Rapid Discriminative Identification of the Two Predominant Echinococcus Species from Canine Fecal Samples in the Tibetan Region of China by Loop-Mediated Isothermal Amplification-Lateral Flow Dipstick Assay.

Echinococcosis poses a significant concern in the fields of public health and veterinary care as it can be transmitted between animals and humans. The primary endemic subtypes are cystic echinococcosis (CE) and alveolar echinococcosis (AE), which result from infestation by Echinococcus granulosus and Echinococcus multilocularis, respectively. A prominent epidemic of echinococcosis greatly affects the Tibet Autonomous Region (TAR) in China. A new technique called the loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) test is introduced in this research to differentiate between E. granulosus and E. multilocularis using their repetitive genetic sequences. The test is characterized by its portable nature, simple operation, quick result production, high sensitivity, and low susceptibility to aerosol contamination. The LAMP-LFD method demonstrated an exceptional minimal detection limit, reaching levels as low as approximately 1 fg/µL (femtogram per microliter) of genomic DNA. The assay's specificity was assessed, and no cross-reactivity was seen. A total of 982 dog fecal samples were collected from 54 counties in the TAR region between July 2021 and June 2022. The established method underwent validation using a commercially available ELISA kit. The agreement rate between the LAMP-LFD and ELISA methods was 97.25%, with a sensitivity of 96.05% and a specificity of 97.35%. The assay described in this study improves specificity by using a double-labeled probe, and it reduces the risk of false-positive results caused by aerosol contamination through the use of a sealed device. This makes it a suitable choice for quickly and accurately identifying the two main types of Echinococcus in field settings.

A live attenuated RHΔompdcΔuprt mutant of Toxoplasma gondii induces strong protective immunity against toxoplasmosis in mice and cats.

BACKGROUND: Toxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis. It is essential to develop an effective anti-T. gondii vaccine for the control of toxoplasmosis, and this study is to explore the immunoprotective effects of a live attenuated vaccine in mice and cats. METHODS: First, the ompdc and uprt genes of T. gondii were deleted through the CRISPR-Cas9 system. Then, the intracellular proliferation and virulence of this mutant strain were evaluated. Subsequently, the immune responses induced by this mutant in mice and cats were detected, including antibody titers, cytokine levels, and subsets of T lymphocytes. Finally, the immunoprotective effects were evaluated by challenge with tachyzoites of different strains in mice or cysts of the ME49 strain in cats. Furthermore, to discover the effective immune element against toxoplasmosis, passive immunizations were carried out. GraphPad Prism software was used to conduct the log-rank (Mantel-Cox) test, Student's t test and one-way ANOVA. RESULTS: The RHΔompdcΔuprt were constructed by the CRISPR-Cas9 system. Compared with the wild-type strain, the mutant notably reduced proliferation (P < 0.05). In addition, the mutant exhibited virulence attenuation in both murine (BALB/c and BALB/c-nu) and cat models. Notably, limited pathological changes were found in tissues from RHΔompdcΔuprt-injected mice. Furthermore, compared with nonimmunized group, high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (IFN-γ, IL-4, IL-10, IL-2 and IL-12) in mice were detected by the mutant (P < 0.05). Remarkably, all RHΔompdcΔuprt-vaccinated mice survived a lethal challenge with RHΔku80 and ME49 and WH6 strains. The immunized sera and splenocytes, especially CD8+ T cells, could significantly extend (P < 0.05) the survival time of mice challenged with the RHΔku80 strain compared with naïve mice. In addition, compared with nonimmunized cats, cats immunized with the mutant produced high levels of antibodies and cytokines (P < 0.05), and notably decreased the shedding numbers of oocysts in feces (95.3%). CONCLUSIONS: The avirulent RHΔompdcΔuprt strain can provide strong anti-T. gondii immune responses, and is a promising candidate for developing a safe and effective live attenuated vaccine.

Proteomic profiling of serum extracellular vesicles identifies diagnostic markers for echinococcosis.

Echinococcosis is a parasitic disease caused by the metacestodes of Echinococcus spp. The disease has a long latent period and is largely underdiagnosed, partially because of the lack of effective early diagnostic approaches. Using liquid chromatography-mass spectrometry, we profiled the serum-derived extracellular vesicles (EVs) of E. multilocularis-infected mice and identified three parasite-origin proteins, thioredoxin peroxidase 1 (TPx-1), transitional endoplasmic reticulum ATPase (TER ATPase), and 14-3-3, being continuously released by the parasites into the sera during the infection via EVs. Using ELISA, both TPx-1 and TER ATPase were shown to have a good performance in diagnosis of experimental murine echinococcosis as early as 10 days post infection and of human echinococcosis compared with that of control. Moreover, TER ATPase and TPx-1 were further demonstrated to be suitable for evaluation of the prognosis of patients with treatment. The present study discovers the potential of TER ATPase and TPx-1 as promising diagnostic candidates for echinococcosis.

