Genetic distribution, characterization, and function of Escherichia coli type III secretion system 2 (ETT2).

Many Gram-negative bacteria use type Ⅲ secretion system (T3SS) to inject effector proteins and subvert host signaling pathways, facilitating the growth, survival, and virulence. Notably, some bacteria harbor multiple distinct T3SSs with different functions. An extraordinary T3SS, the Escherichia coli Type III Secretion System 2 (ETT2), is widespread among Escherichia coli (E. coli) strains. Since many ETT2 carry genetic mutations or deletions, it is thought to be nonfunctional. However, increasing studies highlight ETT2 contributes to E. coli pathogenesis. Here, we present a comprehensive overview of genetic distribution and characterization of ETT2. Subsequently, we outline its functional potential, contending that an intact ETT2 may retain the capacity to translocate effector proteins and manipulate the host's innate immune response. Given the potential zoonotic implications associated with ETT2-carrying bacteria, further investigations into the structure, function and regulation of ETT2 are imperative for comprehensive understanding of E. coli pathogenicity and the development of effective control strategies.

The aroA and luxS Double-Gene Mutant Strain Has Potential to Be a Live Attenuated Vaccine against Salmonella Typhimurium.

Salmonella Typhimurium (S. Typhimurium) is a zoonotic pathogen posing a threat to animal husbandry and public health. Due to the emergence of antibiotic-resistant strains, alternative prevention and control strategies are needed. Live attenuated vaccines are an ideal option that provide protection against an S. Typhimurium pandemic. To develop a safe and effective vaccine, double-gene mutations are recommended to attenuate virulence. In this study, we chose aroA and luxS genes, whose deletion significantly attenuates S. Typhimurium's virulence and enhances immunogenicity, to construct the double-gene mutant vaccine strain SAT52ΔaroAΔluxS. The results show that the mutant strain's growth rate, adherence and invasion of susceptible cells are comparable to a wild-type strain, but the intracellular survival, virulence and host persistence are significantly attenuated. Immunization assay showed that 106 colony-forming units (CFUs) of SAT52ΔaroAΔluxS conferred 100% protection against wild-type challenges; the bacteria persistence in liver and spleen were significantly reduced, and no obvious pathological lesions were observed. Therefore, the double-gene mutant strain SAT52ΔaroAΔluxS exhibits potential as a live attenuated vaccine candidate against S. Typhimurium infection.

The functions of EF-hand proteins from host and zoonotic pathogens.

EF-hand proteins not only regulate biological processes, but also influence immunity and infection. In this review, we summarize EF-hand proteins' functions in host and zoonotic pathogens, with details in structures, Ca2+ affinity, downstream targets and functional mechanisms. Studies entitled as EF-hand-related but with less solid features were also discussed. We believe it could raise cautions and facilitate proper research strategy for researchers.

A lytic phage to control multidrug-resistant avian pathogenic Escherichia coli (APEC) infection.

The inappropriate use of antibiotics has led to the emergence of multidrug-resistant strains. Bacteriophages (phages) have gained renewed attention as promising alternatives or supplements to antibiotics. In this study, a lytic avian pathogenic Escherichia coli (APEC) phage designated as PEC9 was isolated and purified from chicken farm feces samples. The morphology, genomic information, optimal multiplicity of infection (MOI), one-step growth curve, thermal stability, pH stability, in vitro antibacterial ability and biofilm formation inhibition ability of the phage were determined. Subsequently, the therapeutic effects of the phages were investigated in the mice model. The results showed that PEC9 was a member of the siphovirus-like by electron microscopy observation. Biological characterization revealed that it could lyse two serotypes of E. coli, including O1 (9/20) and O2 (6/20). The optimal multiplicity of infection (MOI) of phage PEC9 was 0.1. Phage PEC9 had a latent period of 20 min and a burst period of 40 min, with an average burst size of 68 plaque-forming units (PFUs)/cell. It maintained good lytic activity at pH 3-11 and 4-50°C and could efficiently inhibit the bacterial planktonic cell growth and biofilm formation, and reduce bacterial counts within the biofilm, when the MOI was 0.01, 0.1, and 1, respectively. Whole-genome sequencing showed that PEC9 was a dsDNA virus with a genome of 44379 bp and GC content of 54.39%. The genome contains 56 putative ORFs and no toxin, virulence, or resistance-related genes were detected. Phylogenetic tree analysis showed that PEC9 is closely related to E. coli phages vB_EcoS_Zar3M, vB_EcoS_PTXU06, SECphi18, ZCEC10, and ZCEC11, but most of these phages exhibit different gene arrangement. The phage PEC9 could successfully protect mice against APEC infection, including improved survival rate, reduced bacterial loads, and organ lesions. To conclude, our results suggest that phage PEC9 may be a promising candidate that can be used as an alternative to antibiotics in the control of APEC infection.

