A novel extraction-free dual HiFi-LAMP assay for detection of methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a leading cause of bacteremia and blood stream infections. Methicillin-resistant S. aureus (MRSA) that first appeared in 1961 often caused hospital-acquired infections (HAIs) and community-acquired infections (CAIs) and was associated with high mortality rate. Accurate and rapid point-of-care testing (POCT) of MRSA is crucial for clinical management and treatment of MRSA infections, as well as the prevention and control of HAIs and CAIs. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of methicillin-susceptible S. aureus and MRSA. The dual HiFi-LAMP assay can detect 30 copies/reaction of nuc and mecA genes with detection limits of 147 and 158 copies per 25 µL reaction, respectively. A retrospective clinical evaluation with 107 clinical S. aureus isolates showed both sensitivity and specificity of 100%. A prospective clinical evaluation with 35 clinical samples revealed a specificity of 100% and a sensitivity of 92.3%. The dual HiFi-LAMP assay can detect almost all S. aureus samples (141/142; 99.3%) within 20 min, implying that the entire HiFi-LAMP assay (including sample process) can be completed within 40 min, extremely significantly shorter than 3-5 days by the traditional clinical microbial culture and antibiotic susceptibility testing. The novel extraction-free dual HiFi-LAMP assay can be used as a robust POCT tool to promote precise diagnosis and treatment of MRSA infections in hospitals and to facilitate surveillance of MRSA at hospital and community settings.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) was associated with high mortality rate and listed as a "priority pathogen" by the World Health Organization. Accurate and rapid point-of-care testing (POCT) of MRSA is critically required for clinical management and treatment of MRSA infections. Some previous LAMP-based POCT assays for MRSA might be questionable due to their low specificity and the lack of appropriate evaluation directly using clinical samples. Furthermore, they are relatively tedious and time-consuming because they require DNA extraction and lack multiplex detection capacity. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of MRSA and methicillin-susceptible S. aureus. The assay has high specificity and sensitivity and can be completed within 40 min. Clinical evaluation with real clinical samples and clinical isolates showed excellent performance with 100% specificity and 92.3%-100% sensitivity. The novel extraction-free assay may be a robust POCT tool to promote precise diagnosis of MRSA infections and facilitate surveillance of MRSA at hospital and community settings.

Rapid detection of four major HFMD-associated enteroviruses by multiplex HiFi-LAMP assays.

Hand, foot, and mouth disease (HFMD) caused by various enteroviruses is a major public health concern globally. Human enterovirus 71(EVA71), coxsackievirus A16 (CVA16), coxsackievirus A6 (CVA6), and coxsackievirus A10 (CVA10) are four major enteroviruses responsible for HFMD. Rapid, accurate, and specific point-of-care (POC) detection of the four enteroviruses is crucial for the prevention and control of HFMD. Here, we developed two multiplex high-fidelity DNA polymerase loop-mediated isothermal amplification (mHiFi-LAMP) assays for simultaneous detection of EVA71, CVA16, CVA6, and CVA10. The assays have good specificity and exhibit high sensitivity, with limits of detection (LOD) of 11.2, 49.6, 11.4, and 20.5 copies per 25 µL reaction for EVA71, CVA16, CVA6, and CVA10, respectively. The mHiFi-LAMP assays showed an excellent clinical performance (sensitivity 100.0%, specificity 83.3%, n = 47) when compared with four singleplex RT-qPCR assays (sensitivity 93.1%, specificity 100%). In particular, the HiFi-LAMP assays exhibited better performance (sensitivity 100.0%, specificity 100%) for CVA16 and CVA6 than the RT-qPCR assays (sensitivity 75.0-92.3%, specificity 100%). Furthermore, the mHiFi-LAMP assays detected all clinical samples positive for the four enteroviruses within 30 min, obviously shorter than about 1-1.5 h by the RT-qPCR assays. The new mHiFi-LAMP assays can be used as a robust point-of-care testing (POCT) tool to facilitate surveillance of HFMD at rural and remote communities and resource-limited settings.

Rapid detection of human influenza A viruses by HFman probe-based loop-mediated isothermal amplification assays.

