development. RESULTS: In total, we identified 7329 genes that are expressed during various developmental stages, with 3342 genes exhibiting differential expression during development. Using K-means clustering along with co-expression analysis, we identified clusters functionally enriched for oocyte meiosis, cell cycle, and signaling pathway. Among the 53 predicted ApiAP2 transcription factors, ETH2_0411800 was found to be exclusively expressed during sporogony. The ETH2_0411800 overexpression and knockout strains did not exhibit significant differences in oocyst size or output compared to the parental strain, while the resulting ETH2_0411800 knockout parasite showed a relatively small oocyst output. CONCLUSIONS: The findings of our research suggest that ETH2_0411800 is not essential for the growth and development of E. tenella. Our study provides insights into the gene expression dynamics and is a valuable resource for exploring the roles of transcription factor genes in regulating the development of Eimeria parasites.

Forward genetic analysis of monensin and diclazuril resistance in Eimeria tenella.

Worldwide distributed coccidiosis is caused by infection of both Eimeria species and Cystoisospora in the host intestine and causes huge economic losses to the livestock industry, especially the poultry industry. The control of such diseases relies mainly on chemoprophylaxis with anticoccidials, which has led to a very common drug resistance in this field. However, the genetic mechanisms underlying resistance to many anticoccidial drugs remain unknown. In this study, strains of E. tenella resistant to 250 mg/kg monensin were generated and characterized. Forward genetic approaches based on pooled genome sequencing, including experimental evolution and linkage group selection, were used to locate candidate targets responsible for resistance to monensin and diclazuril in E. tenella. A total of 16 nonsynonymous mutants in protein-coding genes were identified in monensin-resistant strains, and two genomic regions with strong selection signals were also detected in diclazuril-resistant strains. Our study reveals the genetic characterization of the experimental evolution and linkage group selection in Eimeria species, and also provides important information that contributes to the understanding of the molecular mechanism of drug resistance in coccidia.

Transcriptome profile of halofuginone resistant and sensitive strains of Eimeria tenella.

The antiparasitic drug halofuginone is important for controlling apicomplexan parasites. However, the occurrence of halofuginone resistance is a major obstacle for it to the treatment of apicomplexan parasites. Current studies have identified the molecular marker and drug resistance mechanisms of halofuginone in Plasmodium falciparum. In this study, we tried to use transcriptomic data to explore resistance mechanisms of halofuginone in apicomplexan parasites of the genus Eimeria (Apicomplexa: Eimeriidae). After halofuginone treatment of E. tenella parasites, transcriptome analysis was performed using samples derived from both resistant and sensitive strains. In the sensitive group, DEGs associated with enzymes were significantly downregulated, whereas the DNA damaging process was upregulated after halofuginone treatment, revealing the mechanism of halofuginone-induced parasite death. In addition, 1,325 differentially expressed genes (DEGs) were detected between halofuginone resistant and sensitive strains, and the DEGs related to translation were significantly downregulated after halofuginone induction. Overall, our results provide a gene expression profile for further studies on the mechanism of halofuginone resistance in E. tenella.

Tissue-resident, memory CD8+ T cells are effective in clearing intestinal Eimeria falciformis reinfection in mice.

Eimeria, a cousin of malarial parasites, causes coccidiosis that results in huge losses in the poultry industry. Although live coccidiosis vaccines have been developed and used widely for the successful control of the disease, the mechanism underlying protective immunity remains largely unknown. Using Eimeria falciformis as a model parasite, we observed that tissue-resident memory CD8+ T (Trm) cells accumulated in cecal lamina propria following E. falciformis infection in mice, especially after reinfection. In convalescent mice challenged with a second infection, E. falciformis burden diminished within 48-72 h. Deep-sequencing revealed that CD8+ Trm cells were characterized by rapid up-regulation of effector genes encoding pro-inflammatory cytokines and cytotoxic effector molecules. While FTY720 (Fingolimod) treatment prevented the trafficking of CD8+ T cells in peripheral circulation and exacerbated primary E. falciformis infection, such treatment had no impact on the expansion of CD8+ Trm cells in convalescent mice receiving secondary infection. Adoptive transfer of cecal CD8+ Trm cells conferred immune protection in naïve mice, indicating that these cells provide direct and effective protection against infection. Overall, our findings not only explain a protective mechanism of live oocyst-based anti-Eimeria vaccines but also provide a valuable correlate for assessing vaccines against other protozoan diseases.

EtcPRS Mut as a molecular marker of halofuginone resistance in Eimeria tenella and Toxoplasma gondii.

