101 research outputs found
Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay
Salmonella enterica serovar Gallinarum biovars Pullorum (S. Pullorum) and Gallinarum (S. Gallinarum) can result in pullorum disease and fowl typhoid in avian species, respectively, and cause considerable economic losses in poultry in many developing countries. Conventional Salmonella serotyping is a time-consuming, labor-intensive and expensive process, and the two biovars cannot be distinguished using the traditional serological method. In this study, we developed a rapid and reliable one-step multiplex polymerase chain reaction (PCR) assay to simultaneously identify and discriminate the biovars Pullorum and Gallinarum. The multiplex PCR method focused on three specific genes, stn, I137_08605 and ratA. Based on bioinformatics analysis, we found that gene I137_08605 was present only in S. Pullorum and S. Gallinarum, and a region of difference in ratA was deleted only in S. Pullorum after comparison with that of S. Gallinarum and other Salmonella serovars. Three pairs of primers specific for the three genes were designed for the multiplex PCR system and their selectivity and sensitivity were determined. The multiplex PCR results showed that S. Pullorum and S. Gallinarum could be identified and discriminated accurately from all tested strains including 124 strains of various Salmonella serovars and 42 strains of different non-Salmonella pathogens. In addition, this multiplex PCR assay could detect a minimum genomic DNA concentration of 67.4 pg/ÎĽL, and 100 colony forming units. The efficiency of the multiplex PCR was evaluated by detecting natural-occurring Salmonella isolates from a chicken farm. The results demonstrated that the established multiplex PCR was able to identify S. Gallinarum and S. Pullorum individually, with results being consistent with traditional serotyping and biochemical testing. These results demonstrated that a highly accurate and simple biovar-specific multiplex PCR assay could be performed for the rapid identification and discrimination of Salmonella biovars Gallinarum and Pullorum, which will be useful, particularly under massive screening situations
Identification by PCR signature-tagged mutagenesis of attenuated Salmonella Pullorum mutants and corresponding genes in a chicken embryo model
A key feature of the fowl-specific pathogen Salmonella Pullorum is its vertical transmission to progeny via the egg. In this study, PCR signature-tagged mutagenesis identified nine genes of a strain of S. Pullorum that contributed to survival in the chicken embryo during incubation. The genes were involved in invasion, cell division, metabolism and bacterial defence. The competition index in vivo and in vitro together with a virulence evaluation for chicken embryos of all nine mutant strains confirmed their attenuation
Accurate identification and discrimination of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum by a multiplex PCR based on the new genes of torT and I137_14430
Most cases of chicken salmonellosis are caused by Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum, which lead to a significant morbidity and fatality rate. Although the conventional Kaufmann-White scheme is the reliable method for the serotyping of Salmonella, it does not distinguish between closely related biotypes like S. Pullorum and S. Gallinarum. Herein, we conducted a single one-step multiplex PCR assay that can identify and distinguish between S. Pullorum and S. Gallinarum in an accurate manner. This PCR method was based on three genes, including torT for S. Pullorum identification, I137_14430 for S. Gallinarum identification, and stn as the genus-level reference gene for Salmonella. By comparing S. Pullorum to S. Gallinarum and other serovars of Salmonella, in silico study revealed that only the former has a deletion of 126 bp-region in the carboxyl terminus of torT. The I137_14430 gene does not exist in S. Gallinarum. However, it is present in all other Salmonella serotypes. The multiplex PCR approach utilizes unique sets of primers that are intended to specifically target these three different genes. The established PCR method was capable of distinguishing between the biovars Pullorum and Gallinarum from the 29 distinct Salmonella serotypes as well as the 50 distinct pathogens that are not Salmonella, showing excellent specificity and exclusivity. The minimal amount of bacterial cells required for PCR detection was 100 CFU, while the lowest level of genomic DNA required was 27.5 pg/ÎĽL for both S. Pullorum and S. Gallinarum. After being implemented on the clinical Salmonella isolates collected from a poultry farm, the PCR test was capable of distinguishing the two biovars Pullorum and Gallinarum from the other Salmonella strains. The findings of the PCR assay were in line with those of the traditional serotyping and biochemical identification methods. This new multiplex PCR could be used as a novel tool to reinforce the clinical diagnosis and differentiation of S. Pullorum and S. Gallinarum, particularly in high-throughput screening situations, providing the opportunity for early screening of infections and, as a result, more effective management of the illness among flocks
