OBJECTIVETo investigate the prevalence of cagA, vacA, and iceA genotypes in the isolated strains of Helicobacter pylori (H.pylori) from children with gastroduodenal diseases in Jiangxi, China, as well as the association between cagA, vacA, and iceA genotypes and the type of gastroduodenal diseases.

METHODSThe samples of gastric antral mucosa were collected from 316 children with gastroduodenal diseases in Jiangxi, and a total of 107 strains of H.pylori were isolated. The genomic DNA of these strains was extracted, and PCR was used to determine the ureA, cagA, vacA, and iceA genotypes.

RESULTSOf all the 107 isolated strains of H.pylori, the detection rates of ureA and cagA genes were 100% (107/107) and 94.4% (101/107) respectively. The overall detection rate of vacA gene was 100% (107/107), and the detection rates of vacAs1a, vacAs1c, vacAm1, and vacAm2 genes were 74.8% (80/107), 25.2% (27/107), 29.9% (32/107), and 69.2% (74/107) respectively, with both vacAm1 and vacAm2 genes detected in 0.9% (1/107) of all H.pylori strains. In the chimera of vacA gene, the detection rates of vacAs1a/m1, vacAs1a/m2, vacAs1c/m1, and vacAs1c/m2 genes were 26.2% (28/107), 51.4% (55/107), 3.7% (4/107), and 17.8% (19/107) respectively (P<0.001). The detection rates of iceA1 and iceA2 genes were 79.4% (85/107) and 9.3% (10/107), respectively (P<0.001), and both iceA1 and iceA2 genes were detected in 7.5% (8/107) of all strains. The detection rates of the genotypes of H.pylori showed no significant differences between the peptic ulcer, chronic gastritis, and duodenal bulbar inflammation groups (P>0.05).

CONCLUSIONSThe dominant genotypes of H.pylori are cagA, vacAs1a/m2, and iceA1, and there are mixed infections with H.pylori strains of different genotypes in children with gastroduodenal disease from Jiangxi, China. The genotypes of H.pylori are not associated with the type of gastroduodenal disease.

Adolescent ; Antigens, Bacterial ; genetics ; Bacterial Outer Membrane Proteins ; genetics ; Bacterial Proteins ; genetics ; Child ; Child, Preschool ; Female ; Gastritis ; microbiology ; Genotype ; Helicobacter pylori ; classification ; genetics ; isolation & purification ; Humans ; Infant ; Male ; Peptic Ulcer ; microbiology

Vibrios are universal conditioned-pathogenic bacteria in marine culture environment, and the outbreak of vibrio disease resulted in a serious damage to aquaculture. Considering that vibrio disease in aquatic species, especially fishes, usually originated from mixed infection of different species (serotypes or subspecies) of vibrios, it is important to select the potential cross-protective protein antigens as candidates of polyvalent or combined vaccines. In present research, several strains of vibrios were isolated from infected large yellow croaker (Pseudosciaena crocea) and subsequently identified as six strains of V. harveyi, one V. parahaemolyticus and one V. alginolyticus by physiological, biochemical and molecular biological methods. Their outer membrane proteins (OMPs) were extracted and the SDS-PAGE and Western blotting results show that three immuno-blots with common molecular weight presented at approximate 45 kDa, 35 kDa and 22 kDa on their OMP electrophoretogram, indicating the existence of antigens with cross-protection in their OMPs. With the aids of combination of two-dimensional electrophoresis (2-D) and Western blotting and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), a deduced porin (GenBank Accession No. ZP_01260407) from V. alginolyticus and a maltoporin precursor (GenBank Accession No. NP_801154) from V. parahaemolyticus were able to react with polyclonal antibody to whole V. harveyi, suggesting these two proteins could act as the cross-protective antigens and the vaccines prepared with these porins would be probable to bring cross protection to three different vibrios.
Animals ; Antigens, Bacterial ; immunology ; Bacterial Outer Membrane Proteins ; immunology ; Cross Reactions ; Fish Diseases ; microbiology ; Perciformes ; microbiology ; Vibrio ; classification ; immunology ; isolation & purification ; pathogenicity ; Vibrio Infections ; microbiology

OBJECTIVEYersinia enterocolitica is an extracellular pathogen and its related antigens interact with the host immune system. We investigated the difference in immunological characteristics between a highly pathogenic and poorly pathogenic strain of Y. enterocolitica.