A rare CHD5 haplotype and its interactions with environmental factors predicting hepatocellular carcinoma risk.

BACKGROUND: CHD5 is a conventional tumour-suppressing gene in many tumours. The aim of this study was to determine whether CHD5 variants contribute to the risk of hepatocellular carcinoma (HCC). METHODS: Gene variants were identified using next-generation sequencing targeted on referenced mutations followed by TaqMan genotyping in two case-control studies. RESULTS: We discovered a rare variant (haplotype AG) in CHD5 (rs12564469-rs9434711) that was markedly associated with the risk of HCC in a Chinese population. A logistical regression model and permutation test confirmed the association. Indeed, the association quality increased in a gene dose-dependent manner as the number of samples increased. In the stratified analysis, this haplotype risk effect was statistically significant in a subgroup of alcohol drinkers. The false-positive report probability and multifactor dimensionality reduction further supported the finding. CONCLUSIONS: Our results suggest that the rare CHD5 gene haplotype and alcohol intake contribute to the risk of HCC. Our findings can be valuable to researchers of cancer precision medicine looking to improve diagnosis and treatment of HCC.

The single-nucleotide polymorphisms in CHD5 affect the prognosis of patients with hepatocellular carcinoma.

Previous studies showed that the low expressions of chromodomain-helicase-DNA-binding protein 5 (CHD5) were intensively associated with deteriorative biologic and clinical characteristics as well as outcomes in many tumors. The aim of this study is to determine whether CHD5 single nucleotide polymorphisms (SNPs) contribute to the prognosis of hepatocellular carcima (HCC). The SNPs were selected according to their linkage disequilibrium (LD) in the targeted next-generation sequencing (NGS) and then genotyped with TaqMan probers. We revealed a rare haplotype AG in CHD5 (SNPs: rs12564469-rs9434711) was markedly associated with HCC prognosis. The univariate and multivariate regression analyses revealed the patients with worse overall survival time were those with tumor metastasis and haplotype AG, as well as cirrhosis, poor differentiation and IV-TNM stage. Based on the available public databases, we discovered the significant association between haplotype AG and CHD5 mRNA expressions only existed in Chinese. These data proposed that the potentially genetic haplotype might functionally contribute to HCC prognosis and CHD5 mRNA expressions.

[Rapid discriminating hogwash oil and edible vegetable oil using near infrared optical fiber spectrometer technique].

In the present study, a new method using near infrared spectroscopy combined with optical fiber sensing technology was applied to the analysis of hogwash oil in blended oil. The 50 samples were a blend of frying oil and "nine three" soybean oil according to a certain volume ratio. The near infrared transmission spectroscopies were collected and the quantitative analysis model of frying oil was established by partial least squares (PLS) and BP artificial neural network The coefficients of determina- tion of calibration sets were 0.908 and 0.934 respectively. The coefficients of determination of validation sets were 0.961 and 0.952, the root mean square error of calibrations (RMSEC) was 0.184 and 0.136, and the root mean square error of predictions (RMSEP) was all 0.111 6. They conform to the model application requirement. At the same time, frying oil and qualified edible oil were identified with the principal component analysis (PCA), and the accurate rate was 100%. The experiment proved that near infrared spectral technology not only can quickly and accurately identify hogwash oil, but also can quantitatively detect hog- wash oil. This method has a wide application prospect in the detection of oil.

Proteomic analysis of purified Newcastle disease virus particles.

BACKGROUND: Newcastle disease virus (NDV) is an enveloped RNA virus, bearing severe economic losses to the poultry industry worldwide. Previous virion proteomic studies have shown that enveloped viruses carry multiple host cellular proteins both internally and externally during their life cycle. To address whether it also occurred during NDV infection, we performed a comprehensive proteomic analysis of highly purified NDV La Sota strain particles. RESULTS: In addition to five viral structural proteins, we detected thirty cellular proteins associated with purified NDV La Sota particles. The identified cellular proteins comprised several functional categories, including cytoskeleton proteins, annexins, molecular chaperones, chromatin modifying proteins, enzymes-binding proteins, calcium-binding proteins and signal transduction-associated proteins. Among these, three host proteins have not been previously reported in virions of other virus families, including two signal transduction-associated proteins (syntenin and Ras small GTPase) and one tumor-associated protein (tumor protein D52). The presence of five selected cellular proteins (i.e., ß-actin, tubulin, annexin A2, heat shock protein Hsp90 and ezrin) associated with the purified NDV particles was validated by Western blot or immunogold labeling assays. CONCLUSIONS: The current study presented the first standard proteomic profile of NDV. The results demonstrated the incorporation of cellular proteins in NDV particles, which provides valuable information for elucidating viral infection and pathogenesis.