Salmonella Enteritidis activates inflammatory storm via SPI-1 and SPI-2 to promote intracellular proliferation and bacterial virulence
Salmonella Enteritidis is an important intracellular pathogen, which can cause gastroenteritis in humans and animals and threaten life and health. S. Enteritidis proliferates in host macrophages to establish systemic infection. In this study, we evaluated the effects of Salmonella pathogenicity island-1 (SPI-1) and SPI-2 to S. Enteritidis virulence in vitro and in vivo, as well as the host inflammatory pathways affected by SPI-1 and SPI-2. Our results show that S. Enteritidis SPI-1 and SPI-2 contributed to bacterial invasion and proliferation in RAW264.7 macrophages, and induced cytotoxicity and cellular apoptosis of these cells. S. Enteritidis infection induced multiple inflammatory responses, including mitogen-activated protein kinase (ERK-mediated) and Janus kinase-signal transducer and activator of transcript (STAT) (STAT2-mediated) pathways. Both SPI-1 and SPI-2 were necessary to induce robust inflammatory responses and ERK/STAT2 phosphorylation in macrophages. In a mouse infection model, both SPIs, especially SPI-2, resulted in significant production of inflammatory cytokines and various interferon-stimulated genes in the liver and spleen. Activation of the ERK- and STAT2-mediated cytokine storm was largely affected by SPI-2. S. Enteritidis ΔSPI-1-infected mice displayed moderate histopathological damage and drastically reduced bacterial loads in tissues, whereas only slight damage and no bacteria were observed in ΔSPI-2- and ΔSPI-1/SPI-2-infected mice. A survival assay showed that ΔSPI-1 mutant mice maintained a medium level of virulence, while SPI-2 plays a decisive role in bacterial virulence. Collectively, our findings indicate that both SPIs, especially SPI-2, profoundly contributed to S. Enteritidis intracellular localization and virulence by activating multiple inflammatory pathways
MSE-Nets: Multi-annotated Semi-supervised Ensemble Networks for Improving Segmentation of Medical Image with Ambiguous Boundaries
Medical image segmentation annotations exhibit variations among experts due
to the ambiguous boundaries of segmented objects and backgrounds in medical
images. Although using multiple annotations for each image in the
fully-supervised has been extensively studied for training deep models,
obtaining a large amount of multi-annotated data is challenging due to the
substantial time and manpower costs required for segmentation annotations,
resulting in most images lacking any annotations. To address this, we propose
Multi-annotated Semi-supervised Ensemble Networks (MSE-Nets) for learning
segmentation from limited multi-annotated and abundant unannotated data.
Specifically, we introduce the Network Pairwise Consistency Enhancement (NPCE)
module and Multi-Network Pseudo Supervised (MNPS) module to enhance MSE-Nets
for the segmentation task by considering two major factors: (1) to optimize the
utilization of all accessible multi-annotated data, the NPCE separates
(dis)agreement annotations of multi-annotated data at the pixel level and
handles agreement and disagreement annotations in different ways, (2) to
mitigate the introduction of imprecise pseudo-labels, the MNPS extends the
training data by leveraging consistent pseudo-labels from unannotated data.
Finally, we improve confidence calibration by averaging the predictions of base
networks. Experiments on the ISIC dataset show that we reduced the demand for
multi-annotated data by 97.75\% and narrowed the gap with the best
fully-supervised baseline to just a Jaccard index of 4\%. Furthermore, compared
to other semi-supervised methods that rely only on a single annotation or a
combined fusion approach, the comprehensive experimental results on ISIC and
RIGA datasets demonstrate the superior performance of our proposed method in
medical image segmentation with ambiguous boundaries
Multi-scale analysis of schizophrenia risk genes, brain structure, and clinical symptoms reveals integrative clues for subtyping schizophrenia patients
Analysis linking directly genomics, neuroimaging phenotypes and clinical measurements is crucial for understanding psychiatric disorders, but remains rare. Here, we describe a multi-scale analysis using genome-wide SNPs, gene-expression, grey matter volume (GMV) and the Positive and Negative Syndrome Scale scores (PANSS) to explore the etiology of schizophrenia. With 72 drug-naive schizophrenic first episode patients (FEPs) and 73 matched heathy controls, we identified 108 genes, from schizophrenia risk genes, that correlated significantly with GMV, which are highly co-expressed in the brain during development. Among these 108 candidates, 19 distinct genes were found associated with 16 brain regions referred to as hot clusters (HCs), primarily in the frontal cortex, sensory-motor regions and temporal and parietal regions. The patients were subtyped into three groups with distinguishable PANSS scores by the GMV of the identified HCs. Furthermore, we found that HCs with common GMV among patient groups are related to genes that mostly mapped to pathways relevant to neural signaling, which are associated with the risk for schizophrenia. Our results provide an integrated view of how genetic variants may affect brain structures that lead to distinct disease phenotypes. The method of multi-scale analysis that was described in this research, may help to advance the understanding of the etiology of schizophrenia
