Generation of an attenuated strain oral vaccine candidate using a novel double selection platform in Escherichia coli.

Live attenuated bacteria delivered orally are interesting tools for mucosal immunization. The objective of this study was to construct a novel counter-selection platform based on an attenuated wild-type Escherichia coli (E. coli) strain and to utilize it for the delivery of LTR192G-STaA13Q fusion protein as an oral vaccine. First, a counter-selectable marker, namely, PRPL-Kil, was inserted into an attenuated wild-type E. coli strain through the use of the red and G-DOC homologous recombination systems to construct the counter-selection platform, and PRPL-Kil was subsequently replaced by the LT192-STa13 fusion gene to construct the oral vaccine O142 (yaiT::LT192-STa13) (ER-A). Subsequently, BALB/c mice were orogastrically inoculated with ER-A. Our results showed that ER-A could induce the production of specific IgA and IgG against fimbriae (F41) and enterotoxins (LT and STa), with neutralizing activity in BALB/c mice. In addition, assays of cellular immune responses showed that the stimulation index (SI) values of immunized mice were significantly higher than those of control mice (P<0.05), and revealed a marked shift toward Th2-mediated immunity. These findings suggest that ER-A is a suitable candidate for an oral vaccine strain to protect animals from enter toxigenic Escherichia coli (ETEC) infection.

Simultaneous oral immunization of mice with live attenuated Escherichia coli expressing LT192-STa 13 and LT 192-STb fusion immunogen, respectively, for polyvalent vaccine candidate.

Previous epidemiological study showed that most of the porcine enterotoxin Escherichia coli (ETEC) strains harbor multiple enterotoxins but lack any of the fimbriae or non-fimbrial adhesion genes. Therefore, effective ETEC vaccines need to aim directly at all the enterotoxin antigens. The objective of this study was to evaluate the simultaneous immune effect of two live attenuated E. coli strains expressing LTR192G-STaA13Q and LTR192G-STb fusion immunogen, respectively. The results showed that both local mucosal and systemic immune responses against LT, STa, STb, and F41 were induced in BALB/c mice immunized orally with the recombinant E. coli strains ER-A and ER-B simultaneously. In addition, results of cellular immune responses showed that stimulation index (SI) values of immunized mice were significantly higher than control mice (P < 0.05) and a marked shift toward type-2 helper T lymphocyte (Th 2) immunity. Moreover, the induced antibodies demonstrated neutralizing effects on LT, STa, and STb producing E. coli infection. These data indicated that the use of recombinant E. coli ER-A and ER-B could be a valuable strategy for future polyvalent vaccine development of ETEC.

Construction and evaluation of a fluorescence-based live attenuated Escherichia coli delivery system for generating oral vaccine candidate.

Enter toxigenic Escherichia coli (ETEC) is a major pathogen of swine industry that can have a substantial impact on morbidity and mortality. Therefore, it is necessary to develop effective vaccines for the prevention of ETEC infection. Live attenuated bacteria delivery system are effective tools for mucosal immunization. The purpose of this study was to construct a novel delivery system that can present the LTR192G-STb fusion protein as oral vaccine candidate. Firstly, the PRPL-mKate2 fluorescent cassette was inserted into the genome (yaiT pseudogene) of an attenuated E. coli by homologous recombination methods to construct the delivery system O142(yaiT::PRPL-mKate2). Secondly, the oral vaccine O142(yaiT:: LT192-STb) (ER-B) was derived for replacing the PRPL-mKate2 by LT192-STb fusion gene, and then it was tested for its feasibility as oral vaccine candidate. Subsequently, BALB/c mice were orogastrically immunized with ER-B. Results showed that mice orally immunized with ER-B produced high levels of specific IgA and IgG antibodies. The induced antibodies demonstrated neutralizing effects to enter toxins LT and STb. In addition, results of cellular immune responses showed that stimulation index values of immunized mice were significantly higher than the control group (P < 0.05) and with a marked shift towards Th 2 immunity. These data indicated that the recombinant E. coli ER-B could be a valuable candidate of future vaccines against ETEC infection.

Analysis and application of a neutralizing linear epitope on liable toxin B of enterotoxin Escherichia coli.

Heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) is one of the major virulence factors for causing diarrhea in piglets, and LT is a strong immunogen. Thus, LT represents an important target for development of vaccines and diagnostic tests. In this study, bioinformatic tools were used to predict six antigenic B cell epitopes in the B subunit of LT protein (LTB) of ETEC strains. Then, seven antigenic B cell epitopes of LTB were identified by polyclonal antisera (polyclonal antibody (PAb)) using a set of LTB-derived peptides expressed as maltose-binding protein (MBP) fusion protein. In addition, one LTB-specific monoclonal antibody (MAb) was generated and defined its corresponding epitope as mentioned above. This MAb was able to specifically bind with native LT toxin and has no cross-reaction with LT-II (type II heat-labile enterotoxin), Stx1 (Shiga toxin I), Stx2 (Shiga toxin II), STa (heat-stable enterotoxin I), and STb (heat-stable enterotoxin II) toxins. Further, this MAb was able to interrupt LT toxin specific binding to GM1 receptor, indicating that the corresponding epitope is the specific binding region to GM1 receptor. Moreover, in vitro and in vivo assay showed that the MAb was able to neutralize the native LT toxin. Diarrheal suckling pigs challenged with LT-positive ETEC strain recovered when an enema with this purified MAb was administered. This study will provide the foundation for further studies about the interaction between LT toxin and GM1 receptor and about the developing of epitope-based vaccines and specific therapeutic agent.

KGScope: Interactive Visual Exploration of Knowledge Graphs With Embedding-Based Guidance.

Knowledge graphs have been commonly used to represent relationships between entities and are utilized in the industry to enhance service qualities. As knowledge graphs integrate data from a variety of sources, they can also be useful references for data analysts. However, there is a lack of effective tools to make the most of the rich information in knowledge graphs. Existing knowledge graph exploration systems are ineffective because they did not consider various user needs and characteristics of knowledge graphs. Exploratory approaches specifically designed to uncover and summarize insights in knowledge graphs have not been well studied yet. In this article, we propose KGScope that supports interactive visual explorations and provides embedding-based guidance to derive insights from knowledge graphs. We demonstrate KGScope with usage scenarios and assess its efficacy in supporting the exploration of knowledge graphs with a user study. The results show that KGScope supports knowledge graph exploration effectively by providing useful information and helping explore the entire network.

Development of isothermal amplification methods for rapid and sensitive detection of heat-labile enterotoxin producing Escherichia coli.

The objective of this study was to establish a novel isothermal amplification method for detection of heat-labile enterotoxin (LT-I)-producing Escherichia coli. Loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and isothermal multiple-self-matching-initiated amplification (IMSA) were developed and evaluated. Optimal conditions, specificity, and sensitivity tests were performed and compared to qPCR findings. All three methods could produce ladder-like products with LT-I positive samples, while no products were generated with the negative controls. The amplified products could be directly visualized as negative or positive in the isothermal amplification (IAM) tube, which saved time and prevented the possibility of cross-contamination. The detection limits of each assay were similar, and all three assays could directly detect the DNA of Escherichia coli in clinical samples successfully. This is the first report on the application of CPA and IMSA methods for the detection of LT-I. The findings suggest that the three assays may be important tools for the rapid detection of enterotoxigenic Escherichia coli (ETEC) in the clinic.

Establishment and application of isothermal multiple-self-matching-initiated amplification (IMSA) in detecting Type II heat-labile enterotoxin of Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) constitutes a major cause of diarrhea in young children and animals, particularly in poor regions of the world, as well the traveler's diarrhea in adult individuals. Type II heat-labile enterotoxin (LT-II) from ETEC can cause profuse watery diarrhea, posing a potential threat to public health and animal husbandry. In the present study, isothermal multiple-self-matching-initiated amplification (IMSA) was established to rapidly detect LT-II producing ETEC. The specificity and sensitivity were assessed, and clinical samples were tested. The established IMSA method had good specificity for the detection of LT-II gene with a limit of detection of 25 CFU/mL, i.e. 2 times higher than that of real-time PCR and other two isothermal amplifications (loop-mediated isothermal amplification, LAMP and cross-primer isothermal amplification, CPA). Meanwhile, in 103 clinical Escherichia coli strains isolated from diarrhea samples, 9 strains with LT-II+ gene were detected (8.73%), corroborating real-time PCR, LAMP and CPA data. Therefore, the IMSA technology applied for the detection of LT-II producing ETEC has a good application prospect for screening clinical samples in primary medical units or common laboratories.