METHODSWe used SDS-PAGE and western blotting to characterize lipopolysaccharide (LPS), Yersinia outer membrane proteins (Yops), membrane proteins, and whole-cell proteins from poorly pathogenic Y. enterocolitica bio-serotype 2/O:9, isolated from China, and highly pathogenic bio-serotype 1B/O:8, isolated from Japan.

RESULTSThese two strains of Y. enterocolitica had different LPS immune response patterns. Comparison of their Yops also showed differences that could have accounted for their differences in pathogenicity. The membrane and whole-cell proteins of both strains were similar; immunoblottting showed that the 35 kD and perhaps the 10 kD proteins were immunogens in both strains.

CONCLUSIONThe major antigens of the two strains eliciting the host immune response were the LPS and membrane proteins, as shown by comparing protein samples with reference and purified preparations.

Animals ; Antigens, Bacterial ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Female ; Gene Expression Regulation, Bacterial ; physiology ; Lipopolysaccharides ; metabolism ; Rabbits ; Yersinia enterocolitica ; classification ; metabolism

OBJECTIVETo explore the distribution characteristics of the types of M protein gene (emm) in group A streptococcus (GAS) isolated from children in Beijing in the year 2011.

METHODSDuring May to July in 2011, a total of 3315 patients who were diagnosed scarlet fever or pharyngeal infection by doctors in pediatric outpatient and emergency units of 36 hospitals, were selected as subjects. Their throat swab samples were collected and isolated the strains of GAS. Gene emm was then amplified and sequenced by PCR method, and the differences in types of gene emm between different populations and diseases were compared.

RESULTSA total of 633 strains of GAS were isolated from the 3315 throat swab samples, 610 strains out of which were gene emm positive and were recruited in the study. Out of the 610 recruited strains, 448 (73.4%) were isolated from scarlet fever patients, the other 162 (26.6%) were isolated from pharyngeal infection patients; 397 (65.1%) were from urban, the other 213 (34.9%) were from suburb; 240 (39.4%) were from patients aging between 1 - 5 years old, the other 369(60.6%) were from patients aging 6 - 18 years old. A total of 8 types of gene emm (scarlet fever: 6 types, pharyngeal infection: 4 types) and 21 subtypes of gene emn (scarlet fever: 16 subtypes, pharyngeal infection: 10 subtypes) were identified. Three new subtypes were found in the study, naming emm1.63, emm12.62 and st5144.20. Among them, emm1.63 was found both in scarlet fever and pharyngeal infection patients, while emm12.62 and st5144. 20 were only found in pharyngeal infection patients. Among all the types of gene-emm, emm12 accounted for the highest percentage as 80.5% (491/610) and then followed by emm1 (18.0% (110/610)). Among all the subtypes, the dominant subtype was emm12.00, accounting for 69.0% (421/610), following by emm1.00 (16.9% (103/610)) and emm12.19 (6.1% (37/610)). All the above types and subtypes of gene emm were the most prevalent strains in scarlet fever patients and pharyngeal infection patients. Significant differences in the distribution of prevalent strains were observed among various aging patients and regions. The constituent ratios of emm1, emm1.00 and emm12.19 were higher in patients from suburb (emm1: 22.1% (47/213), emm1.00: 19.2% (40/213), emm12.19: 8.0% (17/213)) than those in urban areas (emm1: 15.9% (63/397), emm1.00: 15.6% (62/397), emm12.19: 5.0% (20/397)). The difference showed statistical significance (P < 0.05). The constituent ratio of emm1.00 was higher among patients aging 6-18 years old (19.2% (71/369)) than those aging 1 - 5 years old (13.3% (32/240)). The difference also showed statistical significance (χ(2) = 8.45, P < 0.05).

CONCLUSIONAmong the types of gene emm in GAS isolated from children in Beijing in year 2011, the most prevalent two were emm12 and emm1, and the most prevalent emm subtypes were emm12.00, emm1.00 and emm12.19. A significant difference in their distribution between various aging patients and isolated places can be obviously found.

Adolescent ; Antigens, Bacterial ; classification ; genetics ; Bacterial Outer Membrane Proteins ; classification ; genetics ; Carrier Proteins ; classification ; genetics ; Child ; Child, Preschool ; China ; Female ; Genes, Bacterial ; Genotype ; Humans ; Infant ; Male ; Microbial Sensitivity Tests ; Streptococcus pyogenes ; genetics ; isolation & purification

OBJECTIVETo investigate cagA, vacA and iceA genotypes of Helicobacter pylori (H. pylori) isolated from children suffering from gastric and duodenal diseases in Shanghai and to explore a possible genotype-phenotype correlation.