Causal discovery approach with reinforcement learning for risk factors of type II diabetes mellitus.

BACKGROUND: Statistical correlation analysis is currently the most typically used approach for investigating the risk factors of type 2 diabetes mellitus (T2DM). However, this approach does not readily reveal the causal relationships between risk factors and rarely describes the causal relationships visually. RESULTS: Considering the superiority of reinforcement learning in prediction, a causal discovery approach with reinforcement learning for T2DM risk factors is proposed herein. First, a reinforcement learning model is constructed for T2DM risk factors. Second, the process involved in the causal discovery method for T2DM risk factors is detailed. Finally, several experiments are designed based on diabetes datasets and used to verify the proposed approach. CONCLUSIONS: The experimental results show that the proposed approach improves the accuracy of causality mining between T2DM risk factors and provides new evidence to researchers engaged in T2DM prevention and treatment research.

Effects of low-dose intravenous heparin therapy in aneurysmal subarachnoid hemorrhage: a randomized controlled clinical trial protocol.

BACKGROUND: Heparin anticoagulation therapy is a widely used method to prevent cerebral vasospasm (CV) and venous thrombosis in patients with subarachnoid hemorrhage caused by ruptured cerebral aneurysms. Subcutaneous heparin injection is considered safe and effective, whereas continuous intravenous heparin infusion is still being debated due to the risk of bleeding. Although most retrospective studies have confirmed the safety and effectiveness of unfractionated heparin (UFH) after aneurysm embolization therapy and its ability to reduce CV, there is still no randomized clinical trial comparing UFH and subcutaneous low-molecular-weight heparin (LMWH) injection in this population. Therefore, this study aims to compare the clinical outcomes associated with these two treatment approaches. METHODS: The study is an open-label, single-center, randomized controlled trial and aims to recruit 456 patients, with 228 patients in each group. The primary outcome was CV; the second outcomes measures are occurrence of bleeding events, ischemic events, heparin-induced thrombocytopenia, deep vein thrombosis, cerebral venous circulation time, brain edema score, and hydrocephalus incidence. ETHICS AND DISSEMINATION: This study protocol obtained ethical approval from the Ethics Committee of Baoan People's Hospital, Shenzhen, Guangdong (approval number: BYL20220805). This work will be published in peer-reviewed international medical journals and presented at medical conferences. TRIAL REGISTRATION: ClinicalTrials ID: NCT05696639. Registered on March 30, 2023.

Characteristics, pathogenic mechanism, zoonotic potential, drug resistance, and prevention of avian pathogenic Escherichia coli (APEC).

