Search

Objective: In addition to Streptococcus suis serotype 2, Streptococcus suis serotype 9 (SS9) is also a currently prevalent serotype and a zoonotic pathogen. In our previous study, SS9 DNA nuclease (SsnA) was considered as a candidate virulence factor. To clarify the impact of SsnA on SS9 virulence, we constructed ssnA mutant (ΔssnA) and studied its biological functions., Methods: We evaluated the virulence of wild type strain and ΔssnA in a zebrafish infection model and compared the adherence rate to HEp-2 cells, the survival rate in pig blood, and enzymatic activity between wild type stain and ΔssnA., Results: In a zebrafish infection experiment, the 50% lethal dose value of ΔssnA was 11.2-fold higher than that of wild type strain. The adherence rate of ΔssnA to HEp-2 cells was only 60.61% of the wild strain level. The survival rate of ΔssnA in pig blood was declined to 71.88% of wild strain level. The enzymatic activity assay showed that SsnA can degrade both linear and circular DNA., Conclusion: SsnA contributes to SS9 virulence in a zebrafish infection model, the adherence to HEp-2 cells, and the survival in pig blood. SsnA is indeed an essential virulence factor for SS9.

Objective: To isolate and identify Streptococcus agalactiae phages and screen candidate phages to control infection caused by bovine S. agalactiae., Methods: We used two methods for isolation of S. agalactiae phages, namely (1) isolation of phages from milk and environmental samples, and (2) isolation of phages via induction of lysogens with Mitomycin C. Double-layer agar culture method was used to purify phages. Then the newly obtained phages, with S. agalactiae phage JX01 isolated from mastitis milk, were comparatively analyzed in the following aspects: morphology of phages by transmission electron microscopy, host range of phages to 55 S. agalactiae strains and other Streptococcus strains, phages DNA using EcoR I, Xba I, Pst I and Sal I, the optical multiplicity of infection, absorption curve and one step growth curve, and the stability of phages at different storage conditions., Results: The comparative analysis of the 3 novel phages LYGO9, HZ04 and pA11 (induced from S. agalctiae bovine clinical isolate HAJL2011070601) with JX01 showed that the 4 phages were classified as the member of Siphovirdae family. EcoR I, Sal I, Xba I and Pst I separately digested the 4 phages DNA provided 4, 3, 3 and 2 profiles, respectively. This suggested that they were different strains. All the 4 phages specifically infected bovine S. agalactiae isolates. LYGO9, pA11, JX01 and HZ04 could lyse 12, 13, 20 and 23 of 42 tested bovine S. agalctiae isolates, respectively. This clearly indicated that these 4 phages are closely related., Conclusion: The 3 new phages which specifically lyse bovine S. agalactiae isolates are siphovirus phages. Phage LYGO9 was shown having a short latent period and a larger burst size.

Objective: Streptococcus suis (S. suis) is an emerging zoonotic pathogenic bacterium capable of infecting piglets and human and with sporadic infections in a variety of mammalian species. The aim of this study is to investigate the prevalence of S. suis in wild cats., Methods: We isolated an S. suis strain from a wild cat. We tested the serotype of the isolated strain by anti-serum agglutination and PCR. We determined the sequence type (ST) of the isolated strain by multilocus sequence typing tests (MLST). We constructed the 16S rRNA phylogenetic tree of the isolation and S. suis strains in NCBI database to demonstrated genetic relationship of different strains. We measured the antibiotic resistance of the isolated strain by triple disk diffusion method. We detected the virulence of the isolated strain by mice infection experiments., Results: We isolated an S. suis strain m70 from a wild cat, which belongs to serotype 9. MLST showed that m70 fell into a new ST. The 16S rRNA phylogenetic tree of m70 and S. suis strains in NCBI database demonstrated that m70 was in a separate cluster. m70 was resistant to tetracycline, intermediate to erythromycin, and sensitive to ampicillin, corresponding to clinical S. suis isolates in China. The mortality of mice infected with 10(8) CFU of m70 was achieved 60%-80% (3/5-4/5). The mean LD50 of mice infected with m70 was 5.1 x 10(7) CFU, while the mean LD50 of virulent S. suis strain HA9801 was 3.9 x 10(7) CFU. There is no significant difference between the LD50 of the two strains (P < 0.05)., Conclusion: We isolated an S. suis strain from a wild cat, which belongs to the prevalent serotype and was a virulent strain, indicating the potential of transmission of S. suis from wild cats to humans, especially some prevalent serotype strains.