The control of coccidiosis, causing huge economic losses in the poultry industry, is facing the stagnation of the development of new drugs and the emergence of drug resistance. Thus, the priority for coccidiosis control is to decipher the effect mechanisms and resistance mechanisms of anticoccidial drugs. In this study, we mined and validated a molecular marker for halofuginone resistance in Eimeria tenella through forward and reverse genetic approaches. We screened whole-genome sequencing data and detected point mutations in the ETH2_1020900 gene (encoding prolyl-tRNA synthetase, PRS). Then, we introduced this mutated gene into E. tenella and Toxoplasma gondii and validated that overexpression of this mutated gene confers resistance to halofuginone in vivo and in vitro. These results together show that mutations A1852G and A1854G on the ETH2_1020900 gene are pivotal to halofuginone resistance in E. tenella, encouraging the exploration of mechanisms of drug resistance against other anticoccidial drugs in eimerian parasites.

Evidence of high-efficiency cross fertilization in Eimeria acervulina revealed using two lines of transgenic parasites.

Eimeria species are apicomplexan parasites with a direct life cycle consisting of a replicative phase involving multiple rounds of asexual replication in the intestine or other organs including kidneys, liver, and gallbladder, depending on the species, followed by a sexual phase or gamogony involving the development and fertilization of gametes, an essential process for Eimeria transmission. Recent advances in the genetic manipulation of these parasites made it possible to conduct genetic crosses combined with genomic approaches to elucidate the genetic determinants of Eimeria development, virulence, drug resistance, and immune evasion. Here, we employed genetic techniques to generate two transgenic Eimeria acervulina lines, EaGAM56 and EaHAP2, each expressing two unique fluorescent proteins, with one controlled by a constitutive promotor for cross-efficiency analysis and the other by a male or female gametocyte stage-specific promoter to observe sexual development. The expression of fluorescent proteins in the transgenic lines was analyzed in different developmental stages of the E. acervulina life cycle by immunoblotting and by examination of frozen sections using fluorescence microscopy. The effect of infective doses on cross-fertilization was further investigated by conducting several genetic crosses between the two transgenic lines at different doses and ratios. Two transgenic lines expressing constitutive and gametocyte-specific fluorescence proteins were generated and characterized. These transgenic parasites display synchronous development in chickens, comparable with that of the wild type. Genetic crosses between the two transgenic parasites showed that a high rate of oocysts co-expressing the two reporters could be achieved following inoculation with high doses of infective oocysts. We further showed that the proportion of co-transfected oocysts can be modulated by altering the ratio of the transgenic parental lines. Higher infective doses and similar numbers of functional gametocytes from the parents increase the rate of cross-fertilization. Our data highlight the usefulness of genetic manipulation and fluorescently-labeled transgenic gametocytes as tools to study Eimeria development and to elucidate the factors that modulate sexual development. This work sets the stage for the implementation of novel approaches to investigate other aspects of Eimeria pathogenesis, virulence, and drug susceptibility and resistance.

Atractylodes-I Overcomes the Oxidative Stress-induced Colonic Mucosal Epithelial Cells Dysfunction to Prevent Irritable Bowel Syndrome Via Modulating the miR-34a-5p-LDHA Signaling Pathway.

BACKGROUND: Irritable bowel syndrome (IBS) is a known brain-gut disorder. Currently, the molecular and cellular mechanisms of IBS remain unclear. Atractylenolide-I (ATL-I) is a majorly bioactive component extracted from Rhizoma Atractylodes Macrocephalae. METHODS: Studies have revealed that ATL-I functioned as an anti-tumor drug in various cancers. However, the effects and molecular mechanisms of ATL-I on the pathological processes of colonic mucosal epithelial cells (CMECs) during IBS remain unclear. This study reports ATL-I effectively alleviated the oxidative stress-induced colonic mucosal epithelial cell dysfunction. In colonic mucosal tissues from IBS patients, we detected upregulated miR-34a-5p and suppressed glucose metabolism enzyme expressions. Under H2O2 treatment which mimics in vitro oxidative stress, miR-34a-5p was induced and glucose metabolism was inhibited in the colon mucosal epithelial cell line, NCM460. Meanwhile, ATL-I treatment effectively overcame the oxidative stress-induced miR-34a- 5p expression and glucose metabolism in NCM460 cells. RESULT: By bioinformatics analysis, Western blot and luciferase assay, we illustrated that miR-34a-5p directly targeted the 3'UTR region of glucose metabolism key enzyme, lactate dehydrogenase-A (LDHA) in colonic mucosal epithelial cells. Rescue experiments validated that miR-34a-5p inhibited glucose metabolism by targeting LDHA. Finally, we demonstrated that ATL-I treatment reversed the miR-34a-5p-inhibited glucose metabolism and -exacerbated colonic mucosal epithelial cell dysfunction under oxidative stress by modulating the miR-34a-5p-LDHA pathway. CONCLUSION: Summarily, our study reports the roles and mechanisms of ATL-I in the oxidative stress-induced colonic mucosal epithelial cell dysfunction during IBS through regulating the miR-34a-5p-LDHA-glucose metabolism axis.