Although most Escherichia coli (E. coli) strains are commensal and abundant, certain pathogenic strains cause severe diseases from gastroenteritis to extraintestinal infections. Extraintestinal pathogenic E. coli (ExPEC) contains newborn meningitis E. coli (NMEC), uropathogenic E. coli (UPEC), avian pathogenic E. coli (APEC), and septicemic E. coli (SEPEC) based on their original host and clinical symptom. APEC is a heterogeneous group derived from human ExPEC. APEC causes severe respiratory and systemic diseases in a variety of avians, threatening the poultry industries, food security, and avian welfare worldwide. APEC has many serotypes, and it is a widespread pathogenic bacterium in poultry. In addition, ExPEC strains share significant genetic similarities and similar pathogenic mechanisms, indicating that APEC potentially serves as a reservoir of virulence and resistance genes for human ExPEC, and the virulence and resistance genes can be transferred to humans through food animals. Due to economic losses, drug resistance, and zoonotic potential, APEC has attracted heightened awareness. Various virulence factors and resistance genes involved in APEC pathogenesis and drug resistance have been identified. Here, we review the characteristics, epidemiology, pathogenic mechanism zoonotic potential, and drug resistance of APEC, and summarize the current status of diagnosis, alternative control measures, and vaccine development, which may help to have a better understanding of the pathogenesis and resistance of APEC, thereby reducing economic losses and preventing the spread of multidrug-resistant APEC to humans.

Acetoacetyl-CoA transferase ydiF regulates the biofilm formation of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes persistent infection of poultry and multi-system diseases, which seriously endanger the development of the poultry industry. Biofilm allows bacteria to adapt to the natural environment and plays an important role in resistance to the external environment and the pathogenicity of APEC, but the mechanism of its formation and regulatory network have not been clarified. In this study, we used a Tn5 transposon random mutation library constructed with APEC and identified ydiF, a gene that has not previously been recognized in E. coli biofilm formation. To confirm that the ydiF gene really can regulate the formation of APEC biofilm, the ydiF gene deletion strain was constructed using APEC81. Protein association networks prediction results show that ydiF is mainly associated with genes related to the metabolism of sugars and fatty acids. Deletion of the ydiF gene significantly reduces the formation of APEC biofilm and scanning electron microscopy indicated that the degree of adhesion between the bacteria was also reduced. The deletion of the ydiF gene also significantly reduced the motility of APEC81 and through transmission electron microscopy APEC81 was observed to have significantly fewer flagella. However, the colony morphology of APEC81 on Congo red and Coomassie brilliant blue media was unaffected. The results of fluorescence quantification showed that the deletion of the ydiF gene caused a down-regulation in the transcription of genes related to the second messenger, sugar metabolism, and quorum sensing. These results indicate that ydiF plays an important role in biofilm formation and the movement of APEC. In addition, it may be possible to regulate the formation of APEC biofilms by different methods such as by regulating the second messenger and metabolic system.

Identification of novel biofilm genes in avian pathogenic Escherichia coli by Tn5 transposon mutant library.

Avian pathogenic Escherichia coli (APEC) is the main pathogens that inflict the poultry industry. Biofilm as the pathogenic factors of APEC, which can enhance the anti-host immune system of APEC and improve its survival in the environment. In order to screen for new genes related to APEC biofilm. The APEC strain APEC81 was used to construct a mutant library by Tn5 insertion mutagenesis. Moreover the 28 mutant strains with severely weakened biofilm were successfully screened from 1500 mutant strains by crystal violet staining, in which 17 genes were obtained by high-efficiency thermal asymmetric interlaced PCR. The reported genes include 3 flagella genes (fliS, fliD, and fliR), 4 curli fimbriae genes (csgD, csgA, csgF, and csgG) and 3 type 1 fimbriae genes (fimA, fimD, and fimC). The novel genes include 3 coenzyme genes (gltA, bglX, and mltF) and 4 putative protein genes (yehE, 07045, 11735, 11255). To investigate whether these 17 genes co-regulate the biofilm, the 17 identified genes were deleted from APEC strain APEC81. The results showed that except for the 11735 and 11255 genes, the deletion of 15 genes significantly reduced the biofilm formation ability of APEC81 (P < 0.05). The result of rdar (red, dry and rough) colony morphology showed that curli fimbriae genes (csgD, csgA, csgF, and csgG) and other functional genes (fimC, glxK, yehE, 07045, and 11255) affected the colony morphology. In particular, the hypothetical protein YehE had the greatest influence on the biofilm. It was predicted to have the same structure as the type 1 fimbria protein. When yehE was deleted, the fimE transcription was up-regulated, and the fimA and fimB transcription were down-regulated, resulting in a decrease in type 1 fimbriae. Hence, the yehE mutant significantly reduced the biofilm and the adhesion and invasion ability to cells (P < 0.05). This study identified 5 novel genes (gltA, bglX, mltF, yehE, and 07045) related to biofilm formation and confirmed that yehE affects biofilm formation by type 1 fimbriae, which will benefit further study of the mechanism of biofilm regulation in APEC.

