Thermostable vacuum foam dried Newcastle disease vaccine: Process optimization and pilot-scale study.

Vacuum foam drying (VFD) has been shown to improve the thermostability and long-term shelf life of Newcastle Disease Virus (NDV). This study optimized the VFD process to improve the shelf life of NDV at laboratory-scale and then tested the optimized conditions at pilot-scale. The optimal NDV to T5 formulation ratio was determined to be 1:1 or 3:2. Using the 1:1 virus to formulation ratio, the optimal filling volumes were determined to be 13-17% of the vial capacity. The optimized VFD process conditions were determined to be at a shelf temperature of 25℃ with a minimum overall drying time of 44 h. The vaccine samples prepared using these optimized conditions at laboratory-scale exhibited virus titer losses of ≤ 1.0 log10 with residual moisture content (RMC) below 3%. Furthermore, these samples were transported for 97 days around China at ambient temperature without significant titer loss, thus demonstrating the thermostability of the NDV-VFD vaccine. Pilot-scale testing of the NDV-VFD vaccine at optimized conditions showed promising results for up-scaling the process as the RMC was below 3%. However, the virus titer loss was slightly above 1.0 log10 (approximately 1.1 log10). Therefore, the NDV-VFD process requires further optimization at pilot scale to obtain a titer loss of ≤ 1.0 log10. Results from this study provide important guidance for possible industrialization of NDV-VFD vaccine in the future. KEY POINTS: • The process optimization and scale-up test of thermostable NDV vaccine prepared through VFD is reported for the first time in this study. • The live attenuated NDV-VFD vaccine maintained thermostability for 97 days during long distance transportation in summer without cold chain conditions. • The optimized NDV-VFD vaccine preparations evaluated at pilot-scale maintained acceptable levels of infectivity after preservation at 37℃ for 90 days, which demonstrated the feasibility of the vaccine for industrialization.

A pyrylium salt-based fluorescent probe for the highly sensitive detection of methylamine vapour.

The amine vapour produced by microorganisms is an important indicator of food spoilage. Amines are also ubiquitous in the chemical industry. The development of molecule-based ion sensors has been a pivotal issue that is currently receiving considerable attention. In this work, a new pyrylium salt-based fluorescent probe MTPY has been developed as a rapid, highly sensitive, and selective sensor for methylamine vapour. MTPY exhibits an obvious fluorescence response from yellow to cyan towards CH3NH2 vapour. The calibration curve of titration analysis shows a linear relationship of the fluorescence intensity at 514 nm versus the methylamine concentration in the range of 0.1-2 ppm. In addition to the linearity (R2 = 0.974) and short response time with a low detection limit (2.6 ppt, 8.4 × 10-8 M), the sensing mechanism was traced using mass spectrometry.

A benzophenoxazine-based NIR fluorescent probe for the detection of hydrogen sulfide and imaging in living cells.

Hydrogen sulfide (H2S) plays a vital role in regulating many important physiological functions. However, H2S-containing industrial wastewater is inevitably pumped into the environment which seriously contaminates water supplies and foodstuffs. So it is crucial to develop a single fluorescent probe for H2S detection with high sensitivity and selectivity. Herein, a colorimetric and turn-on near infrared (NIR) fluorescent probe NRDNP based on benzophenoxazine was designed and synthesized by thiolysis of 2,4-dinitrophenyl (DNP) ether as the specific reaction site strategy to achieve highly specific H2S detection in living systems. The studies demonstrated that the probe NRDNP exhibited excellent sensing performance toward H2S with an about 80-fold NIR fluorescence enhancement, a rapid response within 10 min, excellent sensitivity with a detection limit of 19 nM and good selectivity. Furthermore, the NRDNP is an optical sensor which visually changes in terms of the fluorescence colour/intensity upon sensing gaseous H2S molecules. NRDNP has low cytotoxicity and has been successfully applied in the fluorescence imaging of H2S in living cells, suggesting that it would be an effective tool for H2S detection in living systems.

Quorum sensing-1 signaling of N-hexanoyl-L-homoserine lactone contributes to virulence in avian pathogenic Escherichia coli.