Objective: From the previous comparative genomic analysis, we found a specific unknown 10 kb sequence (including 11 Open reading Frames) in Chinese piscine strain GD201008-001 genome. To study the role of 10 kb in the pathogenicity of piscine S. agalactiae, the 10 kb sequence was deleted from the GD201008-001 genome., Methods: The isogenic mutant Δ10 kb was constructed by using the temperature-sensitive Streptococcus-E. coli shuttle vector pSET4s. We compared the growth characteristics, adherence to HEp-2 cell and bacterial virulence in a zebrafish infection model between wild strain and mutant. Meanwhile the expressions of the known virulence genes from GD201008-001 and Δ10 kb were also quantified by real-time PCR., Results: The Δ10 kb showed no significant differences in bacterial morphology and adherence to HEp-2 cells compared with the wild-type strain, but the speed of growth was slightly slower than the wild strain. Furthermore the 50% lethal dose of Δ10 kb was decreased up to 10-fold (P < 0.001) of the parental strain in a zebrafish infection model, and the expressions of the virulence genes, PI-2b and neul, were significantly increased in the mutant., Conclusion: These findings demonstrated that the 10 kb sequence of piscine Streptococcus agalactiae exerts a significant effect on bacterial virulence and probably regulates the virulence genes expression of GD20 1008-001.

Streptococcus suis (S. suis) is an important zoonosis and pathogen that can carry prophages. In this review, we focus on the recent advances in our understanding of lytic phage and lysogenic phage of S. suis, including the morphology of S. suis lytic phage, the functions of lysin and terminase large subunit encoded by S. suis lytic phage, comparative genomics of S. suis prophages, lysogenic. conversion between S. suis lytic phage and prophage. Furthermore, prospective evolution of interactions between phage and host was discussed.

Objective: The effect of flhDC, fliA, fliD and fliE genes involved in moving of Escherichia coli (E. coli) on the motility of lysogened strain by Stx2-encoding phage phiMin27 was explored by gene knockout and phage lysogenic conversion., Methods: Using the lambda Red recombinase system, the mutant strains of E. coli MG1655 named MG1655 deltaflhDC, MG1655 deltafliA, MG1655 deltafliD and MG1655 deltafliE were constructed. Then the corresponding complemented strains by ligating amplified targeted genes into the low copy vector pUC18 at the BamHI and Hind III sites and transforming these plasmids into mutant strains were acquired. By lysogenic infection of Stx2-encoding phage phiMin27, the lysogens for mutants named MG1655 deltaflhDCphiMin27, MG1655 deltafliAdeltaMin27, MG1655 deltafliDphiMin27 and MG1655 deltafliEphiMin27 were achieved. Subsequently, the motility of wild strain, the mutants, the complemented strains and the lysogens were detected. The changes of expression of the other genes involved in motility between wild strain and the lysogens before and after flhDC deletion by qRT-PCR were analyzed., Results: Lysogenic infection of Stx2-encoding phage phiMin27 could promote the expression of fliA and fliD gene and enhance the motility of MG1655. For flhDC deletion, higher expression of fliA and fliD gene of MG1655 appeared, but the motility had no change. However, lysogen for MG1655 deltaflhDC lost the swimming motility. By gene transcriptional level detection, the expression of fliA and fliD gene of MG1655 deltaflhDCphiMin27 was down-regulated significantly compared with MG1655 deltaflhDC, and no marked variation was observed for fliE gene. The single deletion of fliA, fliD and fliE gene had no effect on the motility of E. coli MG1655 and lysogened strain by Stx2-encoding phage phiMin27., Conclusion: The results show that fliA and fliD gene together participated the regulation for flagella motility and flhDC gene could affect the motility of the lysogened strain by phage. It provides the theoretical basis for further research on the mutual regulation between phage lysogenization and host genes.