METHODSFrom May 2007 to January 2008, 59 children were confirmed with Hp infection by gastroscopy. Biopsied specimens were taken from the gastric antrum. cagA, vacA and iceA genes were determined by PCR. The histological changes in the gastric mucosa were evaluated. The levels of IFN-gamma and IL-4 in the gastric mucosa were measured using ELISA.

RESULTScagA, vacAs1/m1, vacAs1/m2, iceA1 and iceA2 were found in 65%, 19%, 40%, 63% and 19% of H. pylori strains, respectively. Both iceA1 and iceA2 were detected in 9% of strains. There were no statistical differences in the distribution of various genotypes between the children with chronic gastritis and peptic ulcer. No association was observed between the genotypes and the degree of inflammation of gastric mucosa. There were no significant differences in levels of IFN-gamma and IL-4 in the gastric mucosa infected by different genotypes of H. pylori strains.

CONCLUSIONScagA/vacAs1/m2/iceA1 may be the commonest genotype combination of H.pylori in children from Shanghai. That there was no association between H.pylori genotypes and clinical variables suggests the potential role of host and environment factors in the development of clinical diseases at a later life.

Adolescent ; Antigens, Bacterial ; genetics ; Bacterial Outer Membrane Proteins ; genetics ; Bacterial Proteins ; genetics ; Child ; Child, Preschool ; Female ; Gastritis ; microbiology ; Genotype ; Helicobacter pylori ; classification ; genetics ; Humans ; Interferon-gamma ; analysis ; Interleukin-4 ; analysis ; Male ; Peptic Ulcer ; microbiology

To predict the B cell epitopes for major outer membrane protein (MOMP) of Chlamydia trachomatis (CT), the secondary structure of CT MOMP was predicted by the methods of GOR based on the sequence of amino acids of E serotype CT MOMP. By combining the comprehensive analysis of transmembrane domain, hydrophilicity profile, surface probability, antigenic index and average flexibility, the B cell predominant epitopes of CT MOMP were further predicted. The N-terminal No. 73-81, 217-225, 377-386, 261-270 and 161-175 were the predominant B cell epitopes. Prediction of the B cell epitopes for the CT MOMP by the multi-parameters is helpful for the identification of B cell epitopes.
Amino Acid Sequence ; Antigens, Bacterial ; immunology ; Chlamydia trachomatis ; classification ; immunology ; Epitopes, B-Lymphocyte ; immunology ; Molecular Sequence Data ; Porins ; immunology ; Protein Conformation ; Protein Structure, Secondary ; Serotyping

OBJECTIVETo study the relationship of the types of Helicobacter pylori (H. pylori) strains with the classification and the severity of chronic gastro-duodenal diseases in children.

METHODSOne hundred and fifteen children with chronic upper gastrointestinal symptoms who were diagnosed as H. pylori infection by gastroscopy were enrolled in this study. H. pylori strains were serotyped by immunoblot technique. The gastric biopsy specimens of all patients were studied histologically.

RESULTSType I H. pylori strains were confirmed in 84 cases (73.0%), intermediate type strains in 21 cases (18.3%), and type II strains in 10 cases (8.7%). Type I H. pylori strains infection caused a moderate gastric mucosal inflammation in 83 cases and a severe inflammation in 1 case. Intermediate type H. pylori strains infection caused a moderate gastric mucosal inflammation in 21 cases. Type II H. pylori strains infection caused a mild gastric mucosal inflammation in 2 cases and a moderate inflammation in 8 cases. Different types of H. pylori strains resulted in different severity of gastric mucosal inflammation (x2=15.444, P < 0.01). The gastric mucosal inflammation due to type I H. pylori strains was the most severe, while the inflammation due to type II H. pylori strains was relatively mild. The incidence of nodulus lymphaticus of gastric mucosa due to type I, type II and intermediate type H. pylori strains infection was 76.2%, 47.6% and 40.0%, respectively (x2=10.171, P < 0.01). The classification of chronic gastro-duodenal diseases was not associated with the types of H. pylori strains.

