Qiuchun Li

Identification of Salmonella enterica Serovar Pullorum Antigenic Determinants Expressed In Vivo

ABSTRACT Salmonella enterica serovar Pullorum affecting poultry causes pullorum disease and results in severe economic loss in the poultry industry. Currently, it remains a major threat in countries with poor poultry surveillance and no efficient control measures. As S. Pullorum could induce strong humoral immune responses, we applied an immunoscreening technique, the in vivo-induced antigen technology (IVIAT), to identify immunogenic bacterial proteins expressed or upregulated during S. Pullorum infection. Convalescent-phase sera from chickens infected with S. Pullorum were pooled, adsorbed against antigens expressed in vitro, and used to screen an S. Pullorum genomic expression library. Forty-five proteins were screened out, and their functions were implicated in molecular biosynthesis and degradation, transport, metabolism, regulation, cell wall synthesis and antibiotic resistance, environmental adaptation, or putative functions. In addition, 11 of these 45 genes were assessed for their differential expression by quantitative real-time reverse transcription-PCR (RT-PCR), revealing that 9 of 11 genes were upregulated to different degrees under in vivo conditions, especially the regulator of virulence determinants, phoQ. Then, four in vivo-induced proteins (ShdA, PhoQ, Cse3, and PbpC) were tested for their immunoreactivity in 28 clinical serum samples from chickens infected with S. Pullorum. The rate of detection of antibodies against ShdA reached 82% and was the highest among these proteins. ShdA is a host colonization factor known to be upregulated in vivo and related to the persistence of S. Typhimurium in the intestine. Furthermore, these antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis, and more potential roles, such as diagnostic, therapeutic, and preventive uses, need to be developed in future studies.

Genetic analysis of Salmonella enterica serovar Gallinarum biovar Pullorum based on characterization and evolution of CRISPR sequence

Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is the cause of pullorum disease, characterized by white diarrhea, which leads to high mortality in poultry. In this study, we aimed to assess the genetic diversity of 655 S. Pullorum strains from 1962 to 2015 in China, Europe, and South America. A sequence typing scheme based on clustered regularly interspaced short palindromic repeats (CRISPR) was used to reveal the genetic relationships among these strains in this study. Overall, a total of 20 Pullorum sequence types (PSTs) of CRISPR were identified in the 655 isolates with PST7 (74%, 486/655) and PST3 (13%, 86/655) to be the most two frequent PSTs belonging to two different lineages, which confirmed the genetic conservation of S. Pullorum strains isolated from six provinces and two direct-controlled municipalities (Beijing and Shanghai) in China. However, the identification of seven new PSTs distributed in strains isolated since 2001 implied that genetic variation continues to develop in S. Pullorum. Interestingly, the whole-genome single-nucleotide polymorphism typing (WGST) of 96 strains out of the 655 isolates divided them into four lineages based on SNP analysis of core genomic sequence and exhibit good correspondence with the CRISPR subtyping method. Notably, 22 out of 26 isolates from Europe and South America were distributed in five distinctive PSTs (with no Chinese strains). Additionally, CRISPR data of spacers and their arrangement exhibit subtle but distinct specificity between different strains, and the dynamic adaptive nature of CRISPR loci provides critical insights into the evolution of S. Pullorum as the bacteria are influenced by their environment.

Molecular cloning, characterization and expression of goose Toll-like receptor 5.

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that are vital to activation of the innate immune system in response to invading pathogens through their recognition of pathogen-associated molecular patterns (PAMPs). TLR5 is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the goose TLR5 gene using rapid amplification of cDNA ends (RACE). The open reading frame (ORF) of goose TLR5 cDNA is 2583 bp in length and encodes an 860 amino acid protein. The entire coding region of the TLR5 gene was successfully amplified from genomic DNA and contained a single exon. The putative amino acid sequence of goose TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat (LRR) domains, a leucine-rich repeat C-terminal (LRR-CT) domain, a transmembrane domain and an intracellular Toll-interleukin-1 receptor (TIR) domain. The amino acid sequence of goose TLR5 shared 50.5% identity with human (Homo sapiens), 49.8% with mouse (Mus musculus) and 82.7% with chicken (Gallus gallus). The goose TLR5 gene was highly expressed in the spleen, liver and brain; moderately expressed in PBMCs, kidney, lung, heart, bone marrow, small intestine and large intestine; and minimally expressed in the cecum. HEK293 cells transfected with goose TLR5 and NF-κB-luciferase containing plasmids significantly responded to flagellin from Salmonella typhimurium indicating that it is a functional TLR5 homologue. In response to infection with S. enterica serovar Enteritidis (SE), the level of TLR5 mRNA significantly increased over the control in PBMCs at 1 d post infection (p.i.) and was slightly elevated in the spleen at 1 d or 3 d p.i. IL-6 was expressed below control levels in PBMCs but was upregulated in the spleen. In contrast to IL-6, an evident decrease in the expression level of IL-8 was observed in both PBMCs and spleens at 1 d or 3 d p.i. SE challenge also resulted in an increase in the mRNA expression of IL-18 and IFN-γ in PBMCs and the spleen. These results imply that the expression of goose TLR5 is differentially regulated in various tissues and may participate in the immune response against bacterial pathogens.