Since China abandoned the zero-COVID policy at the end of 2022, a wave of severe Flu pandemic emerged in China. Rapid and accurate diagnosis of Influenza A virus (IAV) is critical for clinical management and therapeutic decision-making of patients with fever. Here, we reported a novel IAV HF-LAMP assay, which can be performed with purified RNA or directly using clinical samples. The assays with purified RNA and clinical samples have high sensitivity with limit of detection (LOD) of 9.6 copies/reaction, 9900 copies/mL, and short sample-to-answer times of 36 and 50 min, respectively. Both assays showed high specificity and significantly higher IAV detection rate than the rapid antigen detection (RAD) assays. Furthermore, we found the vast majority (91.2 %) of children with fever during the pandemic were infected by IAV, and current IAV infection has a very narrow detectable window. The novel IVA HF-LAMP assays will provide robust tools to facilitate early diagnosis of IAV infection in current and future seasonal influenza epidemics.

Structural Design and Optimization of the Milling Force Measurement Tool System Embedded with Thin-Film Strain Sensors.

A milling force measurement tool system is designed with an elastic beam structure, which is divided into a two-end ring hoop compression sensor mode and a two-end square hoop compression sensor mode to improve the strain sensitivity. A simplified mechanical model of the elastic beam is established, and the relationship between the strain and force of the elastic beam under the action of three cutting force components is investigated, which can act a guide for subsequent milling force measurement tool system calibration tests. Thin-film strain sensors occupy a central position in the milling force measurement tool system, which consists of a substrate, transition layer, insulating layer and resistance grid layer. The resistance grid layer has a particularly significant effect on the thin-film strain sensor's performance. In order to further improve the sensitivity of thin-film strain sensors, the shapes of the substrate, the transition layer, the insulating layer and the resistance grid layer are optimized and studied. A new thin-film strain sensor is designed with a resistance grid beam constructed from an insulating layer and a resistive grid layer double-end-supported on the transition layer. The flow of the wet-etching process of thin-film strain sensors is studied and samples are obtained. The surface microforms of the sensor samples are observed by extended depth-of-field microscopy, confocal microscopy and atomic force microscopy. It can be seen that the boundary of the resistance grid layer pattern is tidy and has high dimensional accuracy, thus enabling the basic achievement of the expected effect of the design. The electrical performance of the samples is tested on an experimental platform that we built, and the results show that the resistive sensitivity coefficient of the samples is increased by about 20%, to 51.2%, compared with that of the flat thin-film strain sensor, which fulfils the design's requirements.

Functional annotation map of natural compounds in traditional Chinese medicines library: TCMs with myocardial protection as a case.

The chemical complexity of traditional Chinese medicines (TCMs) makes the active and functional annotation of natural compounds challenging. Herein, we developed the TCMs-Compounds Functional Annotation platform (TCMs-CFA) for large-scale predicting active compounds with potential mechanisms from TCM complex system, without isolating and activity testing every single compound one by one. The platform was established based on the integration of TCMs knowledge base, chemome profiling, and high-content imaging. It mainly included: (1) selection of herbal drugs of target based on TCMs knowledge base; (2) chemome profiling of TCMs extract library by LC‒MS; (3) cytological profiling of TCMs extract library by high-content cell-based imaging; (4) active compounds discovery by combining each mass signal and multi-parametric cell phenotypes; (5) construction of functional annotation map for predicting the potential mechanisms of lead compounds. In this stud TCMs with myocardial protection were applied as a case study, and validated for the feasibility and utility of the platform. Seven frequently used herbal drugs (Ginseng, etc.) were screened from 100,000 TCMs formulas for myocardial protection and subsequently prepared as a library of 700 extracts. By using TCMs-CFA platform, 81 lead compounds, including 10 novel bioactive ones, were quickly identified by correlating 8089 mass signals with 170,100 cytological parameters from an extract library. The TCMs-CFA platform described a new evidence-led tool for the rapid discovery process by data mining strategies, which is valuable for novel lead compounds from TCMs. All computations are done through Python and are publicly available on GitHub.

Evolution of the Probe-Based Loop-Mediated Isothermal Amplification (LAMP) Assays in Pathogen Detection.

Loop-mediated isothermal amplification (LAMP), as the rank one alternative to a polymerase chain reaction (PCR), has been widely applied in point-of-care testing (POCT) due to its rapid, simple, and cost-effective characteristics. However, it is difficult to achieve real-time monitoring and multiplex detection with the traditional LAMP method. In addition, these approaches that use turbidimetry, sequence-independent intercalating dyes, or pH-sensitive indicators to indirectly reflect amplification can result in false-positive results if non-specific amplification occurs. To fulfill the needs of specific target detection and one-pot multiplex detection, a variety of probe-based LAMP assays have been developed. This review focuses on the principles of these assays, summarizes their applications in pathogen detection, and discusses their features and advantages over the traditional LAMP methods.