A comparative study of microRNAs in different stages of Eimeria tenella.

Apicomplexan parasites have divergent biogenesis machinery for small RNA generation. Analysis has shown that parasites in Plasmodium and Cryptosporidium as well as many species in Leishmania or Trypanosoma do not have a complete machinery in small RNA biogenesis. Recently, the miRNA-generating system of Toxoplasma has been identified as plant/fungal-like and its miRNAome has been elucidated. However, the microRNA (miRNA) expression profiles and their potential regulatory functions in different stages of Eimeria tenella remain largely unknown. In this study, we characterized the RNA silencing machinery of E. tenella and investigated the miRNA population distribution at different life stages by high-throughput sequencing. We characterized the expression of miRNAs in the unsporulated oocyst, sporulated oocyst and schizogony stages, obtaining a total of 392 miRNAs. We identified 58 differentially expressed miRNAs between USO (unsporulated oocysts) and SO (sporulated oocysts) that were significantly enriched for their potential target genes in the regulation of gene expression and chromatin binding, suggesting an epigenetic modulation of sporulating by these miRNAs. In comparing miRNA expression at endogenous and exogenous developmental stages, twenty-four miRNAs were identified differently expressed. Those were mainly associated with the regulation of genes with protein kinase activity, suggesting control of protein phosphorylation. This is the first study about the evolution of miRNA biogenesis system and miRNA control of gene expression in Eimeria species. Our data may lead to functional insights into of the regulation of gene expression during parasite life cycle in apicomplexan parasites.

Antibiotic Changes Host Susceptibility to Eimeria falciformis Infection Associated with Alteration of Gut Microbiota.

Eimeria falciformis is a murine-infecting coccidium that mainly infects the cecum and colon where it coexists with a large number of endogenous bacteria. Here, we found that mice treated with a broad-spectrum antibiotic cocktail including ampicillin, neomycin, metronidazole, and vancomycin had less oocyst production and milder pathological consequences after E. falciformis infection than mice without antibiotics, regardless of the inoculation doses. Furthermore, we showed that antibiotic treatment reduced parasitic invasion and prolonged asexual stage during E. falciformis infection, which may result in alleviating the infection. Interestingly, when further defining different antibiotic combinations for E. falciformis infection, it was shown that mice treated with ampicillin plus vancomycin had substantially attenuated E. falciformis infections as measured by cecal parasite counts and histopathological features. In contrast, treatment with metronidazole plus neomycin was beneficial to E. falciformis infection. Analyses of gut microbiota revealed various changes in bacterial composition and diversity following antibiotic treatments that were associated with host susceptibility to E. falciformis infection. Together, these findings suggest that gut microbiota may regulate the course and pathogenicity of E. falciformis infection, while the mechanisms need to be further investigated, especially for the development of coccidial vaccines for use in farm animals.

Early Transcriptional Response to Monensin in Sensitive and Resistant Strains of Eimeria tenella.

Eimeria parasites are the causative agents of coccidiosis, a common parasitic disease in poultry and livestock that causes significant economic losses to the animal husbandry industry. Ionophore coccidiostats, such as monensin and salinomycin, are widely used for prophylaxis of coccidiosis in poultry. Unfortunately, widespread drug resistance has compromised their efficacy. As a result, there is an increasing need to understand the targets and resistance mechanisms to anticoccidials. However, how Eimeria parasite genes respond to ionophores remains unclear. In this study, resistance to monensin was induced in E. tenella through serial generations of selection. Both sensitive and resistant E. tenella sporozoites were treated with 5 µg/ml monensin for 0, 2, and 4 h, respectively. Gene transcription profiles were then compared by high-throughput sequencing. The results showed that protein translation-related genes were significantly downregulated after drug induction. A total of 1,848 DEGs were detected in the sensitive strain after 2 h of exposure, whereas only 31 were detected in the resistant strain. Among these DEGs in the sensitive strain, genes associated with protein degradation were significantly upregulated, supporting the autophagy-like parasite killing theory. Then, 4 h of exposure resulted in additional 626 and 621 DEGs for sensitive and resistant strains, respectively. This result implies that the gene transcription in sensitive strain is more susceptible to monensin treatment. Our results provide gene expression landscapes of E. tenella following monensin treatment. These data will contribute to a better understanding of the mechanism of drug resistance to polyether ionophores in coccidia.