The Transcription Regulator YgeK Affects Biofilm Formation and Environmental Stress Resistance in Avian Pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is one of the most common pathogens in poultry and a potential gene source of human extraintestinal pathogenic E. coli (ExPEC), leading to serious economic losses in the poultry industry and public health concerns. Exploring the pathogenic mechanisms underpinning APEC and the identification of new targets for disease prevention and treatment are warranted. YgeK is a transcriptional regulator in APEC and is localized to the type III secretion system 2 of E. coli. In our previous work, the transcription factor ygeK significantly affected APEC flagella formation, bacterial motility, serum sensitivity, adhesion, and virulence. To further explore ygeK functions, we evaluated its influence on APEC biofilm formation and resistance to environmental stress. Our results showed that ygeK inactivation decreased biofilm formation and reduced bacterial resistance to environmental stresses, including acid and oxidative stress. In addition, the multi-level regulation of ygeK in APEC was analyzed using proteomics, and associations between differentially expressed proteins and the key targets of ygeK were investigated. Overall, we identified ygeK's new function in APEC. These have led us to better understand the transcriptional regulatory ygeK and provide new clues about the pathogenicity of APEC.

Avian Pathogenic Escherichia coli through Pfs Affects the Tran-Scription of Membrane Proteins to Resist ß-Lactam Antibiotics.

Avian pathogenic Escherichia coli (APEC) is a causative agent of colibacillosis, one of the principal causes of morbidity and mortality in poultry worldwide. Nowadays, antibiotics are mainly used to prevent and control poultry colibacillosis, but the situation of drug resistance is serious. 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) is involved in methylation reactions, polyamine synthesis, vitamin synthesis, and quorum sensing (QS) pathways. In this study, compared with the APEC wild-type strain DE17, the pfs deletion strain DE17Δpfs was more susceptible to ß-lactam antibiotics (amoxicillin, ceftazidime, cefuroxime) by drug sensitivity test and minimum inhibitory concentration (MIC), and the MIC of the DE17Δpfs was half that of the DE17. Quorum sensing signal molecule AI-2 is involved in antibiotic resistance. In the case of pfs inactivation, the DE17Δpfs cannot synthesize AI-2, so it is necessary to add AI-2 to study whether it affects APEC resistance. When the exogenous AI-2 was added, the MIC of all APEC did not change. Transcriptome sequencing indicated that the transcription levels of a lot of outer membrane protein genes and metabolic genes had changed due to the deletion of pfs. Moreover, the transcription levels of the efflux pump gene tolC and penicillin binding protein (fstI and mrcA) were significantly reduced (p < 0.05), while the transcription levels of the porin protein genes (ompF, ompC, and ompD) were significantly increased (p < 0.05). In addition, it was also found that the outer membrane permeability of the DE17Δpfs was significantly increased (p < 0.05). The results indicated that pfs does not affect APEC strain DE17 resistance to ß-lactam antibiotics through AI-2, but pfs affects the sensitivity of APEC to ß-lactam antibiotics by affecting antibiotic-related genes. This study can provide a reference for screening new drug targets.

Effect of nutritional and environmental conditions on biofilm formation of avian pathogenic Escherichia coli.