Evaluation of the Salmonella enterica Serovar Pullorum Pathogenicity Island 2 Mutant as a Candidate Live Attenuated Oral Vaccine

ABSTRACT Salmonella enterica serovar Pullorum (S. Pullorum) is a highly adapted pathogen that causes pullorum disease (PD), an important systemic disease of poultry that causes severe economic losses in developing countries. In the interests of developing a safe and immunogenic oral vaccine, the efficacy of a Salmonella pathogenicity island 2 (SPI2)-deleted mutant of S. Pullorum (S06004ΔSPI2) was evaluated in chickens. S06004ΔSPI2 was severely less virulent than the parental wild-type strain S06004 as determined by the 50% lethal dose (LD50) for 3-day-old chickens when injected intramuscularly. Two-day-old chickens immunized with a single oral dose of S06004ΔSPI2 showed no differences in body weight or clinical symptoms compared with those in the negative-control group. S06004ΔSPI2 bacteria were not isolated from livers or spleens of immunized chickens after a short period of time, and specific humoral and cellular immune responses were significantly induced. Immunized chickens were challenged with S. Pullorum strain S06004 and Salmonella enterica serovar Gallinarum (S. Gallinarum) strain SG9 at 10 days postimmunization (dpi), and efficient protection against the challenges was observed. None of the immunized chickens died, the clinical symptoms were slight and temporary following challenge in immunized chickens compared with those in the control group, and these chickens recovered by 3 to 5 dpi. Overall, these results demonstrate that S06004ΔSPI2 can be used as a live attenuated oral vaccine.

Genetic analysis and CRISPR typing of Salmonella enterica serovar Enteritidis from different sources revealed potential transmission from poultry and pig to human

Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the most prevalent serotypes in Salmonella isolated from poultry and the most commonly reported cause of human salmonellosis. In this study, we aimed to assess the genetic diversity of 329 S. Enteritidis strains isolated from different sources from 2009 to 2016 in China. Clustered regularly interspaced short palindromic repeat (CRISPR) typing was used to characterize these 262 chicken clinical isolates, 38 human isolates, 18 pig isolates, six duck isolates, three goose isolates and two isolates of unknown source. A total of 18 Enteritidis CRISPR types (ECTs) were identified, with ECT2, ECT8 and ECT4 as the top three ECTs. CRISPR typing identified ECT2 as the most prevalent ECT, which accounted for 41% of S. Enteritidis strains from all the sources except duck. ECT9 and ECT13 were identified in both pig and human isolates and revealed potential transmission from pig to human. A cluster analysis distributed 18 ECTs, including the top three ECTs, into four lineages with LI as the predominant lineage. Forty-eight out of 329 isolates were subjected to whole genome sequence typing, which divided them into four clusters, with Cluster I as the predominant cluster. Cluster I included 92% (34/37) of strains located in LI identified from the CRISPR typing, confirming the good correspondence between both typing methods. In addition, the CRISPR typing also revealed the close relationship between ECTs and isolated areas, confirming that CRISPR spacers might be obtained by bacteria from the unique phage or plasmid pools in the environment. However, further analysis is needed to determine the function of CRISPR-Cas systems in Salmonella and the relationship between spacers and the environment.

Automatic Combination of Microfluidic Nanoliter-Scale Droplet Array with High-Speed Capillary Electrophoresis.

In this paper, we developed a novel approach for interfacing a microfluidic two-dimensional droplet array to a high-speed capillary electrophoresis (HSCE) system. Picoliter-scale sample injection (ca. 200 pL) from a nanoliter-scale droplet array covered by nonvolatile oil was automatically achieved using the spontaneous injection mode, without the interference from the cover oil and the need of special droplet extraction interface as in previously reported systems. The system was applied in consecutive separations of 25 different samples of amino acids with a whole separation time less than 15 min, as well as on-line monitoring of in-droplet derivatizing reaction of amino acids by fluorescein isothiocyanate (FITC) over 3 hours. High separation speed (up to 100 samples per hour) and high separation efficiency (up to 9.22 × 105 N/m) were achieved.