Objective: There are one-way or two-way cross-reactions among Streptococcus suis serotype 1, 2, 1/2 and 14, the reason to which was unknown., Methods: The capsular polysaccharides of serotype 14 and 1/2 were purified on Sephacryl S-300 column and identified by phenol-sulphuric acid method and dot-ELISA. The molecular weight of the serotype 14 and 1/2 capsular polysaccharides was revealed as 487.38 kDa and 512.72 kDa by high performance gel permeation chromatography, respectively., Results: The monosaccharide composition of serotype 14 and 1/2 capsular polysaccharides was determined as Glc/Gal/GlcNAc/Rha/Neu5Ac (1: 2.94 : 1.35 : 0.24 : 0.37) and Glc/Gal/GlcNAc/GalNAc/Rha/Neu5Ac (1 : 1.67 : 1.05 : 0.93: 0.72 : 0.7) by pre-column derivatization high performance liquid chromatography, fluorescent labeling HPLC and NMR, respectively. These were compared with the composition of serotype 1 and 2 capsular polysaccharides. Glc, GlcN, Gal and Neu5Ac was contained in the capsular polysaccharides of serotype 1, 2 14 and 1/2. But there is no prominent correlation between the monosaccharide composition and cross-reactions. The cross-reactions among them could be induced by the structure of the capsular polysaccharides and/or the other components on the cell wall.

Objective: The aims of this study were to find out the pathogen causing high mortality of the crawfish population in Anhui over the past years. To provide the strategy for controlling the spread of the disease, an investigation was conducted to trace the origin of the pathogen., Methods: Crawfish samples were tested by nested PCR, to diagnose the White Spot Syndrome Virus (WSSV). Healthy crawfish were inoculated with the supernatant of tissue homogenates. The viral suspension was purified using sucrose density gradients. The gill samples infected with purified virus were then subject to the transmission electron microscopy (TEM). The possible sources, including pond water, feedstuff, crabs, water plants, were investigated., Results: Crawfish samples and commercial feeds exhibited all positive for WSSV. Challenge model on healthy crawfish recovered similar symptoms as the naturally infected ones on 7 to 9 days after inoculation. The viral particles were observed under TEM., Conclusion: Our findings indicated that WSSV was the causative agent that led to high mortality in the crawfish population in this area. These results demonstrated that the exotic viruses are derived from the regions where the frozen feeds were contaminated with WSSV-infected shell debris.

Objective: To learn about the function and gene phylogenetic relationship of the conserved regions in capsular polysaccharides synthesis (CPS) locus,, Methods: based on the CPS locus sequence of Streptococcus suis serotype 1, 2, 7 and 9 and the results to the cross-hybridization experiments, the CPS locus of Streptococcus suis was hypothesized as cassette-like structure which is similar to Streptococcus pneumoniae. PCR, sequencing and southern blotting was used to certify the hypothesis., Results: The CPS locus of Streptococcus suis was cassette-like structure. The 5'-end of the CPS locus contained 4 regulatory genes which are highly similar in all the serotypes. The flanking genes of the CPS locus are conservative. The flanking gene aroA, which is at the downstream to the CPS locus, was selected as the objective gene to develop the PCR to amplify the serotype-specific regions. The orfY, orfX, cpsA, cpsB, cpsC, cpsD and aroA are conserved with high sequence identity in different serotypes.

The study on bacterial capsular polysaccharide is deeper with the development of the molecular biology, saccharide chemistry and immunology. Not only the character and structure of bacterial capsular polysaccharide was researched, but also the genes related to the synthesis, regulation and pathogenicity. This review focuses on the chemical structure, synthesis genes, mechanisms of the diversity, synthesis regulation, function, pathogenicity and application of the bacterial capsular polysaccharide. The research hot spots are also summarized to supply the basic theory and threads to study and apply bacterial capsular polysaccharide.