CONCLUSIONSType I strains were the leading cause of H. pylori infection in children. All of types of H. pylori strains can cause pathohistologic changes of gastric mucosa. Type I H. pylori strains infection can result in the most severe gastric mucosal inflammation and the highest incidence of nodulus lymphaticus. The immunoblot serotyping of H.pylori strains may be useless for the classification of chronic upper gastrointestinal diseases but it is helpful for the evaluation of the severity of the diseases in children.

Adolescent ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; genetics ; Bacterial Proteins ; genetics ; Child ; Child, Preschool ; Chronic Disease ; Female ; Gastric Mucosa ; pathology ; Gastrointestinal Diseases ; microbiology ; pathology ; Helicobacter Infections ; complications ; diagnosis ; Helicobacter pylori ; classification ; Humans ; Male

OBJECTIVETo analyze the genetic differences of Orientia tsutsugamushi (Ot) Sta56 gene between Shandong isolates and other strains deposited in GenBank.

METHODSPCR and restriction fragment length polymorphism (RFLP) were used to amplify the complete sequence of Ot-Sta56 gene. RFLP profiles of Ot were predicted by a computer program according to their complete sequences of Ot-Sta56 gene. PCR amplicon from XDM2 strain was sequenced and analyzed by Clustal X (1.8) and PHYLIP software.

RESULTSThe complete sequences (about 1.6 kbp) of Ot-Sta56 gene were amplified from B16 strain (isolated from patients), FXS2 strain (isolated from A. agrarius) and XDM2 strain. Four species of restriction endonucleases (Hha I, Hinf I, Hae III, Pst I) were used to digest the PCR amplicons from the 3 isolates. When comparing with the RFLP profiles of prototype Ot, the RFLP profiles of PCR amplicons from the 3 isolates were similar to those of Japan Kawasaki strain, but were quite different from the international reference strains Gilliam, Karp, Kato. Results from DNA sequence analysis showed that the complete sequence of Ot-Sta56 gene homology to Japan Kawasaki strain of XDM2 strain was 97%, and deduced amino acid sequence was 92%.

CONCLUSIONData from the complete sequence of Sta56 gene indicated that the genotypes of Ot isolates in Shandong province were similar, but with distinction from the Kawasaki strain.

Amplified Fragment Length Polymorphism Analysis ; Antigens, Bacterial ; genetics ; Bacterial Proteins ; genetics ; Bacterial Typing Techniques ; classification ; DNA, Bacterial ; genetics ; Genes, Bacterial ; Membrane Proteins ; genetics ; Orientia tsutsugamushi ; genetics ; isolation & purification ; Polymorphism, Restriction Fragment Length ; Sequence Analysis, DNA

OBJECTIVEThe determination of antigenicity and immunogenicity of Leptospira interrogans genus-specific outer envelope proteins (OEPs) will offer evidence for developing universal leptospiral genetic engineering vaccine and detection kit.

METHODSIn this study, Ni-NTA affinity chromatography is used to purify the recombinant products rLipL21, rOmpL1/1, rOmpL1/2, rLipL32/1, rLipL32/2, rLipL41/1 and rLipL41/2 expressed by the major genotypes of four leptospiral OEPs of 15 serogroups. SDS-PAGE is applied to examine the expression and purity of the recombinant proteins. Rabbits are intracutaneously immunized with the recombinant proteins to obtain antisera. Microscope agglutination test (MAT) is used to measure the cross inmmunoagglutination titers of antisera. The OMPs of the reference standard strains belonging to 15 serogroups of L. interrogans in China and L. biflexa strain Patoc I are prepared using salt-denature method. By each of the antisera as the first antibody, Western blot assay is established to detect the natural expressions and immunoreactivity of the four OEPs.

RESULTSThe outputs of rLipL21, rLipL32/1, rLipL32/2, rLipL41/1l, rLipL41/2, rOmpL1/1 and rOmpL1/2 are 10%, 40%, 35%, 15%, 10%, 30% and 15%, respectively. Each the purified recombinant proteins shows a single fragment after SDS-PAGE. Each the rabbit antisera displays extensive cross immunoreactivity between the products expressed by different genotypes of the same gene and the MAT titers ranging from 1:2-1:128. All the four OEPs can be detectable in the OEPs preparations. However, LipL21 is found to exist only in L. interrrogans.

CONCLUSIONThe results of this study indicate that all the four OEPs are superficial genus-specific antigens of Leptospira which can be used as the candidate antigens of leptospiral universal vaccine and detection kit.