Avian pathogenic E. coli (APEC) caused avian colibacillosis is mostly common in poultry industry worldwide. APEC virulence factors lead to pathogenesis and the quorum sensing (QS) system is actively involved in the regulation of these virulence factors. Signaling molecules in QS are known as autoinducers (AIs). In QS-1, E. coli encodes a single LuxR homolog, i.e., SdiA, but does not express the LuxI homolog, an acyl-homoserine lactone (AHL) synthase of producing AI-1. Avian pathogenic E. coli (APEC) regulates its virulence genes expression in response to exogenous AHLs, but regulatory mechanisms of AHL and QS-1 are still unknown. This study targeted the APEC CE129 isolate as the reference strain, and the Yersinia enterocolitica yenI gene was expressed into APEC CE129. CE129/pyenI was conferred the ability to produce AHL signal. The CE129 SdiA mutant strain with an in-frame sdiA (AHL receptor) gene deletion was constructed by a λRed recombination system, which lost the ability to sense AHL. The goal of this study was to explore the function of QS-1 upon virulence and elucidate the regulatory effect of QS-1/AHL signals in the APEC strain. Adherence and invasion assays revealed that QS-1 affected APEC adherence and survival ability. APEC biofilm formation was also suppressed under C6HSL. Interestingly, APEC exhibited different phenotypes of acid tolerance and flagella expression when compared to enterotoxigenic E. coli or enterohemorrhagic E. coli (ETEC and EHEC, respectively). These findings enhance our understanding of the QS mechanism.

Role of long polar fimbriae type 1 and 2 in pathogenesis of mammary pathogenic Escherichia coli.

Escherichia coli is a leading cause of bovine mastitis worldwide. The bacteria can rapidly grow in milk and elicit a strong lipopolysaccharide (LPS)/toll-like receptor-4 (TLR4)-dependent inflammatory response. Recently, the long polar fimbriae (LPF) were identified as a promising virulence factor candidate widely distributed in mammary pathogenic E. coli (MPEC) strains. Mammary pathogenic E. coli possess 2 lpf loci encoding LPF1 and LPF2, respectively. By deleting the major fimbrial subunit gene, lpfA, we found that both LPF1 and LPF2 contribute to MPEC adhesion, invasion, and biofilm formation in vitro. The lpf1A and lpf2A mutants showed reduced cytotoxicity in our in vitro cell infection model. Furthermore, we observed that LPF2 induced a mild TLR4-independent proinflammatory response. The median lethal dose (LD50) of both ∆lpf2A and ∆lpf1A∆lpf2A mutants to BALB/c mice increased by 0.38 and 0.15 logs, respectively, whereas that of wild-type strain MPJS13 was 8.69 logs. In contrast, LPF1 deficiency significantly enhanced the LPS/TLR4-mediated inflammatory response in mammary epithelial cells, and the LD50 of the mutant decreased to 8.18 logs. In conclusion, our data suggested that LPF are important in MPEC colonization of mammary cells and may provide a benefit to bacterial intracellular survival that induces persistent bovine mastitis.

Long polar fimbriae contribute to pathogenic Escherichia coli infection to host cells.

Long polar fimbria (LPF) is one of the few fimbrial adhesins of enterohemorrhagic Escherichia coli (E. coli) O157:H7 associated with colonization on host intestine, and both two types of LPF (including LPF1 and LPF2) play essential roles during the bacterial infection process. Though the fimbriae had been well studied in intestinal pathogenic E. coli strains, new evidences from our research revealed that it might be the key virulence for bovine mastitis pathogenic E. coli (MPEC) as well. This article summarizes the current knowledge on the LPF in E. coli, focusing on its genetic characteristics, prevalence, expression regulation, and adherence mechanism in different pathotypes of E. coli strains.

Binding determinants in the interplay between porcine aminopeptidase N and enterotoxigenic Escherichia coli F4 fimbriae.

The binding of F4+ enterotoxigenic Escherichia coli (ETEC) and the specific receptor on porcine intestinal epithelial cells is the initial step in F4+ ETEC infection. Porcine aminopeptidase N (APN) is a newly discovered receptor for F4 fimbriae that binds directly to FaeG adhesin, which is the major subunit of the F4 fimbriae variants F4ab, F4ac, and F4ad. We used overlapping peptide assays to map the APN-FaeG binding sites, which has facilitated in the identifying the APN-binding amino acids that are located in the same region of FaeG variants, thereby limiting the major binding regions of APN to 13 peptides. To determine the core sequence motif, a panel of FaeG peptides with point mutations and FaeG mutants were constructed. Pull-down and binding reactivity assays using piglet intestines determined that the amino acids G159 of F4ab, N209 and L212 of F4ac, and A200 of F4ad were the critical residues for APN binding of FaeG. We further show using ELISA and confocal microscopy assay that amino acids 553-568, and 652-670 of the APN comprise the linear epitope for FaeG binding in all three F4 fimbriae variants.