Objective: We investigated the distribution of autotransporter adhesin gene B11 and other putative virulence genes of APEC IMT5155, which contributed to study the pathogenic mechanism of Avian pathogenic Escherichia coli (APEC)., Methods: PCR and Dot blot were used to detect the distribution of putative virulence genes in E. coli strains isolated from different country (101 Chinese strains and 121 German strains), which were from different sources (human, avian and pig). The association of E. coli phylogenetic group and distribution of putative virulence genes was also analyzed., Results: The putative virulence genes were most prevalent among APEC isolates, 36.4% (32/88) of APEC isolates harboring A1, 53.4% (47/88) for A8, 63.6% (56/88) for A10, 37.5% (33/88) for B11, 59.1% (52/88) for F3. The isolates positive for putative virulence genes were linked to E. coli phylogenetic group B2. Among Neonatal meningitis E. coli isolates, 60%, 80% and 80% were positive for putative virulence genes D1, E9 and F11, respectively. However, all the Neonatal meningitis E. coli strains were negative for B11., Conclusion: Autotransporter adhesin gene B11 and other putative virulence genes were associated to APEC. However, putative virulence genes D1, E9 and F11 were associated to Neonatal meningitis E. coli, which indicated that APEC might be a virulence gene reservoir for Neonatal meningitis E. coli.

Objective: To characterize the genes of Campylobacter jejuni isolates and to genotype the strains from chicken, cow, duck, pig and red-crowned Crane during 2006 to 2008 by multilocus sequence typing (MLST)., Methods: To choose 7 pairs Campylobacter jejuni home-keeping gene, gltA, aspA, glnA, glyA, pgm, tkt and uncA as target gene, PCR was applied to detect 42 samples in East China between 2006-2008. Sequencing, analysis and comparation of the genes were done., Result: Typing the 42 isolates by MLST and finding 24 new sequence typing types., Conclusion: MLST is a useful method to evuluate Campylobacter jejuni genes and gene evolution.

Objective: The enzyme elastase is an important virulent factor of the opportunistic pathogen Aeromonas hydrophila. To know better about the molecular characterization of this enzyme, we purified the elatase and investigated its property and activity., Methods: We cloned a structural gene ahyB encoding for the extracellular elastase of A. hydrophila J-1 and expressed it in E. coli BL21 by using pET-32a as vector. We purified the recombinant enzyme using His Bind Resin Purification Kit and recovered the elastolytic activity of the purified protein by incubation in a buffer containing 6 mol/L guanidine HCl and subsequent removal of denaturant by dilution. In addition, we also purified the native enzyme from the culture supernatant of A. hydrophila J-1 by 30 to 60% ammonium sulfate fractionation, anion exchange chromatography and sephaceryl chromatography. We compared the properties of the two elastase preparations., Results: Native enzyme showed a pH optimum at 8.5, but recombinant enzyme at 10.0. Compared with the native enzyme, the recombinant enzyme was more stable for heat. Both the elastase preparations showed some identical properties concerning inhibitors,which were both mainly inhibited by EDTA, the cation chelator, and OPA, a Zn2+ /Fe2+ protease inhibitor. However,the recombinant elastase had higher tolerance than native enzyme against the inhibitors. And the metal cation Zn2+ and Fe2+ strongly inhibited the enzyme activity., Conclusion: The recombinant product showed the similar enzymatic characteristics as the native enzyme from A. hydrophila J-1 and the two elastases belonged to the zinc-and-iron-dependant metalloendopeptidase. This is the first report on the recombinant expression and subsequent molecular chracterization analysis of A. hydrophila elastase.

