scholarly journals Genome-Wide Identification of Virulence Genes in Erysipelothrix rhusiopathiae: Use of a Mutant Deficient in a tagF Homolog as a Safe Oral Vaccine against Swine Erysipelas

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
Yoshihiro Shimoji ◽  
Yohsuke Ogawa ◽  
Manae Tsukio ◽  
Kazumasa Shiraiwa ◽  
Sayaka Nishikawa ◽  
...  
Keyword(s):  
Clinical Symptoms ◽  
Oral Vaccine ◽  
Lethal Dose ◽  
Erysipelothrix Rhusiopathiae ◽  
Gene Encoding ◽  
Lethal Challenge ◽  
Content Type ◽  
Genome Wide ◽  
Transposon Mutants ◽  
Swine Erysipelas

ABSTRACT Swine erysipelas is caused by the Gram-positive pathogen Erysipelothrix rhusiopathiae. The swine erysipelas live vaccine in Japan, the E. rhusiopathiae Koganei 65-0.15 strain (Koganei), has been reported to cause arthritis and endocarditis. To develop a vaccine with increased safety, we used a virulent Fujisawa strain to construct transposon mutants for a total of 651 genes, which covered 38% of the coding sequence of the genome. We screened the mutants for attenuation by inoculating mice with 108 CFU of each mutant and subsequently assessed protective capability by challenging the surviving mice with 103 CFU (102 times the 50% lethal dose) of the Fujisawa strain. Of the 23 attenuated mutants obtained, 6 mutants were selected and evaluated for protective capability in pigs by comparison to that of the Koganei strain. A mutant in the ERH_0432 (tagF) gene encoding a putative CDP-glycerol glycerophosphotransferase was found to be highly attenuated and to induce humoral and cell-mediated immune responses in conventional pigs. An in-frame deletion mutant of the gene, the Δ432 mutant, was constructed, and attenuation was further confirmed in germfree piglets; three of four piglets subcutaneously inoculated with 109 CFU of the Δ432 mutant showed no apparent clinical symptoms, whereas all four of the Koganei-inoculated piglets died 3 days after inoculation. It was confirmed that conventional pigs inoculated orally or subcutaneously with the Δ432 strain were almost completely protected against lethal challenge infection. Thus, the tagF homolog mutant of E. rhusiopathiae represents a safe vaccine candidate that can be administered via the oral and subcutaneous routes.

scholarly journals Clonal Lineages of Erysipelothrix rhusiopathiae Responsible for Acute Swine Erysipelas in Japan Identified by Using Genome-Wide Single-Nucleotide Polymorphism Analysis

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Yohsuke Ogawa ◽  
Kazumasa Shiraiwa ◽  
Yoshitoshi Ogura ◽  
Tadasuke Ooka ◽  
Sayaka Nishikawa ◽  
...  
Keyword(s):  
Polymorphism Analysis ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Erysipelothrix Rhusiopathiae ◽  
Single Nucleotide ◽  
Content Type ◽  
Genome Wide ◽  
Wide Range ◽  
Swine Erysipelas ◽  
Lineage Iv