Construction and sequencing of an infectious clone of the goose embryo-adapted Muscovy duck parvovirus vaccine strain FZ91-30.

BACKGROUND: Muscovy duck parvovirus (MDPV) is the etiological agent of Muscovy duckling parvoviral disease, which is characterized by diarrhea, locomotive dysfunction, stunting, and death in young ducklings, and causes substantial economic losses in the Muscovy duck industry worldwide. FZ91-30 is an attenuated vaccine strain that is safe and immunogenic to ducklings, but the genomic information and molecular mechanism underlining the attenuation are not understood. METHODS: The FZ91-30 strain was propagated in 11-day-old embryonated goose eggs, and viral particles were purified from the pooled allantoic fluid by differential centrifugation and ultracentrifugation. Single-stranded genomic DNA was extracted and annealed to form double-stranded DNA. The dsDNA digested with NcoI resulted two sub-genomic fragments, which were then cloned into the modified plasmid pBluescript II SK, respectively, generating plasmid pBSKNL and pBSKNR. The sub-genomic plasmid clones were sequenced and further combined to construct the plasmid pFZ that contained the entire genome of strain FZ91-30. The complete genome sequences of strain FM and YY and partial genome sequences of other strains were retrieved from GenBank for sequence comparison. The plasmid pFZ containing the entire genome of FZ91-30 was transfected in 11-day-old embryonated goose eggs via the chorioallantoic membranes route to rescue infectious virus. A genetic marker was introduced into the rescued virus to discriminate from its parental virus. RESULTS: The genome of FZ91-30 consists of 5,131 nucleotides and has 98.9 % similarity to the FM strain. The inverted terminal repeats (ITR) are 456 nucleotides in length, 14 nucleotides longer than that of Goose parvovirus (GPV). The exterior 415 nucleotides of the ITR form a hairpin structure, and the interior 41 nucleotides constitute the D sequence, a reverse complement of the D' sequence at the 3' ITR. Amino acid sequence alignment of the VP1 proteins between FZ91-30 and five pathogenic MDPV strains revealed that FZ91-30 had five mutations; two in the unique region of the VP1 protein (VP1u) and three in VP3. Sequence alignment of the Rep1 proteins revealed two amino acid alterations for FZ91-30, both of which were conserved for two pathogenic strains YY and P. Transfection of the plasmid pFZ in 11-day-old embryonated goose eggs resulted in generation of infectious virus with similar biological properties as compared with the parental strain. CONCLUSIONS: The amino acid mutations identified in the VP1 and Rep1 protein may contribute to the attenuation of FZ91-30 in Muscovy ducklings. Plasmid transfection in embryonated goose eggs was suitable for rescue of infectious MDPV.

The flagellin hypervariable region is a potential flagella display domain in probiotic Escherichia coli strain Nissle 1917.

The most studied probiotic, Escherichia coli strain Nissle 1917 (EcN) possesses flagella of serotype H1. To explore the potential to use EcN flagellin in flagella display applications, we investigated the effect of deleting amino acids in the hypervariable region of flagellin on EcNc (EcN cured of its two cryptic plasmids pMUT1 and pMUT2). Two EcNc flagellin isogenic mutants with deletions of amino acid residual from 277 to 286 and from 287 to 296 in the hypervariable domain were constructed. Both mutants were flagellated, adherent to IPEC-J2 cells, and colonized BALB/c mice. These hypervariable regions may have future utility in the display of heterologous epitopes.

Analysis of the genome sequence of the pathogenic Muscovy duck parvovirus strain YY reveals a 14-nucleotide-pair deletion in the inverted terminal repeats.

