Use of in vivo-induced antigen technology (IVIAT) for the identification of Streptococcus suis serotype 2 in vivo-induced bacterial protein antigens

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus suis </it>serotype 2 (SS2) is a zoonotic agent that causes death and disease in both humans and swine. A better understanding of SS2-host molecular interactions is crucial for understanding SS2 pathogenesis and immunology. Conventional genetic and biochemical approaches used to study SS2 virulence factors are unable to take into account the complex and dynamic environmental stimuli associated with the infection process. In this study, <it>in vivo</it>-induced antigen technology (IVIAT), an immunoscreening technique, was used to identify the immunogenic bacterial proteins that are induced or upregulated <it>in vivo </it>during SS2 infection.</p> <p>Results</p> <p>Convalescent-phase sera from pigs infected with SS2 were pooled, adsorbed against <it>in vitro </it>antigens, and used to screen SS2 genomic expression libraries. Upon analysis of the identified proteins, we were able to assign a putative function to 40 of the 48 proteins. These included proteins implicated in cell envelope structure, regulation, molecule synthesis, substance and energy metabolism, transport, translation, and those with unknown functions. The <it>in vivo</it>-induced changes in the expression of 10 of these 40 genes were measured using real-time reverse transcription (RT)-PCR, revealing that the expression of 6 of the 10 genes was upregulated in the <it>in vivo </it>condition. The strain distribution of these 10 genes was analyzed by PCR, and they were found in the most virulent SS2 strains. In addition, protein sequence alignments of the newly identified proteins demonstrate that three are putative virulence-associated proteins.</p> <p>Conclusion</p> <p>Collectively, our results suggest that these <it>in vivo</it>-induced or upregulated genes may contribute to SS2 disease development. We hypothesize that the identification of factors specifically induced or upregulated during SS2 infection will aid in our understanding of SS2 pathogenesis and may contribute to the control SS2 outbreaks. In addition, the proteins identified using IVIAT may be useful potential vaccine candidates or virulence markers.</p

Determination of the mimic epitope of the M-like protein adhesin in swine Streptococcus equi subsp. zooepidemicus

<p>Abstract</p> <p>Background</p> <p>The M-like protein, also known as SzP, is expressed on the surface of <it>Streptococcus equi </it>subsp. z<it>ooepidemicus </it>(<it>S</it>. z<it>ooepidemicus</it>). Previous studies demonstrated that SzP is similar to M protein of group A <it>Streptococcus </it>in the structure and characteristics of antiphagocytosis. The M protein is an adhesin that can bind to the host cells, however it is not known whether the SzP of <it>S</it>. z<it>ooepidemicus </it>also functions as an adhesin. We conducted an investigation to determine SzP as an adhesin, and one SzP epitope was identified to be responsible for mediating binding to HEp-2 cells.</p> <p>Methods</p> <p>The gene encoding SzP was expressed in <it>E. coli</it>, and the purified recombinant SzP (rSzP) was recognized by rabbit anti-<it>S</it>. z<it>ooepidemicus </it>antibodies using immunoblot. Furthermore, the adherence of <it>S</it>. z<it>ooepidemicus </it>to HEp-2 cells was inhibited by anti-rSzP antibodies in a dose-dependent manner. We employed a random 12-peptide phage display library for screening of immunodominant mimics of the SzP, which were recognized by an anti-SzP specific monoclonal antibody (mAb 2C8). Initial positive phage clones were identified by ELISA, followed by assays to determine the adherence-inhibiting ability of the peptide.</p> <p>Results</p> <p>Ten out of fourteen selected positive clones showed high reactivity that effectively inhibited the binding of mAb 2C8 to rSzP. The motif XSLSRX was highly conserved among six of the ten clones.</p> <p>Conclusion</p> <p>Collectively, our findings suggest that the motif XSLSRX may represent an immunodominant mimic epitope of the SzP of <it>S</it>. z<it>ooepidemicus </it>strain ATCC 35246, and that the same epitope may be used to mediate SzP binding to HEp-2 cells.</p

Overexpression of luxS

LuxS/AI-2 quorum sensing (QS) system involves the production of cell signaling molecules via luxS-based autoinducer-2 (AI-2). LuxS has been reported to plays critical roles in regulating various behaviors of bacteria. AI-2 is a byproduct of the catabolism of S-adenosylhomocysteine (SAH) performed by the LuxS and Pfs enzymes. In our previous study, the function of LuxS in AI-2 production was verified in Streptococcus suis (SS). Decreased levels of SS biofilm formation and host-cell adherence as well as an inability to produce AI-2 were observed in bacteria having a luxS mutant gene. In this study, the level of AI-2 activity exhibits a growth-phase dependence with a maximum in late exponential culture in SS. An SS strain that overexpressed luxS was constructed to comprehensively understand the function of AI-2. Overexpressed luxS was not able to increase the level of pfs expression and produce additional AI-2, and the bacteria were slower growing and produced only slightly more biofilm than the wild type. Thus, AI-2 production is not correlated with luxS transcription. luxS expression is constitutive, but the transcription of pfs is perhaps correlated with AI-2 production in SS