ABSTRACTErysipelothrix rhusiopathiaecauses swine erysipelas, an important infectious disease in the swine industry. In Japan, the incidence of acute swine erysipelas due toE. rhusiopathiaeserovar 1a has recently increased markedly. To study the genetic relatedness of the strains from the recent cases, we analyzed 34E. rhusiopathiaeserovar 1a swine isolates collected between 1990 and 2011 and further investigated the possible association of the live Koganei 65-0.15 vaccine strain (serovar 1a) with the increase in cases. Pulsed-field gel electrophoresis analysis revealed no marked variation among the isolates; however, sequencing analysis of a hypervariable region in the surface-protective antigen A gene (spaA) revealed that the strains isolated after 2007 exhibited the samespaAgenotype and could be differentiated from older strains. Phylogenetic analysis based on genome-wide single-nucleotide polymorphisms (SNPs) revealed that the Japanese strains examined were closely related, showing a relatively small number of SNPs among them. The strains were classified into four major lineages, with Koganei 65-0.15 (lineage III) being phylogenetically separated from the other three lineages. The strains isolated after 2007 and the two older strains constituted one major lineage (lineage IV) with a specificspaAgenotype (M203/I257-SpaA), while the recent isolates were further divided into two geographic groups. The remaining older isolates belonged to either lineage I, with the I203/L257-SpaA type, or lineage II, with the I203/I257-SpaA type. These results indicate that the recent increased incidence of acute swine erysipelas in Japan is associated with two sublineages of lineage IV, which have independently evolved in two different geographic regions.IMPORTANCEUsing large-scale whole-genome sequence data fromErysipelothrix rhusiopathiaeisolates from a wide range of hosts and geographic origins, a recent study clarified the existence of three distinct clades (clades 1, 2, and 3) that are found across multiple continents and host species, representing both livestock and wildlife, and an “intermediate” clade between clade 2 and the dominant clade 3 within the species. In this study, we found that theE. rhusiopathiaeJapanese strains examined exhibited remarkably low levels of genetic diversity and confirmed that all of the Japanese and Chinese swine isolates examined in this study belong to clonal lineages within the intermediate clade. We report thatspaAgenotyping ofE. rhusiopathiaestrains is a practical alternative to whole-genome sequencing analysis of theE. rhusiopathiaeisolates from eastern Asian countries.

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

2015 ◽  
Vol 22 (7) ◽  
pp. 706-710 ◽  
Author(s):  
Junlei Yin ◽  
Zhao Cheng ◽  
Xiaochun Wang ◽  
Lijuan Xu ◽  
Qiuchun Li ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Clinical Symptoms ◽  
Oral Vaccine ◽  
Systemic Disease ◽  
Lethal Dose ◽  
Pathogenicity Island ◽  
Economic Losses ◽  
Control Group ◽  
Content Type ◽  
Short Period

ABSTRACTSalmonella entericaserovar 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 aSalmonellapathogenicity island 2 (SPI2)-deleted mutant ofS. 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 withS. Pullorum strain S06004 andSalmonellaenterica 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.

Cloning, expression and immunogenic characterization of theapxIIAgene ofActinobacillus pleuropneumoniae

2007 ◽  
Vol 4 (1) ◽  
pp. 63-67
Author(s):  
Yan Ke-Xia ◽  
Liu Jian-Jie ◽  
Wu Bin ◽  
Tang Xi-Biao ◽  
Cai Li-Jun ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Lethal Dose ◽  
Serum Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gene Encoding ◽  
Lethal Challenge ◽  
Sds Page ◽  
Prokaryotic Expression Vector ◽  
Protection Rate ◽  
Protein Group

AbstractThe structural gene encoding ApxII toxin (apxIIA) was amplified from the genomic DNA ofActinobacillus pleuropneumoniae(APP) strain HB08 (serotype 2) and cloned into the prokaryotic expression vector pET-28a. SDS-PAGE and Western blot analysis showed that theapxIIAgene was expressed inEscherichia coliBL21 (DE3) and the expressed products could react with ApxII antibodies. The recombinant ApxIIA was purified from the inclusion bodies. Kunming mice were intraperitoneally vaccinated twice, with an interval of 2 weeks, using unfolded/refolded recombinant proteins, the native ApxII toxin extracted from the cultural supernatant of a strain of APP serotype 7 (APP-7) or phosphate-buffered saline (PBS). Serum antibody was examined by ApxIIA-specific enzyme-linked immunosorbent assay (ELISA) 2 weeks after every vaccination. Two weeks after the second vaccination, mice were challenged intraperitoneally with a lethal dose of APP-7 (1.08 × 108cfu per mouse). The protection rate reached 91.7% in the native ApxII group, 83.3% in the refolded recombinant protein group and 58.3% in the unfolded recombinant protein group, while all mice in the PBS group died within 36 h after challenge. Our data revealed that the refolded recombinant ApxIIA had excellent immunogenicity and could elicit protection against a lethal challenge of APP.