Subroutine Embedding and Finite Element Simulation of the Improved Constitutive Equation for Ti6Al4V during High-Speed Machining.

The Johnson-Cook (J-C) constitutive model is not suitable for Ti-6Al-4V alloy in the high-speed cutting finite element simulation, as it has no response dynamic recrystallization softening effect under heavy impact and high temperature. In this paper, an improved constitutive model considering the recrystallization effect was established, and the parameters were fitted with the data of flow stress-strain of the Split Hopkinson Pressure Bar (SHPB) test. The relevant theories of cutting finite element simulation were studied, such as nonlinear constitutive elastic-plastic deformation, strain state, and material yield. A subroutine that included the Recht shear failure instability criterion and the improved model was coded in Fortran and embedded in the finite element simulation software AdvantEdge FEM, along with the return mapping stress integration algorithm. The simulated stress of the improved model dropped dramatically from 460 MPa to 220 MPa when the temperature rises from 950 °C to 1000 °C, and its decline reached 46.7%, while the J-C model only decreased by 10%. Comparative studies indicate that the stress change of the improved constitutive simulation is closer to the SHPB test results than the J-C constitutive, and the new one is more suitable when it expresses the high temperature and heavy impact in the high-speed milling.

Rapid point-of-care detection of BK virus in urine by an HFman probe-based loop-mediated isothermal amplification assay and a finger-driven microfluidic chip.

Background: BK virus (BKV)-associated nephropathy (BKVN) is one of the leading causes of renal dysfunction and graft loss in renal transplant recipients. Early monitoring of BKV in urine is crucial to minimize the deleterious effects caused by this virus on preservation of graft function. Methods: We report a simple, rapid, sensitive loop-mediated isothermal amplification (LAMP) assay using an HFman probe for detecting BKV in urine. To evaluate the performance of the assay, a comparison of the HFman probe-based LAMP (HF-LAMP) assay with two qPCR assays was performed using urine samples from 132 HIV-1 infected individuals. We further evaluated the performance of HF-LAMP directly using the urine samples from these HIV-1 infected individuals and 30 kidney transplant recipients without DNA extraction. Furthermore, we combined the HF-LAMP assay with a portable finger-driven microfluidic chip for point-of-care testing (POCT). Results: The assay has high specificity and sensitivity with a limit of detection (LOD) of 12 copies/reaction and can be completed within 30 min. When the DNA was extracted, the HF-LAMP assay showed an equivalent and potentially even higher sensitivity (93.5%) than the qPCR assays (74.2-87.1%) for 132 urine samples from HIV-1 infected individuals. The HF-LAMP assay can be applied in an extraction-free format and can be completed within 45 min using a simple heat block. Although some decreased performance was seen on urine samples from HIV-1 infected individuals, the sensitivity, specificity, and accuracy of the extraction-free BKV HF-LAMP assay were 95%, 100%, and 96.7% for 30 clinical urine samples from kidney transplant recipients, respectively. Conclusion: The assay has high specificity and sensitivity. Combined with a portable finger-driven microfluidic chip for easy detection, this method shows great potential for POCT detection of BKV.

Pathogenic mechanism of Eimeria tenella autophagy activation of chicken embryo cecal epithelial cells induced by Eimeria tenella.

Eimeria tenella mainly invades and develops into cecal epithelial cells of chickens, resulting in cecal epithelial cell damage. Infectious intracellular pathogens possibly act by influencing the autophagy process after invading cells. The interaction between E. tenella and the autophagy of host cells was explored by infecting E. tenella with chick embryo cecal epithelial cells. Transmission electron microscopy, laser confocal microscopy, and Western blot analysis were used to demonstrate that E. tenella infection could induce autophagy in host cells. Results showed that infection with E. tenella induced the formation of autophagosomes in cells. The expression of ATG 5, Beclin-1, and LC3B-II proteins were significantly (P < 0.01) increased after E. tenella infected host cells. Expression of p62 protein levels were significantly (P < 0.01) decreased in host cells infected with E. tenella. Chloroquine (CQ) significantly (P < 0.01) increased the expression levels of LC3B-II and P62 in E. tenella-infected host cells. Rapamycin (RAPA) induced autophagy in host cells, thus reducing the intracellular infection of E. tenella. By contrast, the infection rate of E. tenella increased in cells treated with 3-Methyladenine (3-MA). Hence, E. tenella sporozoite infection could induce autophagy activation in chick embryo cecal epithelial cells, and enhanced autophagy could reduce the infection rate of E. tenella.