A gene knock-in method used to purify plasmid pSPI12 from Salmonella enterica serovar Pullorum and characterization of IpaJ

A small plasmid with 4080 bp long, designated pSPI12, was purified from Salmonella enterica serovar Pullorum using a gene knock-in method by inserting a kanamycin resistance cassette in the plasmid. The G+C content of the plasmid was 51.8%, which is in the range of Salmonella genomic DNA. A sequence analysis revealed that pSPI12 had 99.1% homology to pSFD10, which was first reported in the vaccine strain S. enterica serovar Chloreaesuis C500, but not prevalent among other strains of S. Chloreaesuis. The plasmid has seven open reading frames (ORFs), with one ORF containing a putative virulence-related protein, which had 49% homology with invasion plasmid antigen J protein (IpaJ) secreted by type III secretion system of Shigella flexneri. The putative IpaJ protein was expressed and purified as a His-tagged fusion protein reacted with convalescent sera against S. Pullorum, confirming its identification as an immunogen of the pathogen. In addition, the gene was upregulated for 1h post-infection of HD-11 cells with the pathogen by a quantitative real-time reverse transcription PCR assay. The results suggest that IpaJ may be a virulent protein involved in the early stage of infection by S. Pullorum.

Could FlhF be a key element that controls Campylobacter jejuni flagella biosynthesis in the initial assembly stage

The disordered arrangement of flagella biosynthetic genes, combined with a simplified regulatory mechanism, has made elucidating the process of Campylobacter jejuni flagellation difficult. FlhF is a recently identified element that controls the assembly of the flagella, although its function mechanism and regulatory preference are not well defined at present. In this study, we found that inactivation of FlhF caused the transcription of most flagella genes down-regulated. The importance of FlhF was systematically evaluated by analyzing changes in the transcription profiles between wild-type and flhF mutant strains, which showed that FlhF affects late flagella genes obviously. FlhF is constitutively expressed during C. jejuni growth, demonstrating that it is a class I flagella element that participates in early flagella assembly. In addition, the early flagella component FlhB was not localized to the cell pole in the flhF mutant. Thus, flagella assembly was impeded at the initial stage. We propose a model in which FlhF helps target the early flagella components to the cell pole, functioning prior to the formation of the flagella export apparatus, and thus places FlhF at the top of the flagella regulatory cascade hierarchy. Inactivation of FlhF impeded flagella assembly at the initial stage and decreased transcription of flagella genes through a feed-back control mechanism, leading to FlhF having a significant influence on the expression of late flagella components and resulting in the aflagellate C. jejuni phenotype. Our present study has uncovered how FlhF influences C. jejuni flagella biosynthesis, which will be helpful in understanding the C. jejuni flagella biosynthetic pathway and bacterial flagellation in general.

Construction and characterization of a cigR deletion mutant of Salmonella enterica serovar Pullorum

ABSTRACT Salmonella enterica serovar Pullorum (S. Pullorum) is the causative agent of pullorum disease (PD) and results in severe economic losses to the poultry industry. As a Salmonella type III secretion system 2 (T3SS2) effector and predicted membrane protein, CigR is encoded by the cigR gene within Salmonella pathogenicity island 3 (SPI3). In order to research the influence of the cigR gene on S. Pullorum, a cigR mutant of S. Pullorum S06004 was constructed by the lambda Red recombination system, and then its characterization was analysed. Lack of cigR did not affect the growth and biochemical properties, but resulted in decreased biofilm formation. The mutant strain was stable with the deletion of the cigR gene. Macrophage infection assay and in vivo competition assay showed that the mutant strain increased the replication and/or survival ability in the HD11 cell line and in chickens compared to that of the parent strain, the median lethal dose (LD50) of the mutant strain was one-fifth of the parent strain for 2-day-old chickens when injected intramuscularly. These results demonstrate CigR plays roles in biofilm formation and pathogenicity of S. Pullorum, deletion of cigR can significantly decrease biofilm formation and significantly increase virulence.

Complete Genome Sequence of Salmonella enterica Serovar Choleraesuis Vaccine Strain C500 Attenuated by Chemical Mutation

ABSTRACT Salmonella enterica serovar Choleraesuis strain C500 is a live vaccine attenuated by chemical methods. Here, we report the complete genome sequence of the strain, which may be helpful for elucidating the attenuation mechanism of the vaccine strain.