Transcriptome analysis of a newly established mouse model of Toxoplasma gondii pneumonia.

BACKGROUND: Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii. Toxoplasma gondii infection of the lungs can lead to severe pneumonia. However, few studies have reported Toxoplasma pneumonia. Most reports were clinical cases due to the lack of a good disease model. Therefore, the molecular mechanisms, development, and pathological damage of Toxoplasma pneumonia remain unclear. METHODS: A mouse model of Toxoplasma pneumonia was established by nasal infection with T. gondii. The model was evaluated using survival statistics, lung morphological observation, and lung pathology examination by hematoxylin and eosin (H&E) and Evans blue staining at 5 days post-infection (dpi). Total RNA was extracted from the lung tissues of C57BL/6 mice infected with T. gondii RH and TGME49 strains at 5 dpi. Total RNA was subjected to transcriptome analysis by RNA sequencing (RNA-seq) followed by quantitative real-time polymerase chain reaction (qRT-PCR) validation. Transcript enrichment analysis was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to assess the biological relevance of differentially expressed transcripts (DETs). RESULTS: C57BL/6 mice infected with T. gondii via nasal delivery exhibited weight loss, ruffled fur, and respiratory crackles at 5 dpi. The clinical manifestations and lethality of RH strains were more evident than those of TGME49. H&E staining of lung tissue sections from mice infected with T. gondii at 5 dpi showed severe lymphocytic infiltration, pulmonary edema, and typical symptoms of pneumonia. We identified 3167 DETs and 1880 DETs in mice infected with the T. gondii RH and TGME49 strains, respectively, compared with the phosphate-buffered saline (PBS) control group at 5 dpi. GO and KEGG enrichment analyses of DETs showed that they were associated with the immune system and microbial infections. The innate immune, inflammatory signaling, cytokine-mediated signaling, and chemokine signaling pathways displayed high gene enrichment. CONCLUSION: In this study, we developed a new mouse model for Toxoplasma pneumonia. Transcriptome analysis helped to better understand the molecular mechanisms of the disease. These results provided DETs during acute T. gondii lung infection, which expanded our knowledge of host immune defenses and the pathogenesis of Toxoplasma pneumonia.

Rapid and visual detection of Staphylococcus aureus in milk using a recombinase polymerase amplification-lateral flow assay combined with immunomagnetic separation.

AIMS: The aim of this study was to develop a novel approach using lateral flow recombinase polymerase amplification (RPA-LF) combined with immunomagnetic separation (IMS) for the rapid detection of Staphylococcus aureus in milk. METHODS AND RESULTS: Under optimum conditions, the average capture efficiency values for S. aureus strains (104 colony-forming units [CFU] per ml) was above 95.0% in PBST and ~80% in milk within 45 min with 0.7 mg immunomagnetic beads. The RPA-LF assay, which comprised DNA amplification via RPA at 39°C for 10 min and visualization of the amplicons through LF strips for 5 min, detected S. aureus within 15 min. The method only detected S. aureus and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. Sensitivity experiments confirmed a detection limit of RPA-LF assay as low as 600 fg per reaction for the S. aureus genome (corresponding to approximately 36 CFU of S. aureus), which was about 16.7-fold more sensitive than that of the conventional polymerase chain reaction method. When RPA-LF was used in combination with IMS to detect S. aureus inoculated into artificially contaminated milk, it exhibited a detection limit of approximately 40 CFU per reaction. CONCLUSIONS: The newly developed IMS-RPA-LF method enabled detection of S. aureus at levels as low as 40 CFU per reaction in milk samples without culture enrichment for an overall testing time of only 70 min. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly developed IMS-lateral flow RPA-LF assay effectively combines sample preparation, amplification and detection into a single platform. Because of its high sensitivity, specificity and speed, the IMS-RPA-LF assay will have important implications for the rapid detection of S. aureus in contaminated food.