Genomic information about Muscovy duck parvovirus is still limited. In this study, the genome of the pathogenic MDPV strain YY was sequenced. The full-length genome of YY is 5075 nucleotides (nt) long, 57 nt shorter than that of strain FM. Sequence alignment indicates that the 5' and 3' inverted terminal repeats (ITR) of strain YY contain a 14-nucleotide-pair deletion in the stem of the palindromic hairpin structure in comparison to strain FM and FZ91-30. The deleted region contains one "E-box" site and one repeated motif with the sequence "TTCCGGT" or "ACCGGAA". Phylogenetic trees constructed based the protein coding genes concordantly showed that YY, together with nine other MDPV isolates from various places, clustered in a separate branch, distinct from the branch formed by goose parvovirus (GPV) strains. These results demonstrate that, despite the distinctive deletion, the YY strain still belongs to the classical MDPV group. Moreover, the deletion of ITR may contribute to the genome evolution of MDPV under immunization pressure.

Membrane cholesterol plays an important role in enteropathogen adhesion and the activation of innate immunity via flagellin-TLR5 signaling.

Lipid rafts are cholesterol- and sphingolipid-rich ordered microdomains distributed in the plasma membrane that participates in mammalian signal transduction pathways. To determine the role of lipid rafts in mediating interactions between enteropathogens and intestinal epithelial cells, membrane cholesterol was depleted from Caco-2 and IPEC-J2 cells using methyl-ß-cyclodextrin. Cholesterol depletion significantly reduced Escherichia coli and Salmonella enteritidis adhesion and invasion into intestinal epithelial cells. Complementation with exogenous cholesterol restored bacterial adhesion to basal levels. We also evaluated the role of lipid rafts in the activation of Toll-like receptor 5 signaling by bacterial flagellin. Depleting membrane cholesterol reduced the ability of purified recombinant E. coli flagellin to activate TLR5 signaling in intestinal cells. These data suggest that both membrane cholesterol and lipid rafts play important roles in enteropathogen adhesion and contribute to the activation of innate immunity via flagellin-TLR5 signaling.

More than a locomotive organelle: flagella in Escherichia coli.

The flagellum is a locomotive organelle that allows bacteria to respond to chemical gradients. This review summarizes the current knowledge regarding Escherichia coli flagellin variants and the role of flagella in bacterial functions other than motility, including the relationship between flagella and bacterial virulence.

Escherichia coli type III secretion system 2: a new kind of T3SS?

Type III secretion systems (T3SSs) are employed by Gram-negative bacteria to deliver effector proteins into the cytoplasm of infected host cells. Enteropathogenic Escherichia coli use a T3SS to deliver effector proteins that result in the creation of the attaching and effacing lesions. The genome sequence of the Escherichia coli pathotype O157:H7 revealed the existence of a gene cluster encoding components of a second type III secretion system, the E. coli type III secretion system 2 (ETT2). Researchers have revealed that, although ETT2 may not be a functional secretion system in most (or all) strains, it still plays an important role in bacterial virulence. This article summarizes current knowledge regarding the E. coli ETT2, including its genetic characteristics, prevalence, function, association with virulence, and prospects for future work.

Quorum-sensing gene luxS regulates flagella expression and Shiga-like toxin production in F18ab Escherichia coli.

To investigate the effect of the luxS gene on the expression of virulence factors in Shiga-like toxin producing and verotoxin-producing Escherichia coli, the luxS gene from E. coli 107/86 (wild type, O139:H1:F18ab, Stx2e) was deleted. The successful deletion of luxS was confirmed by bioluminescence assays. The luxS deletion mutant exhibited changed flagella-related phenotypes, like impaired expression of flagella, decreased flagella motility, reduced biofilm formation, and reduced ability to induce pro-immunity response in host cells, which were restored after complementation with the intact luxS gene. The mutant strain also displayed attenuated production of Stx2e. This study provides new information to the crucial function of luxS in regulating Shiga-like toxin producing E. coli virulence.

[Advance on the pathogenicity and immunological application of bacterial flagella--a review].

Being a surface structure of bacteria, flagella have been thought to simply act as the locomotive organelles for a long time. In recent years, as increasing information gathered from studies on the pathogenicity of flagella, we found flagella could contribute to invasion and adhesion to the host cells, playing an important role in the biofilm formation and being correlated with bacterial virulence secretion system. Binding of flagellin and toll-like receptor 5 may stimulate signaling pathway, resulting in the pro-inflammatory response. Meanwhile, flagella act as a new immune adjuvant as well, because of their good immunity character. This article summarizes the current knowledge of bacterial flagella, including their structure, contribution to the pathogenicity of the bacteria, and their potential application in immunity.

Action-Evaluator: A Visualization Approach for Player Action Evaluation in Soccer.