scholarly journals The Live Attenuated Actinobacillus pleuropneumoniae Triple-Deletion Mutant ΔapxICΔapxIICΔapxIV-ORF1Strain, SLW05, Immunizes Pigs against Lethal Challenge with Haemophilus parasuis

2012 ◽  
Vol 20 (2) ◽  
pp. 134-139 ◽  
Author(s):  
Shulin Fu ◽  
Jiwen Ou ◽  
Minmin Zhang ◽  
Juan Xu ◽  
Huazhen Liu ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Lethal Dose ◽  
Interleukin 2 ◽  
Actinobacillus Pleuropneumoniae ◽  
Economic Losses ◽  
Respiratory Pathogens ◽  
Haemophilus Parasuis ◽  
Lethal Challenge ◽  
Content Type ◽  
Novel Approach

ABSTRACTHaemophilus parasuisandActinobacillus pleuropneumoniaeboth belong to the familyPasteurellaceaeand are major respiratory pathogens that cause large economic losses in the pig industry worldwide. We previously constructed an attenuatedA. pleuropneumoniaeserovar 1 live vaccine prototype, SLW05 (ΔapxICΔapxIICΔapxIV-ORF1), which is able to produce nontoxic but immunogenic ApxIA, ApxIIA, and ApxIVA. This triple-deletion mutant strain was shown to elicit protective immunity against virulentA. pleuropneumoniae. In the present study, we investigated whether immunization with SLW05 could also protect against lethal challenge with virulentH. parasuisSH0165 (serovar 5) or MD0322 (serovar 4). The SLW05 strain was found to elicit a strong humoral antibody response in pigs and to confer significant protection against challenge with a lethal dose ofH. parasuisSH0165 or MD0322. IgG subtype analysis revealed that SLW05 induces a bias toward a Th1-type immune response and stimulates interleukin 2 (IL-2) and gamma interferon (IFN-γ) production. Moreover, antisera from SLW05-vaccinated pigs efficiently inhibited bothA. pleuropneumoniaeandH. parasuisgrowth in a whole-blood assay. This is the first report that a live attenuatedA. pleuropneumoniaevaccine with SLW05 can protect against lethalH. parasuisinfection, which provides a novel approach for developing an attenuatedH. parasuisvaccine.

scholarly journals Engineering and Preclinical Evaluation of Attenuated Nontyphoidal Salmonella Strains Serving as Live Oral Vaccines and as Reagent Strains

2011 ◽  
Vol 79 (10) ◽  
pp. 4175-4185 ◽  
Author(s):  
Sharon M. Tennant ◽  
Jin-Yuan Wang ◽  
James E. Galen ◽  
Raphael Simon ◽  
Marcela F. Pasetti ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Lethal Dose ◽  
Invasive Disease ◽  
Oral Immunization ◽  
Oral Vaccines ◽  
Wild Type ◽  
Lethal Challenge ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTWhile nontyphoidalSalmonella(NTS) has long been recognized as a cause of self-limited gastroenteritis, it is becoming increasingly evident that multiple-antibiotic-resistant strains are also emerging as important causes of invasive bacteremia and focal infections, resulting in hospitalizations and deaths. We have constructed attenuatedSalmonella entericaserovar Typhimurium andSalmonella entericaserovar Enteritidis strains that can serve as live oral vaccines and as “reagent strains” for subunit vaccine production in a safe and economical manner. Prototype attenuated vaccine strains CVD 1921 and CVD 1941, derived from the invasive wild-type strainsS. TyphimuriumI77 andS. EnteritidisR11, respectively, were constructed by deletingguaBA, encoding guanine biosynthesis, andclpP, encoding a master protease regulator. TheclpPmutation resulted in a hyperflagellation phenotype. An additional deletion infliDyielded reagent strains CVD 1923 and CVD 1943, respectively, which export flagellin monomers. Oral 50% lethal dose (LD50) analyses showed that the NTS vaccine strains were all highly attenuated in mice. Oral immunization with CVD 1921 or CVD 1923 protected mice against lethal challenge with wild-typeS. TyphimuriumI77. Immunization with CVD 1941 but not CVD 1943 protected mice against lethal infection withS. EnteritidisR11. Immune responses induced by these strains included high levels of serum IgG anti-lipopolysaccharide (LPS) and anti-flagellum antibodies, with titers increasing progressively during the immunization schedule. SinceS. TyphimuriumandS. Enteritidisare the most common NTS serovars associated with invasive disease, these findings can pave the way for development of a highly effective, broad-spectrum vaccine against invasive NTS.

