Yachen Hu

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.

Characterization of CRISPR-Cas system in clinical Staphylococcus epidermidis strains revealed its potential association with bacterial infection sites

Staphylococcus epidermidis is considered as a major cause of nosocomial infections, bringing an immense burden to healthcare systems. Virulent phages have been confirmed to be efficient in combating the pathogen, but the prensence of CRISPR-Cas system, which is a bacterial immune system eliminating phages was reported in few S. epidermidis strains. In this study, the CRISPR-Cas system was detected in 12 from almost 300 published genomes in GenBank and by PCR of cas6 gene in 18 strains out of 130 clinical isolates obtained in Copenhagen. Four strains isolated in 1965-1966 harboured CRISPR elements confirming that this immunity system was not recently acquired by S. epidermidis. In these CRISPR-positive strains, 44 and 12 spacers were found to belong to CRISPR1 and CRISPR2 elements, respectively. However, only 15 spacers displayed homology to reported phages and plasmids DNA. Interestingly, 5 different spacers located in the CRISPR1 locus with homolgy to virulent phage 6ec DNA sequences, and 19 strains each carrying 2 or 3 different spacers recognizing this phage, implied that the CRISPR-Cas immunity could be abrogated by nucleotide mismatch between the spacer and its target phage sequence, while new spacers obtained from the evolved phage could recover the CRISPR interference. In addition, phylogenetic analysis of the 29 CRISPR-positive isolates divided them into four lineages, with 81% human blood isolates as a distinct sub-lineage, suggesting that the CRISPR difference is closely related to diverse habitats. Knowledge of CRISPR and its prevalence may ultimately be applied in the understanding of origin and evolution of CRISPR-positive S. epidermidis strains.

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.

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.

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.

A new PCR assay based on the new gene-SPUL_2693 for rapid detection of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) and biovar Pullorum (S. Pullorum) are gram-negative bacteria, members of the most important infectious pathogens, and have caused common problems in the poultry industry, especially in the developing countries. O- and H-antigen specific anti-sera are commonly for slide and tube agglutination tests to identify Salmonella serovars. However, it is both labor intensive and time consuming, so there is an urgent need for a new technique for the rapid detection of the major Salmonella serovars. In this study, we developed a 1-step PCR assay to identify the serovar Gallinarum. This PCR-based assay was based on the SPUL_2693 gene, which was located in SPI-19 and found by comparing the genomes of the S. Pullorum and S. Gallinarum in the whole data of NCBI. The specificity of this gene was evaluated by bioinformatics analysis, and the results showed that the SPUL_2693 gene exists in all serovar Gallinarum. The specificity and sensitivity of this PCR assay were evaluated in our study. The developed PCR assay was able to distinguish the serovar Gallinarum from 27 different Salmonella serovars and 5 different non-Salmonella pathogens. The minimum limit of genomic DNA of S. Pullorum for PCR detection was 2.143 pg/μL, and the minimum limit number of cells was 6 CFU. This PCR assay was also applied to analyze Salmonella strains isolated from a chicken farm in this study. The PCR assay properly identified the serovar Gallinarum from other Salmonella serovars, and the results were in agreement with the results of a traditional serotyping assay. In general, the newly developed PCR-based assay can be used to accurately judge the presence of the serovar Gallinarum and can be combined with traditional serotyping assays, especially in the case of large quantities of samples.

Virulence of Salmonella enterica serovar Pullorum isolates compared using cell-based and chicken embryo infection models

ABSTRACT To reveal differences in virulence among strains of Salmonella enterica serovar Pullorum (S. Pullorum), we used 2 cell‐based infection models and a chicken embryo infection model in this study. S. Pullorum strain S06004 was used to infect 4 different avian cell lines (HD‐11, DF‐1, LMH, DT‐40), and the results showed that the infection of S06004 in both LMH and HD‐11 cells was more stable than in DF‐1 and DT‐40 cells. Therefore, the HD‐11 and LMH cell lines were used as the appropriate macrophage and epithelial cell models, respectively, to study the infection of S. Pullorum. Fifty strains isolated during the years 1962 to 2010 were then analyzed to compare their infection rates in HD‐11 and LMH cells. The result showed that the infection rates of most strains were very similar to that of S06004, except for S9876 which displayed the highest infection rate among these strains. Based on the cell infection results, 10 strains were selected to be used in the chicken embryo infection model. Sixteen‐day‐old SPF chicken embryos were infected with the pathogen at a dose of 103 CFU/100 &mgr;L via allantoic cavity inoculation. The strains C79‐13, 7101, and S06013 caused death of more than 80% embryos, whereas S09C12 and 6703 resulted less than 20% death. Thus, this study established cell‐based infection models to screen S. Pullorum strains in vitro, and a chick embryo model to evaluate their in vivo virulence.

Analyses of prevalence and molecular typing reveal the spread of antimicrobial-resistant Salmonella infection across 2 breeder chicken farms1

ABSTRACT In this study, Salmonella prevalence and antimicrobial resistance were evaluated at various production stages in 2 geographically separated breeder farms (referred to as G and F). Day‐old chicks for the breeder flock at farm F were purchased from farm G. A total of 219 Salmonella isolates, all identified as Salmonella enterica subsp. enterica serovar Enteritidis, were recovered from 1,430 samples (sick chicken carcasses and/or dead embryos). The isolation rates at breeder farms G and F were 10.53% (56/532) and 18.15% (163/898), respectively. Resistance to 4‐6 antimicrobial agents was the most frequent phenotype during the laying stage at both farms, suggesting that chicks are exposed to higher risk of antimicrobial‐resistant Salmonella infection during this stage of the breeding process. Using clustered regularly interspaced short palindromic repeat (CRISPR) typing, 5 CRISPR patterns were identified, out of which one pattern was shared by the 2 farms. In addition, pulsed‐field gel electrophoresis (PFGE) typing result indicated that 2 clusters (PF‐1 and PF‐2) were shared among the 2 breeder farms, suggesting that strains were transmitted from breeder farm G to farm F via the trade of day‐old chicks. Our findings suggested that the trade of day‐old breeder chicks could be one of the potential Salmonella transmission routes, and antibiotics should be administered with caution during the laying stage.

A rapid method to identify Salmonella enterica serovar Gallinarum biovar Pullorum using a specific target gene ipaJ

ABSTRACT Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is the pathogen of pullorum disease, which leads to severe economic losses in many developing countries. Traditional methods to identify S. enterica have relied on biochemical reactions and serotyping, which are time-consuming with accurate identification if properly carried out. In this study, we developed a rapid polymerase chain reaction (PCR) method targeting the specific gene ipaJ to detect S. Pullorum. Among the 650 S. Pullorum strains isolated from 1962 to 2016 all over China, 644 strains were identified to harbour ipaJ gene in the plasmid pSPI12, accounting for a detection rate of 99.08%. Six strains were ipaJ negative because pSPI12 was not found in these strains according to whole genome sequencing results. There was no cross-reaction with other Salmonella serotypes, including Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum), which show a close genetic relationship with S. Pullorum. This shows that the PCR method could distinguish S. Gallinarum from S. Pullorum in one-step PCR without complicated biochemical identification. The limit of detection of this PCR method was as low as 90 fg/μl or 102 CFU, which shows a high sensitivity. Moreover, this method was applied to identify Salmonella isolated from the chicken farm and the results were consistent with what we obtained from biochemical reactions and serotyping. Together, all the results demonstrated that this one-step PCR method is simple and feasible to efficiently identify S. Pullorum.