Mucosal and Systemic Immune Responses to Influenza H7N9 Antigen HA1–2 Co-Delivered Intranasally with Flagellin or Polyethyleneimine in Mice and Chickens
Consecutive cases of human infection with H7N9 influenza viruses since 2013 in China have prompted efforts to develop an effective treatment. Subunit vaccines introduced by intranasal administration can block an infection at its primary site; flagellin (fliC) and polyethyleneimine (PEI) have been shown to be potent adjuvants. We previously generated the hemagglutinin (HA)1–2-fliC fusion protein consisting of the globular head domain (HA1–2; amino acids 62–284) of HA fused with Salmonella typhimurium fliC. In the present study, we investigated its effectiveness of both flagellin and PEI as mucosal adjuvants for the H7N9 influenza subunit vaccine. Mice immunized intranasally with HA1–2-fliC and HA1–2-PEI showed higher HA1–2-specific immunoglobulin (Ig)G and IgA titers in serum, nasal wash, and bronchial alveolar lavage fluid. Moreover, splenocyte activation and proliferation and the number of HA1–2-specific interferon (IFN)-γ- and interleukin (IL)-4-producing splenocytes were markedly increased in the fliC and PEI groups; in the latter, there were more cells secreting IL-4 than IFN-γ, suggesting that fliC induced T helper type (Th)1 and Th2 immune responses, and PEI induced Th2-biased responses, consistent with the serum antibody isotype pattern (IgG1/IgG2a ratio). Furthermore, virus challenge was performed in a chicken model. The results showed that chickens receiving fliC and PEI adjuvant vaccine exhibited robust immune responses leading to a significant reduction in viral loads of throat and cloaca compared to chickens receiving only HA1–2. These findings provide a basis for the development of H7N9 influenza HA1–2 mucosal subunit vaccines
An avian influenza A (H7N9) virus vaccine candidate based on the fusion protein of hemagglutinin globular head and Salmonella typhimurium flagellin
Corrigendum: Mucosal and Systemic Immune Responses to Influenza H7N9 Antigen HA1–2 Co-Delivered Intranasally With Flagellin or Polyethyleneimine in Mice and Chickens
Quantitative analysis of the developmental potential of cells and tissues based on evolutionary conservation of genes and regulatory regions
Objective·To study the relationship between evolution and the
developmental process from the perspective of DNA sequence conservation, and explore their
inherent principles.Methods·First, conservation rate (CR) was established by analyzing the
conservation of amino acid sequences of coding genes in 100 species to quantify the
evolutionary conservation of genes. The relationship between CR and developmental potential
was verified by using the feature genes involved in embryonic stem cells pathways. Secondly,
cell type-specific genes and their characteristics in conservation were studied by analyzing
the RNA sequencing (RNA-seq) data of the three early germ layers (ectoderm, mesoderm and
endoderm) and their corresponding mature organs (brain, heart, liver, etc). Then, chromatin
immunoprecipitation sequencing (ChIP-seq) data of enhancer histone H3 acetylated at lysine 27
(H3K27ac) from early germ layers and mature organs were collected to search for enhancer sites
and identify super enhancers in various cells and tissues by using the ROSE procedure.
Functional enrichment and signaling pathway analysis of genes was used to examine the identity
correlation between SEs-regulated genes and the corresponding cell characteristics, to clarify
whether the SEs identified in this study were consistent with the characteristics reported in
previous studies. Finally, PhastCons program was used to calculate the DNA conservation score
(CS) of non-coding regulatory regions to study their relationship with developmental
potential.Results·In the coding region of DNA, CR was successfully established to quantify the
conservation of genes. The gene expression data of early germ layers and mature organs showed
that the genes with higher conservation rate were more relevant to the stemness and early
developmental process, and the differences between the tissues from early and late development
could be distinguished by using CR. In the non-coding regions of DNA, it was found that the
conservation of regulatory regions was also correlated with development. The CS of the SE
sequences in the early developmental germ layers was significantly higher than that of the SE
sequences in the corresponding mature organs. However, cell-specific typical enhancers (TEs)
did not show such a trend.Conclusion·During the developmental process, CR of genes expressed
in the coding region decreases, and CS of super-enhancer DNA in the non-coding region
decreases
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