Capsaicin receptor TRPV1 maintains quiescence of hepatic stellate cells in the liver via recruitment of SARM1.

BACKGROUND & AIMS: Capsaicin receptor, also known as transient receptor potential vanilloid 1 (TRPV1), is involved in pain physiology and neurogenic inflammation. Herein, we discovered the presence of TRPV1 in hepatic stellate cells (HSCs) and aimed to delineate its function in this cell type and liver fibrosis. METHODS: TRPV1 expression was examined in liver biopsies from patients with liver fibrosis using quantitative real-time PCR and immunostaining. Its contribution to liver fibrosis was examined in Trpv1-/- mice, upon lentiviral delivery of the TRPV1 gene, and in human and mouse primary HSCs, using patch clamp, intracellular Ca2+ mobilization determination, FACS analyses and gain/loss of function experiments. Binding of sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) to TRPV1 was determined using mass spectrometry, co-immunoprecipitation, surface plasmon resonance, bioluminescence resonance energy transfer, and NanoBiT. RESULTS: TRPV1 mRNA levels are significantly downregulated in patients with liver fibrosis and mouse models, showing a negative correlation with F stage and α-smooth muscle actin expression, a marker of HSC activation. TRPV1 expression and function decrease during HSC activation in fibrotic livers in vivo or during culture. Genetic and pharmacological inhibition of TRPV1 in quiescent HSCs leads to NF-κB activation and pro-inflammatory cytokine production. TRPV1 requires binding of its N-terminal ankyrin repeat domain to the TIR-His583 (Toll/interleukin-1 receptor) domain of SARM1 to prevent HSCs from pro-inflammatory activation. Trpv1-/- mice display increased HSC activation and more severe liver fibrosis, whereas TRPV1 overexpression is antifibrotic in various disease models. CONCLUSION: The antifibrotic properties of TRPV1 are attributed to the prevention of HSC activation via the recruitment of SARM1, which could be an attractive therapeutic strategy against liver fibrosis. IMPACT AND IMPLICATIONS: We identified the neuronal channel protein TRPV1 as a gatekeeper of quiescence in hepatic stellate cells, a key driver of liver fibrogenesis and chronic liver disease. Physiologically expressed in healthy liver and consistently downregulated during liver fibrosis development, its therapeutic re-expression is expected to have few side effects, making it an attractive target diagnostic tool and drug candidate for industry and clinicians.

Optimization of Processing Parameters and Adhesive Properties of Aluminum Oxide Thin-Film Transition Layer for Aluminum Substrate Thin-Film Sensor.

A thin-film strain micro-sensor is a cutting force sensor that can be integrated with tools. Its elastic substrate is an important intermediate to transfer the strain generated by the tools during cutting to the resistance-grid-sensitive layer. In this paper, 1060 aluminum is selected as the elastic substrate material and aluminum oxide thin film is selected as the transition layer between the aluminum substrate and the silicon nitride insulating layer. The Stoney correction formula applicable to the residual stress of the aluminum oxide film is derived, and the residual stress of the aluminum oxide film on the aluminum substrate is obtained. The influence of Sputtering pressure, argon flow and negative substrate bias process parameters on the surface quality and sputtering power of the aluminum oxide thin film is discussed. The relationship model between process parameters, surface roughness, and sputtering rate of thin films is established. The sputtering process parameters for preparing an aluminum oxide thin film are optimized. The micro-surface quality of the aluminum oxide thin film obtained before and after the optimization of the process parameters and the surface quality of Si3N4 thin film sputtered on alumina thin film before and after the optimization are compared. It is verified that the optimized process parameters of aluminum oxide film as a transition layer can improve the adhesion between the insulating-layer silicon nitride film and the aluminum substrate.

[Application of high content imaging technique in research on effective components of traditional Chinese medicine: a review].