Immunoproteomic analysis of bacterial proteins of Actinobacillus pleuropneumoniae serotype 1

<p>Abstract</p> <p>Background</p> <p><it>Actinobacillus pleuropneumoniae </it>(APP) is one of the most important swine pathogens worldwide. Identification and characterization of novel antigenic APP vaccine candidates are underway. In the present study, we use an immunoproteomic approach to identify APP protein antigens that may elicit an immune response in serotype 1 naturally infected swine and serotype 1 virulent strain S259-immunized rabbits.</p> <p>Results</p> <p>Proteins from total cell lysates of serotype 1 APP were separated by two-dimensional electrophoresis (2DE). Western blot analysis revealed 21 immunoreactive protein spots separated in the pH 4-7 range and 4 spots in the pH 7-11 range with the convalescent sera from swine; we found 5 immunoreactive protein spots that separated in the pH 4-7 range and 2 in the pH 7-11 range with hyperimmune sera from S259-immunized rabbits. The proteins included the known antigens ApxIIA, protective surface antigen D15, outer membrane proteins P5, subunit NqrA. The remaining antigens are being reported as immunoreactive proteins in APP for the first time, to our knowledge.</p> <p>Conclusions</p> <p>We identified a total of 42 immunoreactive proteins of the APP serotype 1 virulent strain S259 which represented 32 different proteins, including some novel immunoreactive factors which could be researched as vaccine candidates.</p

Suppression subtractive hybridization identifies an autotransporter adhesin gene of E. coli IMT5155 specifically associated with avian pathogenic Escherichia coli (APEC)

<p>Abstract</p> <p>Background</p> <p>Extraintestinal pathogenic <it>E. coli </it>(ExPEC) represent a phylogenetically diverse group of bacteria which are implicated in a large range of infections in humans and animals. Although subgroups of different ExPEC pathotypes, including uropathogenic, newborn meningitis causing, and avian pathogenic <it>E. coli </it>(APEC) share a number of virulence features, there still might be factors specifically contributing to the pathogenesis of a certain subset of strains or a distinct pathotype. Thus, we made use of suppression subtractive hybridization and compared APEC strain IMT5155 (O2:K1:H5; sequence type complex 95) with human uropathogenic <it>E. coli </it>strain CFT073 (O6:K2:H5; sequence type complex 73) to identify factors which may complete the currently existing model of APEC pathogenicity and further elucidate the position of this avian pathoype within the whole ExPEC group.</p> <p>Results</p> <p>Twenty-eight different genomic loci were identified, which are present in IMT5155 but not in CFT073. One of these loci contained a gene encoding a putative autotransporter adhesin. The open reading frame of the gene spans a 3,498 bp region leading to a putative 124-kDa adhesive protein. A specific antibody was raised against this protein and expression of the adhesin was shown under laboratory conditions. Adherence and adherence inhibition assays demonstrated a role for the corresponding protein in adhesion to DF-1 chicken fibroblasts. Sequence analyses revealed that the flanking regions of the chromosomally located gene contained sequences of mobile genetic elements, indicating a probable spread among different strains by horizontal gene transfer. In accordance with this hypothesis, the adhesin was found to be present not only in different phylogenetic groups of extraintestinal pathogenic but also of commensal <it>E. coli </it>strains, yielding a significant association with strains of avian origin.</p> <p>Conclusions</p> <p>We identified a chromosomally located autotransporter gene in a highly virulent APEC strain which confers increased adherence of a non-fimbriated <it>E. coli </it>K-12 strain to a chicken fibroblast cell line. Even though flanked by mobile genetic elements and three different genetic regions upstream of the gene, most probably indicating horizontal gene transfer events, the adhesin gene was significantly linked with strains of avian origin. Due to the nucleotide sequence similarity of 98% to a recently published adhesin-related gene, located on plasmid pAPEC-O1-ColBM, the name <it>aatA </it>(APEC autotransporter adhesin A) was adopted from that study.</p> <p>Our data substantiate that AatA might not only be of relevance in APEC pathogenicity but also in facilitating their reservoir life style in the chicken intestine, which might pave the way for future intestinal preventive strategies.</p

Comparative Proteomic Analysis of Streptococcus suis Biofilms and Planktonic Cells That Identified Biofilm Infection-Related Immunogenic Proteins