Biological and transcriptional studies reveal VmeL is involved in motility, biofilm formation and virulence in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a marine pathogen thought to be the leading cause of seafood-borne gastroenteritis globally, urgently requiring efficient management methods. V. parahaemolyticus encodes 12 resistance/nodulation/division (RND) efflux systems. However, research on these systems is still in its infancy. In this study, we discovered that the inactivation of VmeL, a membrane fusion protein within the RND efflux systems, led to reduction of the ability of biofilm formation. Further results displayed that the decreased capacity of Congo red binding and the colony of ΔvmeL is more translucent compared with wild type strains, suggested reduced biofilm formation due to decreased production of biofilm exopolysaccharide upon vmeL deletion. In addition, the deletion of vmeL abolished surface swarming and swimming motility of V. parahaemolyticus. Additionally, deletion of vmeL weakened the cytotoxicity of V. parahaemolyticus towards HeLa cells, and impaired its virulence in a murine intraperitoneal infection assay. Finally, through RNA-sequencing, we ascertained that there were 716 upregulated genes and 247 downregulated genes in ΔvmeL strain. KEGG enrichment analysis revealed that quorum sensing, bacterial secretion systems, ATP-binding cassette transporters, and various amino acid metabolism pathways were altered due to the inactivation of vmeL. qRT-PCR further confirmed that genes accountable to the type III secretion system (T3SS1) and lateral flagella were negatively affected by vmeL deletion. Taken together, our results suggest that VmeL plays an important role in pathogenicity, making it a good target for managing infection with V. parahaemolyticus.

Effect of Acetamizuril on enolase in second-generation merozoites of Eimeria tenella.

As an obligate intracellular apicomplexan parasite, Eimeria tenella (E. tenella) can rapidly invade chicken cecum epithelial cells and cause avian coccidiosis. Enolase, an essential enzyme that catalyzes the reversible conversion of 2-phosphoglycerate into phosphoenolpyruvate, plays a very important role in glycolysis. In this study, each chicken was inoculated with 8×10(4) sporulated E. tenella oocysts suspended in 1ml of distilled water to determine the effects of acetamizuril, a new triazine anticoccidial drug, on enolase in the second-generation merozoites of E. tenella. The chickens were divided into two groups: the untreatment group (challenged with E. tenella oocysts and provided with normal feed) and the treatment group (challenged with E. tenella oocysts and provided with 5mg/kg of acetamizuril by oral gavage at 96h after inoculation). The second-generation merozoites of E. tenella (mz-En) were obtained at 120h after inoculation. Subsequently, quantitative real-time PCR and Western blotting were conducted to detect the enolase changes in mz-En at the transcriptional and translational levels. The results showed that enolase mRNA expression was downregulated, and the translational level was decreased in the treatment group. In addition, the subcellular localization of enolase demonstrated that enolase was distributed primarily at the top of the mz-En and that the fluorescence intensity was weak after treatment with acetamizuril. These findings indicated that enolase may be a promising target to prevent coccidiosis.

[Detection of antimalarial activity for new compounds by isotopic microtest].

Two clone lines (Dd2 and 3D7) of Plasmodium falciparum were cultivated continuously in human erythrocytes at 37 degrees C in RPMI 1640 medium with human serum and subjected to 6% sorbitol treatment 2 times in order to obtain highly synchronized cultures. The second generation parasites after the treatment were diluted with human RBC to be a suspension of P. falciparum-human RBC at 2.5% hematocrit and 0.5% parasitemia, and 2 microCi/ml of 8-3H-hypoxanthine was added. Isotopic microtest was employed to detect the antimalarial activity for 20 new compounds. Results revealed that the 20 compounds showed no anti-malarial activity, while the control drugs, chloroquine and quinine, exhibited high efficacy, indicating that the isotopic microtest is a stable and reproducible assay for screening new antimalarials.

[Comparison of the proteome of the sporutated oocysts of Eimeria tenella diclazuril sensitive strain with diclazuril resistant strain].

Two-dimensional electrophoresis (2-DE) was employed to compare the proteome of Diclazuril-resistance Eimeria tenella with that of sensitive strains for identifying unique proteins of these stains. 5 protein spots were found to express differentially. Four spots which remarkably were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The data obtained from peptide mass fingerprinting were used in NCBInr database search, two protein spots in gel were identified as Eimeria tenella sporulated oocyst TA4 antigen protein, Heat shock 70kD protein, two protein spots were functional proteins of Eukaryote. These proteins are potentially basic work for finding molecular mechanism about drug-resistance of Eimeria tenella and new marker in the detection of resistance of Eimeria tenella.