scholarly journals Protective Immunity against Tularemia Provided by an Adenovirus-Vectored Vaccine Expressing Tul4 of Francisella tularensis

2012 ◽  
Vol 19 (3) ◽  
pp. 359-364 ◽  
Author(s):  
Ravinder Kaur ◽  
Shan Chen ◽  
Maria T. Arévalo ◽  
Qingfu Xu ◽  
Yanping Chen ◽  
...  
Keyword(s):  
Single Dose ◽  
Francisella Tularensis ◽  
Virulent Strain ◽  
Lethal Dose ◽  
Protein Vaccine ◽  
Lethal Challenge ◽  
Live Attenuated Vaccine ◽  
Content Type ◽  
Skin Contact ◽  
Vectored Vaccine

ABSTRACTFrancisella tularensis, a category A bioterrorism agent, is a highly infectious organism that is passed on via skin contact and inhalation routes. A live attenuated vaccine strain (LVS) has been developed, but it has not been licensed for public use by the FDA due to safety concerns. Thus, there exists a need for a safer and improved vaccine. In this study, we have constructed a replication-incompetent adenovirus, Ad/opt-Tul4, carrying a codon-optimized gene for expression of a membrane protein, Tul4, ofF. tularensisLVS. Its ability to protect against lethal challenge and its immunogenicity were evaluated in a murine model. An intramuscular injection of a single dose (1 × 107PFU) of Ad/opt-Tul4 elicited a robust Tul4-specific antibody response. Assays suggest a Th1-driven response. A single dose elicited 20% protection against challenge with 100 × 50% lethal dose (LD50)F. tularensisLVS; two additional booster shots resulted in 60% protection. In comparison, three doses of 5 μg recombinant Tul4 protein did not elicit significant protection against challenge. Therefore, the Ad/opt-Tul4 vaccine was more effective than the protein vaccine, and protection was dose dependent. Compared to LVS, the protection rate is lower, but an adenovirus-vectored vaccine may be more attractive due to its enhanced safety profile and mucosal route of delivery. Furthermore, simple genetic modification of the vaccine may potentially produce antibodies protective against a fully virulent strain ofF. tularensis. Our data support the development and further research of an adenovirus-vectored vaccine against Tul4 ofF. tularensisLVS.

scholarly journals Oral Immunization with NontoxigenicClostridium difficileStrains Expressing Chimeric Fragments of TcdA and TcdB Elicits Protective Immunity againstC. difficileInfection in Both Mice and Hamsters

2018 ◽  
Vol 86 (11) ◽  
Author(s):  
Yuanguo Wang ◽  
Shaohui Wang ◽  
Laurent Bouillaut ◽  
Chunhui Li ◽  
Zhibian Duan ◽  
...  
Keyword(s):  
Vaccine Candidate ◽  
Cysteine Proteinase ◽  
Oral Vaccine ◽  
Lethal Dose ◽  
Disease Onset ◽  
Oral Immunization ◽  
Mucosal Vaccine ◽  
Hamster Model ◽  
Content Type ◽  
Protection Against Infection