Streptococcus suis (SS) is a zoonotic pathogen that causes severe disease symptoms in pigs and humans. Biofilms of SS bind to extracellular matrix proteins in both endothelial and epithelial cells and cause persistent infections. In this study, the differences in the protein expression profiles of SS grown either as planktonic cells or biofilms were identified using comparative proteomic analysis. The results revealed the existence of 13 proteins of varying amounts, among which six were upregulated and seven were downregulated in the Streptococcus biofilm compared with the planktonic controls. The convalescent serum from mini-pig, challenged with SS, was applied in a Western blot assay to visualize all proteins from the biofilm that were grown in vitro and separated by two-dimensional gel electrophoresis. A total of 10 immunoreactive protein spots corresponding to nine unique proteins were identified by MALDI-TOF/TOF-MS. Of these nine proteins, five (Manganese-dependent superoxide dismutase, UDP-N-acetylglucosamine 1-carboxyvinyltransferase, ornithine carbamoyltransferase, phosphoglycerate kinase, Hypothetical protein SSU05_0403) had no previously reported immunogenic properties in SS to our knowledge. The remaining four immunogenic proteins (glyceraldehyde-3-phosphate dehydrogenase, hemolysin, pyruvate dehydrogenase and DnaK) were identified under both planktonic and biofilm growth conditions. In conclusion, the protein expression pattern of SS, grown as biofilm, was different from the SS grown as planktonic cells. These five immunogenic proteins that were specific to SS biofilm cells may potentially be targeted as vaccine candidates to protect against SS biofilm infections. The four proteins common to both biofilm and planktonic cells can be targeted as vaccine candidates to protect against both biofilm and acute infections

Cross-cultural adaptation and psychometric properties of the Chinese version of the Orthorexia Nervosa Inventory

BackgroundOrthorexia nervosa refers to an unhealthy preoccupation with maintaining a perfect diet, which is marked by highly restrictive eating habits, rigid food rituals, and the avoidance of foods perceived as unhealthy or impure. In recent years, the Orthorexia Nervosa Inventory (ONI) has gained recognition as a promising tool for assessing orthorexia tendencies and behaviors, addressing the limitations of existing ON-specific measures. This study aimed to evaluate the psychometric properties of the Chinese version of the ONI.MethodsA total of 717 participants (Mage = 20.11 years, 78.66% female) completed the Orthorexia Nervosa Inventory (ONI) alongside the Chinese version of the Düsseldorf Orthorexia Scale (C-DOS). The ONI was translated into Chinese using the Brislin traditional translation model, following formal authorization from the original author. This translation process included literal translation, back translation, and cultural adaptation to ensure both linguistic and contextual fidelity. Item analysis was employed to assess item differentiation. Scale reliability was determined by measuring internal consistency. Furthermore, exploratory and confirmatory factor analyses were conducted to investigate and confirm the underlying factor structure and overall validity of the scale.ResultsThe Chinese version of the Orthorexia Nervosa Inventory (ONI) consists of 24 items across three dimensions. The overall Cronbach’s alpha coefficient for the scale was 0.956, indicating excellent internal consistency. The Cronbach’s alpha coefficients for the individual dimensions were 0.894, 0.933, and 0.848, respectively, demonstrating high reliability for each dimension. Additionally, McDonald’s ω was 0.957 for the entire scale, reflecting strong stability in internal consistency, with individual dimensions having McDonald’s ω coefficients of 0.895, 0.934, and 0.854. The Spearman-Brown split-half reliability coefficient was 0.931, and McDonald’s ω for the split-half reliability was also 0.931, indicating excellent consistency across the scale’s two halves. The test–retest reliability was 0.987, with a 95% confidence interval ranging from 0.978 to 0.993, suggesting excellent stability over time and strong consistency across different measurement points. All model fit indices fell within acceptable ranges, affirming the structural validity of the Chinese version. The results from both exploratory and confirmatory factor analyses further supported this conclusion.ConclusionThis study successfully translated and culturally adapted the ONI into Chinese, followed by a comprehensive evaluation of its psychometric properties. The findings demonstrate that the Chinese version of the ONI possesses strong reliability and validity. In the context of varying cultural backgrounds and dietary habits, this scale serves as a valid tool for assessing and screening the Chinese ON population

Genotyping and pathobiologic characterization of canine parvovirus circulating in Nanjing, China

Abstract Background Canine parvovirus (CPV) is an important pathogen that causes acute enteric disease in dogs. It has mutated and spread throughout the world in dog populations. We provide an update on the molecular characterization of CPV that circulated in Nanjing, a provincial capital in China between 2009 and 2012. Results Seventy rectal swab samples were collected from the dogs diagnosed with CPV infection in 8 animal hospitals of Nanjing. Sequence analysis of VP2 genes of 31 samples revealed that 29 viral strains belonged to CPV-2a subtype, while other two strains were classified into CPV-2b. To investigate the pathogenicity of the prevalent virus, we isolated CPV-2a and performed the animal experiment. Nine beagles were inoculated with 105.86 of 50% tissue culture infectious doses (TCID50) of the virus. All the experimentally infected beagles exhibited mild to moderate mucoid or watery diarrhea on day 4 post-infection (p.i.). On day 9 p.i., characteristic histopathological lesions were clearly observed in multiple organs of infected dogs, including liver, spleen, kidney, brain and all segments of the small and large intestines, while viral DNA and antigen staining could be detected in the sampled tissues. It is notable that canine parvovirus was isolated in one from two brain samples processed. Conclusion Our results indicated that CPV-2a is the predominant subtype in Nanjing of China. And this virus caused extensive lesions in a variety of tissues, including the brain. </jats:sec