AIMS: To study the effects of environmental stress and nutrient conditions on biofilm formation of avian pathogenic Escherichia coli (APEC). METHODS AND RESULTS: The APEC strain DE17 was used to study biofilm formation under various conditions of environmental stress (including different temperatures, pH, metal ions, and antibiotics) and nutrient conditions (Luria-Bertani [LB] and M9 media, with the addition of different carbohydrates, if necessary). The DE17 biofilm formation ability was strongest at 25°C in LB medium. Compared to incubation at 37°C, three biofilm-related genes (csgD, dgcC, and pfs) were significantly upregulated and two genes (flhC and flhD) were downregulated at 25°C, which resulted in decreased motility. However, biofilm formation was strongest in M9 medium supplemented with glucose at 37°C, and the number of live bacteria was the highest as determined by confocal laser scanning microscopy. The bacteria in the biofilm were surrounded by a thick extracellular matrix, and honeycomb-like or rough surfaces were observed by scanning electron microscopy. Moreover, biofilm formation of the DE17 strain was remarkably inhibited under acidic conditions, whereas neutral and alkaline conditions were more suitable for biofilm formation. Biofilm formation was also inhibited at specific concentrations of cations (Na+ , K+ , Ca2+ , and Mg2+ ) and antibiotics (ampicillin, chloramphenicol, kanamycin, and spectinomycin). The real-time quantitative reverse transcription PCR showed that the transcription levels of biofilm-related genes change under different environmental conditions. CONCLUSIONS: Nutritional and environmental factors played an important role in DE17 biofilm development. The transcription levels of biofilm-related genes changed under different environmental and nutrient conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings suggest that nutritional and environmental factors play an important role in APEC biofilm development. Depending on the different conditions involved in this study, it can serve as a guide to treating biofilm-related infections and to eliminating biofilms from the environment.

Effect of Antibiotics on the Colonization of Live Attenuated Salmonella Enteritidis Vaccine in Chickens.

Salmonellosis, caused by Salmonella Enteritidis, is a prevalent zoonosis that has serious consequences for human health and the development of the poultry sector. The Salmonella Enteritis live vaccine (Sm24/Rif12/Ssq strain) is used to prevent Salmonella Enteritidis around the world. However, in some parts of the world, poultry flocks are frequently raised under intensive conditions, with significant amounts of antimicrobials to prevent and treat disease and to promote growth. To investigate whether antibiotic use influences the colonization of orally administered Salmonella live vaccines, 240 1-day-old specific pathogen-free chicks were randomly divided into 24 groups of 10 animals for this study. The different groups were treated with different antibiotics, which included ceftiofur, amoxicillin, enrofloxacin, and lincomycin-spectinomycin. Each group was immunized 2, 3, 4, and 5 days after withdrawal, respectively. At 5 days after immunization, the blood, liver, and ceca with contents were collected for the isolation of the Salmonella live vaccine strain. The result showed that no Salmonella vaccine strain was isolated in the blood and liver of the chicks in those groups. The highest number of Salmonella vaccine strains was isolated in the cecum from chicks vaccinated 2 days after ceftiofur withdrawal, and no Salmonella vaccine strain was isolated from the cecum in chicks immunized 3 days after ceftiofur withdrawal. Among the chickens immunized 4 days after the withdrawal of amoxicillin, enrofloxacin, and lincomycin-spectinomycin, the number of Salmonella vaccine colonization in the cecum was the highest, which was higher than that of the chickens immunized at other withdrawal interval (2, 3, and 5 days) groups and was higher than that of the chickens without treatment (P < 0.05). This study provides a reference for the effective use of the Salmonella Enteritidis live vaccine and key antibiotics commonly utilized in the poultry industry.

An Association between Inflammation and Cerebral Venous Thrombosis: A Retrospective Study.

OBJECTIVES: Evidence is currently accumulating for the role of inflammation in cerebral venous thrombosis (CVT). Neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), monocyte/high-density lipoprotein ratio (MHR), and systematic immune-inflammation index (SII) are easily obtainable indicators of systemic inflammations. However, there were few studies on the relationship between them and CVT. Therefore, we aimed to evaluate the connection between the occurrence of CVT and the inflammatory markers described. MATERIALS AND METHODS: The samples from 150 participants (including 90 CVT and 60 primary headaches as controls) with similar baseline characteristics were collected in this retrospective study. The NLR, PLR, MHR, SII and file records were employed to compare CVT patients with the control group. RESULTS: The levels of NLR (3.93 [2.27, 7.87] vs. 1.65 [1.31, 2.06], P < 0.001), PLR (149.52 [98.39, 198.82] vs. 107.34 [83.31, 129.47], P < 0.001), SII (896.84 [559.89, 1591.87] vs. 382.45 [273.51, 520.92], P < 0.001) and MHR (0.51 [0.40, 0.64] vs. 0.41 [0.29, 0.53], P = 0.001) were significantly higher in the CVT group. After multivariate logistic regression analysis, the SII degree (13.136, [5.675, 30.407], P < 0.001) and MHR degree (2.620, [1.123, 6.113], P < 0.01) were found as independent predictors of CVT. CONCLUSIONS: NLR, PLR, SII, and MHR may be able to assist in the diagnosis of CVT which confirmed that inflammation played an important role in CVT.