ABSTRACTThe symptoms ofClostridium difficileinfection (CDI) are attributed largely to twoC. difficiletoxins, TcdA and TcdB. Significant efforts have been devoted to developing vaccines targeting both toxins through parenteral immunization routes. However,C. difficileis an enteric pathogen, and mucosal/oral immunization would be particularly useful to protect the host against CDI, considering that the gut is the main site of disease onset and progression. Moreover, vaccines directed only against toxins do not target the cells and spores that transmit the disease. Previously, we constructed a chimeric vaccine candidate, mTcd138, comprised of the glucosyltransferase and cysteine proteinase domains of TcdB and the receptor binding domain of TcdA. In this study, to develop an oral vaccine that can target bothC. difficiletoxins and colonization/adhesion factors, we expressed mTcd138 in a nontoxigenicC. difficile(NTCD) strain, resulting in strain NTCD_mTcd138. Oral immunization with spores of NTCD_mTcd138 provided mice full protection against infection with a hypervirulentC. difficilestrain, UK6 (ribotype 027). The protective strength and efficacy of NTCD_mTcd138 were further evaluated in the acute CDI hamster model. Oral immunization with spores of NTCD_mTcd138 also provided hamsters significant protection against infection with 2 × 104UK6 spores, a dose 200-fold higher than the lethal dose of UK6 in hamsters. These results imply that the genetically modified, nontoxigenicC. difficilestrain expressing mTcd138 may represent a novel mucosal vaccine candidate against CDI.

scholarly journals Characterization and Identification of a Novel Candidate Vaccine Protein through Systematic Analysis of Extracellular Proteins of Erysipelothrix rhusiopathiae

2013 ◽  
Vol 81 (12) ◽  
pp. 4333-4340 ◽  
Author(s):  
Fang Shi ◽  
Yohsuke Ogawa ◽  
Akiyuki Sano ◽  
Tomoyuki Harada ◽  
Jiro Hirota ◽  
...  
Keyword(s):  
Metabolic Enzymes ◽  
Extracellular Proteins ◽  
Vaccine Antigen ◽  
Immunogold Electron Microscopy ◽  
Major Protein ◽  
Erysipelothrix Rhusiopathiae ◽  
Lethal Challenge ◽  
Systematic Analysis ◽  
Content Type

ABSTRACTErysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate fromE. rhusiopathiaecultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins ofE. rhusiopathiaeto search for novel vaccine antigens. A concentrated culture supernatant from theE. rhusiopathiaeFujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of twoE. rhusiopathiaestrains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposedcholine-bindingproteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface ofE. rhusiopathiaeFujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophagesin vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen.

scholarly journals Distinct epigenetic signatures between adult-onset and late-onset depression

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hirotaka Yamagata ◽  
Hiroyuki Ogihara ◽  
Koji Matsuo ◽  
Shusaku Uchida ◽  
Ayumi Kobayashi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
Clinical Symptoms ◽  
Late Onset ◽  
Adult Onset ◽  
Blood Biomarkers ◽  
Major Depressive ◽  
Genome Wide ◽  
Healthy Control ◽  
Epigenetic Signatures

AbstractThe heterogeneity of major depressive disorder (MDD) is attributed to the fact that diagnostic criteria (e.g., DSM-5) are only based on clinical symptoms. The discovery of blood biomarkers has the potential to change the diagnosis of MDD. The purpose of this study was to identify blood biomarkers of DNA methylation by strategically subtyping patients with MDD by onset age. We analyzed genome-wide DNA methylation of patients with adult-onset depression (AOD; age ≥ 50 years, age at depression onset < 50 years; N = 10) and late-onset depression (LOD; age ≥ 50 years, age at depression onset ≥ 50 years; N = 25) in comparison to that of 30 healthy subjects. The methylation profile of the AOD group was not only different from that of the LOD group but also more homogenous. Six identified methylation CpG sites were validated by pyrosequencing and amplicon bisulfite sequencing as potential markers for AOD in a second set of independent patients with AOD and healthy control subjects (N = 11). The combination of three specific methylation markers achieved the highest accuracy (sensitivity, 64%; specificity, 91%; accuracy, 77%). Taken together, our findings suggest that DNA methylation markers are more suitable for AOD than for LOD patients.

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