Underestimated Risk Perception Characteristics of Drivers Based on Extended Theory of Planned Behavior.

Aggressive driving behaviors due to drivers' underestimation of risks are one of the major causes of traffic accidents. Due to the complexity of factors influencing risk perception, the mechanism of risk underestimation remains unclear. In this study, the theory of planned behavior (TPB) was extended by adding a new variable, namely drivers' normlessness, forming an extended TPB (ETPB) framework to analyze the factors influencing risk underestimation and the extent of their influence. A total of 376 drivers' perceived characteristics of risk underestimation were collected through an online survey, and a structural equation model was applied to investigate the effects of normlessness, behavioral attitudes, subjective norm, and perceived behavioral control on the tendency to underestimate the risk. The results showed that the ETPB model can explain the variance in the underestimation risk behavior by 69%; perceptual behavior control, attitude, and subjective norm (in descending order) had significant positive effects on driver's tendency to underestimate risk; the normlessness variable can directly promote attitude and underestimated risk behavior; drivers with low annual mileage, complete insurance coverage, and no prior accident experience were more likely to underestimate driving risk. The study contributes to understanding of risk perception characteristics and provide theoretical basis for reducing underestimated risk behavior.

Tylvalosin demonstrates anti-parasitic activity and protects mice from acute toxoplasmosis.

AIMS: Toxoplasmosis, caused by Toxoplasma gondii (Tg), is one of the most prevalent zoonotic diseases worldwide. Currently, safe and efficient therapeutic options for this disease are still being developed, and are urgently needed. Tylvalosin (Tyl), a broad-spectrum third-generation macrolide, exhibits anti-bacterial, anti-viral, and anti-inflammatory properties. The present study aims to explore the anti-parasitic and immunomodulation activities of Tyl against Tg, and the underlying mechanism. MAIN METHODS: Adhesion, invasion, replication, proliferation, plaque, reversibility, immunofluorescence assays and transmission electron microscopy were utilized to determine the anti-Toxoplasma effect of Tyl. With acute toxoplasmosis model and rabies virus-induced brain inflammation model, the anti-toxoplasmosis and immunomodulation activities of Tyl were assessed by colorimetric assay, histopathological and Oil red O staining, and real-time quantitative PCR. The involved molecular mechanisms were investigated by western blotting and immunohistochemical staining. KEY FINDINGS: Tyl (5 and 10 µg/ml) can inhibit Tg propagation, and damage its ultrastructure irreversibly. The combination of Tyl and Pyrimethamine (Pyr) exhibits a better synergistic effect. Tyl (50 and 100 mg/kg) treatment intraperitoneally can delay mice death and improve survival rate, accompanying the reduced histopathological score and parasite load in the indicated tissues, espically for ileum, liver, spleen, lung and brain. Furthermore, Tg can modulate host phospho-p38 MAPK (pp38), subtilisin/kexin-isozyme-1 (SKI-1)-sterol regulatory element binding protein-1 (SREBP-1) (SKI-1-SREBP-1) pathway and peroxisome proliferators-activated receptor δ (PPARδ), while Tyl is able to reverse these signal pathways close to normal levels. SIGNIFICANCE: Our findings indicate that Tyl exhibits anti-Toxoplasma activity and protects mice from acute toxoplasmosis.

Immunocompetent rabbits infected with Cryptosporidium cuniculus as an animal model for anti-cryptosporidial drug testing.

Cryptosporidium is one of the leading causes of diarrheal disease in humans and animals, which can be severe and deadly in neonates and immunocompromised hosts. Studies on the biology of Cryptosporidium and drug discovery efforts have been hindered by a number of factors including the limited availability of animal models. Here, we report the establishment and characterization of an immunocompetent rabbit model for infection with Cryptosporidium cuniculus. By testing four known anti-cryptosporidial compounds (nitazoxanide, baicalein, curcumin and matrine), we showed that the rabbit could be used as an alternative animal model for evaluating anti-cryptosporidial drug efficacy in vivo.