Animals ; Antibody Formation ; Antigens, Bacterial ; immunology ; Electrophoresis, Polyacrylamide Gel ; Genetic Engineering ; Immunization ; Leptospira interrogans ; classification ; immunology ; Membrane Proteins ; Rabbits ; Recombinant Proteins ; immunology ; Serotyping

BACKGROUNDAlmost half of the world's population suffer from the Helicobacter pylori (H. pylori) infection, but only some individuals develop gastric diseases with clinical symptoms. One reason for the phenomenon may be the different pathogenicity of infected H. pylori strains. The presence of cytotoxin-associated gene A (cagA) and expression of vacuolating cytotoxin activity encoded by vacuolating cytotoxin gene A (vacA) are considered the two major virulent markers of H. pylori. The aim of this study was to detect dominant cagA/vacA genotypes and coinfection frequency of H. pylori in patients with peptic ulceration (PU) or chronic gastritis (CG), and to determine correlations among different cagA/vacA genotypes, coinfection and severity of the diseases.

METHODSFor each of 139 patients in Zhejiang Province who had been diagnosed as PU or CG based on clinical symptoms and gastroscopy, two gastric biopsy specimens (one from antrum and the other from corpus) for H. pylori isolation were taken by two different disinfected biopsy forceps. One hundred and fifty-six H. pylori strains were isolated from both the antrum and corpus biopsy specimens of 78 patients (36 PU and 42 CG). PCRs were performed to detect cagA genes, and signal (s) and middle (m) regions of vacA genes in the H. pylori isolates. The amplified fragments of dominant vacA gene s and m subtypes from representative H. pylori isolates were sequenced after TA cloning. Dominant cagA/vacA genotypes of the H. pylori isolates, coinfection frequency and correlations among the different genotypes, coinfection and severity of the diseases were determined.

RESULTSOf the H. pylori strains isolated from the antrum specimens, 96.2% were cagA gene positive, as were 97.4% of the H. pylori strains isolated from the corpus specimens. Only one s region subtype (s1a) and four m region subtypes m1, m2, m1b and m1b-m2 of vacA gene were found. The proportions of vacA gene subtypes s1a/m1, s1a/m2, s1a/m1b and s1a/m1b-m2 in the 83 strains isolated from the antrum specimens were 7.2%, 61.5%, 30.1% and 1.2%, respectively, while those in the other 84 strains isolated from the corpus specimens were 9.5%, 58.3%, 28.6% and 3.6%, respectively. s1a/m2 (58.3% vs 30.1%, chi(2) = 13.47, P < 0.01) and then s1a/m1b (28.6% vs 9.5%, chi(2) = 9.88, P < 0.01) were the dominant vacA gene subtypes in the H. pylori isolates. The dominant H. pylori genotype was cagA + s1a/m2 (59.0% from antrum specimens and 57.1% from corpus specimens), and followed by cagA + s1a/m1b (28.9% from antrum specimens and 27.4% from corpus specimens). Sixteen of 78 patients (20.5%) were infected with two or three H. pylori strains with different genotypes. However, no statistically significant differences among cagA occurrence, the different vacA subtypes and PU or CG could be found (each P > 0.05). Similarities of the nucleotide sequences from vacA gene s region PCR products of six isolates and from vacA gene m region PCR products of four isolates were 93.2% to 98.3% and 93.8% to 97.6%, respectively, compared to the reported corresponding sequences.

CONCLUSIONSThe dominant genotypes of H. pylori in PU or CG patients in Zhejiang area may be cagA + s1a/m2 and cagA + s1a/m1b. Numerous coinfections with different H. pylori strains in PU or CG patients indicate diversity of the infected H. pylori origins. s and m regions of vacA gene from different H. pylori isolates show high nucleotide sequence similarities. cagA gene positive rate, different vacA gene subtypes and coinfection with different H. pylori strains are not closely associated with severity of the diseases.

Adult ; Amino Acid Sequence ; Antigens, Bacterial ; genetics ; Bacterial Proteins ; genetics ; Chronic Disease ; Female ; Gastritis ; microbiology ; Genotype ; Helicobacter Infections ; microbiology ; Helicobacter pylori ; classification ; genetics ; Humans ; Male ; Molecular Sequence Data ; Peptic Ulcer ; microbiology