Lsr operon is associated with AI-2 transfer and pathogenicity in avian pathogenic Escherichia coli.

The function of Autoinducer-2 (AI-2) which acts as the signal molecule of LuxS-mediated quorum sensing, is regulated through the lsr operon (which includes eight genes: lsrK, lsrR, lsrA, lsrC, lsrD, lsrB, lsrF, and lsrG). However, the functions of the lsr operon remain unclear in avian pathogenic Escherichia coli (APEC), which causes severe respiratory and systemic diseases in poultry. In this study, the presence of the lsr operon in 60 APEC clinical strains (serotypes O1, O2, and O78) was investigated and found to be correlated with serotype and has the highest detection rate in O78. The AI-2 binding capacity of recombinant protein LsrB of APEC (APEC-LsrB) was verified and was found to bind to AI-2 in vitro. In addition, the lsr operon was mutated in an APEC strain (APEC94Δlsr(Cm)) and the mutant was found to be defective in motility and AI-2 uptake. Furthermore, deletion of the lsr operon attenuated the virulence of APEC, with the LD50 of APEC94Δlsr(Cm) decreasing 294-fold compared with wild-type strain APEC94. The bacterial load in the blood, liver, spleen, and kidneys of ducks infected with APEC94Δlsr(Cm) decreased significantly (p < 0.0001). The results of transcriptional analysis showed that 62 genes were up-regulated and 415 genes were down-regulated in APEC94Δlsr(Cm) compared with the wild-type strain and some of the down-regulated genes were associated with the virulence of APEC. In conclusion, our study suggests that lsr operon plays a role in the pathogenesis of APEC.

Development of a multiplex PCR assay for the simultaneous and rapid detection of six pathogenic bacteria in poultry.

Escherichia coli, Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella spp. and Staphylococcus aureus are six bacterial pathogens of avian. However, these pathogens may cause many similar pathological changes, resulting in clinical isolates that are difficult to quickly and simultaneously detect and identify. Here, a multiplex polymerase chain reaction (m-PCR) assay is reported to rapidly identify targets genes (phoA, KMT1, ureR, toxA, invA, and nuc) of these six pathogens in clinical samples. Six pairs of specific primers were designed. The optimal reaction conditions, specificity, and sensitivity of the m-PCR assay were investigated. The results showed that betaine remarkably improved amplification of the target genes. Specific test results showed that all six pathogens were detected by the proposed m-PCR protocol without cross-amplification with viruses or parasites. Sensitivity test results showed that the m-PCR system could amplify the six target genes from bacterial genomes or cultures with template amounts of 500 pg or 2.8-8.6 × 103 colony forming units, respectively. Furthermore, the six bacterial pathogens isolated from the infected tissue samples were successfully identified. The proposed m-PCR assay is a useful tool to monitor and diagnose bacterial infection in birds with high specificity, sensitivity and throughput.

Use of a modified bacterial ghost lysis system for the construction of an inactivated avian pathogenic Escherichia coli vaccine candidate.