High content imaging(HCI) technique that combines automatic high throughput with high-resolution cell imaging, is characterized by abundant data information, high imaging sensitivity, easy visualization and standardization, and is commonly used in the cellular(or subcellular) phenotypic analysis. Abundant phenotypic information can be obtained by using HCI in one experiment, including cellular morphology, cellular structure, and signal transduction pathways of related functions, on the basis of the maintenance of the integrity of cellular structures and functions. Multiple studies have shown that a series of dynamic spatio-temporal interactive change processes were induced by the disturbance of cells by specific factors, making cell phenotypes change accordingly, especially for the slight perturbation response of cells. Generally, the detection of one or several endpoint effect indicators is often difficult to accurately and comprehensively reflect the overall efficacy information of traditional Chinese medicine(TCM) because of its unique characteristics of multi-components and multi-targets. The application of HCI is thus helpful to discover the effective components and their action modes in the complex system of TCM. This paper reviewed the application progress in the HCI technique in the screening of active components and their regulation mechanism to provide references for further research.

An HFman Probe-Based Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Simultaneous Detection of Hantaan and Seoul Viruses.

Hantaviruses are zoonotic pathogens that are widely distributed worldwide. Hantaan virus (HTNV) and Seoul virus (SEOV) are two most common hantaviruses that infect humans and cause hemorrhagic fever with renal syndrome (HFRS). Rapid and sensitive detection of HTNV and SEOV are crucial for surveillance, clinical treatment and management of HFRS. This study aimed to develop a rapid HFman probe-based mulstiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to simultaneously detect HTNV and SEOV. A novel multiplex RT-LAMP assay was developed, and 46 serum samples obtained from clinically suspected patients were used for evaluation. The novel RT-LAMP assay can detect as low as 3 copies/reaction of hantaviruses with a detection limit of 41 and 73 copies per reaction for HTNV and SEOV, respectively. A clinical evaluation showed that the consistencies of the multiplex RT-LAMP with RT-qPCR assay were 100% and 97.8% for HTNV and SEOV, respectively. In view of the high prevalence of HTNV and SEOV in rural areas with high rodent density, a colorimetric visual determination method was also developed for point-of-care testing (POCT) for the diagnosis of the two viruses. The novel multiplex RT-LAMP assay is a sensitive, specific, and efficient method for simultaneously detecting HTNV and SEOV.

Role of EtMIC4 EGF-like in regulating the apoptosis of Eimeria tenella host cells via the EGFR pathway.

This study aimed to explore the role and key point of EtMIC4 EGF-like recombinant protein in regulating the apoptosis of Eimeria tenella host cells via the epidermal growth factor receptor (EGFR) pathway. The cells were treated with EtMIC4 EGF-like protein, EGFR-specific siRNA, or both. Infection and apoptosis rates as well as dynamic changes in the key genes and proteins of the EGFR signaling pathway in the host cells were determined. Results showed that the E. tenella and EtMIC4 EGF-like group had the highest infection rate (P < 0.01). In cells treated with EtMIC4 EGF-like for 4 to 24 h, the apoptosis rate was significantly decreased (P < 0.01) and the relative mRNA expression and protein phosphorylation levels of EGFR, protein kinase B (AKT), and extracellular regulated protein kinases (ERK) were significantly increased (P < 0.01). In E. tenella sporozoites infected for 4 to 96 h, the rate of host cell apoptosis induced by E. tenella infection was significantly (P < 0.01) reduced by EtMIC4 EGF-like. The relative mRNA expression and protein phosphorylation levels of EGFR, AKT, and ERK in the host cells of E. tenella + EtMIC4 EGF-like group were significantly increased (P < 0.01). These results indicated that E. tenella could activate the EGFR pathway through EtMIC4 EGF-like and regulate the expression of key genes in the AKT and ERK signaling pathways, thereby inhibiting cell apoptosis.

Study of the Pattern Preparation and Performance of the Resistance Grid of Thin-Film Strain Sensors.