In soccer, player action evaluation provides a fine-grained method to analyze player performance and plays an important role in improving winning chances in future matches. However, previous studies on action evaluation only provide a score for each action, and hardly support inspecting and comparing player actions integrated with complex match context information such as team tactics and player locations. In this work, we collaborate with soccer analysts and coaches to characterize the domain problems of evaluating player performance based on action scores. We design a tailored visualization of soccer player actions that places the action choice together with the tactic it belongs to as well as the player locations in the same view. Based on the design, we introduce a visual analytics system, Action-Evaluator, to facilitate a comprehensive player action evaluation through player navigation, action investigation, and action explanation. With the system, analysts can find players to be analyzed efficiently, learn how they performed under various match situations, and obtain valuable insights to improve their action choices. The usefulness and effectiveness of this work are demonstrated by two case studies on a real-world dataset and an expert interview.

Flagella from F18+Escherichia coli play a role in adhesion to pig epithelial cell lines.

F18 fimbriae and toxins produced by F18 fimbriae-carrying Escherichia coli (E. coli) strains are known virulence factors responsible for post-weaning diarrhea (PWD) and edema disease (ED). In this study, we showed that fliC isogenic mutants constructed in two reference wild-type F18 fimbriae (F18+) E. coli were markedly impaired in adherence in vitro cell models (p < 0.05). Flagella purified from F18+E. coli could directly bind to cultured piglet epithelial cells and block adherence of F18+E. coli to cells when pre-incubated. In addition, the F18+E. coli fliC deletion mutants up-regulated the expression of type I fimbriae produced by F18+E. coli strains. These results demonstrated that expression of flagella is essential for the adherence of F18+E. coli in vitro.

Both flagella and F4 fimbriae from F4ac+ enterotoxigenic Escherichia coli contribute to attachment to IPEC-J2 cells in vitro.

The role of flagella in the pathogenesis of F4ac+ Enterotoxigenic Escherichia coli (ETEC) mediated neonatal and post-weaning diarrhea (PWD) is not currently understood. We targeted the reference C83902 ETEC strain (O8:H19:F4ac+ LT+ STa+ STb+), to construct isogenic mutants in the fliC (encoding the major flagellin protein), motA (encoding the flagella motor), and faeG (encoding the major subunit of F4 fimbriae) genes. Both the ΔfliC and ΔfaeG mutants had a reduced ability to adhere to porcine intestinal epithelial IPEC-J2 cells. F4 fimbriae expression was significantly down-regulated after deleting fliC, which revealed that co-regulation exists between flagella and F4 fimbriae. However, there was no difference in adhesion between the ΔmotA mutant and its parent strain. These data demonstrate that both flagella and F4 fimbriae are required for efficient F4ac+ ETEC adhesion in vitro.

Flagella and bacterial pathogenicity.

As locomotive organelles, flagella allow bacteria to move toward favorable environments. A flagellum consists of three parts: the basal structure (rotary motor), the hook (universal joint), and the filament (helical propeller). For ages, flagella have been generally regarded as important virulence factors, mainly because of their motility property. However, flagella are getting recognized to play multiple roles with more functions besides motility and chemotaxis. Recent evidence has pinpointed that the bacterial flagella participate in many additional processes including adhesion, biofilm formation, virulence factor secretion, and modulation of the immune system of eukaryotic cells. This mini-review summarizes data from recent studies that elucidated how flagella, as a virulence factor, contribute to bacterial pathogenicity.

Formulation and Evaluation of Lipidized Imiquimod as an Effective Adjuvant.

Adjuvants are essential for the induction of robust immune responses against vaccine antigens. Small-molecule TLR7 agonists hold high potential for this purpose. In this communication, imiquimod (IMQ) bearing a cholesterol lipid moiety derivative, IMQ-Chol, was designed and synthesized as a vaccine adjuvant, which could release parent IMQ molecules in aqueous conditions via amide bond hydrolysis. We performed a series of immunological evaluations by cooperating with the inactivated foot-and-mouth disease virus (FMDV). All of the results confirmed that IMQ-Chol could stimulate the body for a prolonged time to produce strong humoral and cellular immunity with a balanced Th1/Th2 immune response through a TLR7-related MAPK pathway. In addition, the results of the proof-of-concept vaccine indicated IMQ-Chol had a good effect on preventing and treating FMD in pigs.