Establishment and Application of Polymerase Spiral Reaction Amplification for Salmonella Detection in Food.

Salmonella is a common zoonotic and foodborne pathogen that causes high morbidity and mortality in developing countries. In this study, we established and validated a polymerase spiral reaction (PSR) assay which targeted the conserved invasion gene (invA) of Salmonella by SYBR Green I indicator methods. Subsequently, assays for determination of the optimal conditions for optimal specificity and sensitivity of PSR were performed. We performed comprehensive evaluations using loop-mediated isothermal amplification (LAMP) and realtime PCR. A total number of 532 samples of daily food were analyzed by PSR. Twenty-seven bacterial strains were tested in the specificity assay, from which positive results were obtained only for 14-Salmonella strains. However, none of the 13 non-Salmonella strains was amplified. Similarly with LAMP and real-time PCR, the detection limit of the PSR assay was 50 CFU/ml. The PSR method was also successfully applied to evaluate the contamination with Salmonella in 532 samples of daily food, corroborating traditional culture method data. The novel PSR method is simple, sensitive, and rapid and provides new insights into the prevention and detection of foodborne diseases.

Homologous recombination is a force in the evolution of canine distemper virus.

Canine distemper virus (CDV) is the causative agent of canine distemper (CD) that is a highly contagious, lethal, multisystemic viral disease of receptive carnivores. The prevalence of CDV is a major concern in susceptible animals. Presently, it is unclear whether intragenic recombination can contribute to gene mutations and segment reassortment in the virus. In this study, 25 full-length CDV genome sequences were subjected to phylogenetic and recombinational analyses. The results of phylogenetic analysis, intragenic recombination, and nucleotide selection pressure indicated that mutation and recombination occurred in the six individual genes segment (H, F, P, N, L, M) of the CDV genome. The analysis also revealed pronounced genetic diversity in the CDV genome according to the geographically distinct lineages (genotypes), namely Asia-1, Asia-2, Asia-3, Europe, America-1, and America-2. The six recombination events were detected using SimPlot and RDP programs. The analysis of selection pressure demonstrated that a majority of the nucleotides in the CDV individual gene were under negative selection. Collectively, these data suggested that homologous recombination acts as a key force driving the genetic diversity and evolution of canine distemper virus.

Frequency of virulence factors in Escherichia coli isolated from suckling pigs with diarrhoea in China.

Escherichia coli-associated diarrhoea is an important disease adversely affecting the pig industry. This study was conducted to investigate the frequency of virulence factors expressed by E. coli strains isolated from suckling pigs with diarrhoea in China. A total of 381 E. coli strains, obtained from 290 faecal samples from pigs on 38 farms, were tested for fimbriae (K88, K99, 987P, F41, F18, F17), non-fimbrial adhesins (AIDA-I, paa, CS31A, eae, saa), enterotoxin (LT-I, LT-II, STa, STb, EAST1), Shiga toxin (Stx1, Stx2, Stx2e), pathogenicity islands (HPI, LEE), α-haemolysin (hlyA), afa8 gene cluster (afaD, afaE) and sepA genes by PCR. Out of the 381 isolates, 206 carried at least one virulence gene. Of the 206 virulence positive isolates, the virulence factor genes detected were EAST1 (n=120), irp2 (n=59), paa (n=50), STb (n=41), AIDA-I (n=34), LT-I (n=23), ler (n=11), hlyA (n=9), K88 (n=8), eae (n=8), STa (n=7), sepA (n=6), F18 (n=5), afaD (n=3), afaE (n=3), K99 (n=2) and Stx2e (n=1), with most isolates carrying multiple virulence genes. These results demonstrate that relatively few isolates from the study population express K88, K99, LT-I or STa, but that EAST1 (58%), irp2 (29%), AIDA-I (16.5%), paa (24%) and STb (20%) are frequent virulence factors expressed by E. coli strains isolated from suckling pigs with diarrhoea in China.