Vaccination is an effective strategy to prevent avian colibacillosis. Bacterial ghosts (BGs) are prepared by the controlled expression of the phiX174 gene E, which mediates the lysis of Gram-negative bacteria. Staphylococcal nuclease A may be used to produce BGs for further inactivation of host bacteria and elimination of residual genetic material. In this study, the double promoter lysis plasmid (pUC19-ΔcI857-E-rrnB-pL-SN) was successfully constructed and BGs were prepared at 37 °C. The cleavage efficiency of Escherichia coli BGs was 99.9%. Furthermore, to evaluate the immunological effects of the BG vaccines in chickens, a BG vaccine was prepared using the serotype O2 avian pathogenic Escherichia coli deletion strain (DE17ΔluxSΔaroA). The results showed that the BG vaccine was able to achieve over 90% immune protection against virulent challenge using the same serotype O2 strain (DE17 or CE35), while it showed poor cross-protection against serotypes O1 and O78 (data not shown). The enzyme-linked immunosorbent assay results showed that the antibody levels in the immunized groups were higher than in the control group (p < 0.05), with the BG group being the highest. The cytokine tests showed that the levels of interferon-γ in the BG immune group were higher than in the phosphate-buffered saline (PBS) control group (non-immune) (p < 0.01) and the formalin-inactivated vaccine immune group (p < 0.05), and the levels of tumor necrosis factor-α in the BG group were higher than in the formalin-inactivated vaccine (p > 0.05) and the PBS control groups (p < 0.05). In addition, pathological analysis revealed that the PBS control group showed typical fibrinous pericarditis and perihepatitis, whereas the immune group showed no obvious pathological changes. In summary, our findings provide a new strategy for the prevention and control of avian colibacillosis.

The role of the wzy gene in lipopolysaccharide biosynthesis and pathogenesis of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes severe respiratory and systemic diseases in poultry. The wzy gene encodes the O-antigen polymerase (Wzy), which plays an important role in the synthesis of the lipopolysaccharide (LPS) of bacteria. However, the function of the wzy gene in APEC remains unclear. Hence, in this study, a strain harboring a wzy gene mutant (DE17Δwzy) was constructed and the characteristics of this strain were analyzed. The results showed that mutant of wzy changed the phenotype of the LPS and affected serum agglutination of the O-antigen. Decreased motility and biofilm formation was also observed, but the endotoxin titer of the LPS in APEC was not affected. In addition, the wzy mutation significantly decreased the adherence and invasion to DF-1 cells, especially the survival abilities in duck serum and complement. Furthermore, an LD50 assay revealed that the virulence of mutant strain DE17Δwzy was attenuated 132-fold compared with wild-type strain DE17. Moreover, the bacterial load in the blood, liver, spleen, and kidneys of ducks infected with DE17Δwzy was decreased significantly compared with wild-type strain DE17 (p < 0.0001). These results confirmed that the wzy gene is associated with LPS biosynthesis and bacterial pathogenicity in APEC.

Targeted shRNA-loaded liposome complex combined with focused ultrasound for blood brain barrier disruption and suppressing glioma growth.

Our previous studies have demonstrated that focused ultrasound (FUS) combined with DNA-loaded microbubbles (MBs) can induce noninvasive, reversible, local disruption of the blood brain barrier (BBB) and enable targeted exogenous gene transfer into the central nervous system. However, due to low gene loading or the absence of positive targeting, to date, there has been no therapeutic effect of MBs combined with FUS in tumor treatment. In the current study, we adopted a phospholipid complex that exhibited sufficient gene loading and peptide-mediated targeting to delay glioma growth. First, we bound MBs to shBirc5-lipo-NGR, which performed the dual function of tumor cell targeting and effective gene loading. Next, we demonstrated that FUS-aided MB-shBirc5-lipo-NGR exhibited a higher transfection efficiency compared with the control group. Finally, we evaluated the silencing effect of shBirc5 using an apoptosis assay, real time-polymerase chain reaction (PCR), western blotting (WB) in vitro and a volume measurement survival analysis in vivo. The experimental group exhibited a significant therapeutic effect, while the FUS-only, MB-shBirc5-lipo-NGR-only and FUS-aided MB-shControl-lipo-NGR groups displayed no changes in tumor growth or survival time (P < .01). Consequently, our study indicated that MB-shBirc5-lipo-NGR combined with FUS is a promising new RNA interference technique for the treatment of glioma.