The thin-film strain sensor is a cutting-force sensor that can be integrated with cutting tools. The quality of the alloy film strain layer resistance grid plays an important role in the performance of the sensor. In this paper, the two film patterning processes of photolithography magnetron sputtering and photolithography ion beam etching are compared, and the effects of the geometric size of the thin-film resistance grid on the resistance value and resistance strain coefficient of the thin film are compared and analyzed. Through orthogonal experiments of incident angle, argon flow rate, and substrate negative bias in the ion beam etching process parameters, the effects of the process parameters on photoresist stripping quality, etching rate, surface roughness, and resistivity are discussed. The effects of process parameters on etching rate, surface roughness, and resistivity are analyzed by the range method. The effect of substrate temperature on the preparation of Ni Cr alloy films is observed by scanning electron microscope. The surface morphology of the films before and after ion beam etching is observed by atomic force microscope. The influence of the lithography process on the surface quality of the film is discussed, and the etching process parameters are optimized.

An HFman probe-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for HIV-1 detection.

Loop-mediated isothermal amplification (LAMP) is suitable for the development of a rapid and cost-effective nucleic acid technique for point of care (POC) applications. However, LAMP methods often generate non-specific amplification, therefore inevitably resulting in false positive results especially when sequence-independent dyes are used to indirectly reflect the results. In this study, we established and optimized a reverse transcription LAMP (RT-LAMP) assay with a high-fidelity DNA polymerase-mediated fluorescent probe (HFman probe) for human immunodeficiency virus-1 (HIV-1) detection. The assay showed high sensitivity and specificity. Using 101 plasma samples with different HIV-1 viral load, we demonstrated that our assay can detect the major HIV-1 subtypes circulating in China, including CRF01_AE, CRF07_BC, CRF08_BC, CRF55_01B, and unique recombinant forms (URFs). We also compared our assay with an approved commercial real-time quantitative polymerase chain reaction (RT-qPCR) kit and found the sensitivity, specificity and consistency was 88.8%, 100% and 89.1%, respectively. The HFman probe-based RT-LAMP assay is a high specific detection method that is rapid, variant-tolerant and simple to operate, and thus is of great significance for timely disclosure of HIV status and rapid POC diagnosis.

Induction of polyploid Malus prunifolia and analysis of its salt tolerance.

The apple rootstock Malus prunifolia (Willd.) Borkh. is widely used for apple production. Because polyploid plants are often more tolerant to abiotic stress than diploids, we wondered whether polyploidy induction in M. prunifolia might improve its stress tolerance, particularly to high salinity. We used a combination of colchicine and dimethyl sulfoxide (DMSO) to induce chromosome doubling in M. prunifolia and identified the resulting polyploids by stomatal observations and flow cytometry. We found the best way to induce polyploidy in M. prunifolia was to use 2% DMSO and 0.05% colchicine for 2 days for leaves or 0.02% colchicine for stem segments. The results of hydroponic salt treatment showed that polyploid plants were more salt tolerant and had greater photosynthetic efficiency, thicker leaf epidermis and palisade tissues, and shorter but denser root systems than diploids. During salt stress, the polyploid leaves and roots accumulated less Na+, showed upregulated expression of three salt overly sensitive (SOS) pathway genes, and produced fewer reactive oxygen species. The polyploid plants also had considerably higher ABA and jasmonic acid levels than diploid plants under salt stress. Under normal growth conditions, gibberellins (GAs) levels were much lower in polyploid leaves than in diploid leaves; however, after salt treatment, polyploid leaves showed upregulation of essential GAs synthesis genes. In summary, we developed a system for the induction of polyploidy in M. prunifolia and response to salt stress of the resulting polyploids, as reflected in leaf and root morphology, changes in Na+ accumulation, antioxidant capacity and plant hormone levels.

The role of Ca2+ in the injury of host cells during the schizogenic stage of E. tenella.

Cecal epithelial cell damage is a key factor in host injure during the development of E. tenella. The intracellular free Ca2+ of the host cell is closely related to the invasion, development and proliferation of intracellular parasites, and cell damage. To determine the relationship between Ca2+ and host cell damage in the schizogenic stage of E. tenella, we established a chick embryo cecal epithelial cells model of E. tenella infection. Fluorescence staining, flow cytometry, transmission electron microscopy, inhibition and blocking experiments were used to detect the damage effect and mechanism of host cells during the schizogenic stage of E. tenella. The results showed that the host cells cytoskeletal remodeling, cell and organelle structure was destroyed, and apoptosis and necrosis were increased during the schizont stage of E. tenella. Furthermore, the above-mentioned effects of the schizogenic stage of E. tenella on cells can be alleviated by reducing the intracellular Ca2+ concentration in the host cells. These observations indicate that the effect of host cell injury was closely related to Ca2+ during schizont stage of E. tenella.