Phage endolysin targets the integrate cell wall and attack bonds in the peptidoglycan, resulting in degradation of bacteria. It features two or three domain structures, involving one or two catalytic domains and one binding domain. Endolysin is a promising antibiotic agent against gram-positive bacteria pathogen, such as Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus. Compared to antibiotics, it is more specific and tested bacteria show no resistance to lysin. Therefore, it's a feasible measure for solving drug resistant problem. Beyond this, it is highly active and rapid lysis efficiency and has synergy effect when used together or with other antibiotics. Antibody against endolysin will not neutralize its activity. So endolysin treatment may be a new approach for preventing and controlling of bacteria pathogen.

Pigs may play an important role in the evolution and ecology of influenza A virus. The tracheal epithelium of pigs contain both SA alpha 2,6 Gal and SA alpha 2,3 Gal receptors and can be infected with swine, human and avian viruses, therefore, pigs have been considered as an intermediate host for the adaptation of avian influenza viruses to humans or as mixing vessels for the generation of genetically reassortant viruses. Evolution patterns among swine influenza viruses including evolution of host adaptation, antigenic drift and genetic reassortment, and the latter is the main one. Unlike human influenza viruses, swine viruses have different epizootiological patterns in different areas of world, which is enzootic and geographic dependence. Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2, and these include classical swine H1N1, avian-like H1N1, human-like H3N2, reassortant H3N2 and various genotype H1N2 viruses. In Europe, North America and China, influenza A viruses circulating in pigs are distinct in the genetic characteristics and genetic sources. Since 1979, three subtypes, avian-like H1N1, reassortant H1N2 and H3N2 viruses, have been co-circulating in European swine. Before 1998, classical H1N1 viruses were the exclusive cause of swine influenza in North America. However, after that, three triple-reassortant H1N2, H3N2 and H1N1 viruses with genes of human, swine and avian virus began to emerge in pigs. Genetically, the pandemic viruses emerging in human, so called influenza A (H1N1) viruses, contain genes from both Europe and North American SIV lineages. SIV is not the same as Europe and the United States in the prevalence and genetic background in China, mainly classical swine H1N1 and human-like H3N2 type virus. However, in recent years, SIV from Europe and North America have been introduced into Chinese pig herds, so more attention should be given on the evolutionary of SIV in China. Worldwide, more than 50 cases of SIV infection in human have been documented since the 1970s, which indicate that SIV is also an important zoonosis, and the potential of SIV as human pandemic virus or genes donator. In view of SIV in the importance of ecology, as well as a potential threat to human public health, it is recommended to start as soon as possible regular surveillance, paying close attention to its prevalence and molecular evolution. At the same time, we should establish the surveillance network of the whole influenza virus (including human and animal influenza virus) in China, ecologically mastering the prevalence and evolution of influenza viruses, which is of great significance for the protection of animal health and the prevention of human pandemic.

Objective: The trag (transfer gene G) was one of the novel infection-related factors identified by in vivo-induced antigen technology (IVIAT) from Streptococcus suis type 2 expression libraries with swine convalesecent sera in our former research. We detected the distribution of trag in different Streptococcus suis isolates and identify the differential expression of the new infection-related factor between in vivo and in vitro condition., Methods: According to the sequence of trag of North American strain 89/1591, a pair of primers were designed to detect the distribution of trag in total 43 SS isolates. Another pair of primers were designed to amplify the ORF of trag of 5 SS representive strains (ZY05719, HA9801, 98012, SH040805, SH040917). Partial gene of trag was cloned and inserted into expression vector pET28a(+), and induced by IPTG to express recombinant TRAG. The recombinant protein was probed with swine convalescent sera and immune sera respectively., Results: The trag was detected in the most of SS2 isolates (30/32), in SS9 isolates (4/6), and 1 isolate of SS7, while it was not found in SS2 European strain ATCC43765, avirulent strain SS2 T15, 1 isolates of SS1, 1 isolates of SS1/2 and 2 isolates of group C streptococcal strains from pigs. Comparisons between the sequences of TRAG of 5 isolates with that of SS isolates, showed a high homology (>97%) with North American strain 89/1589 and China strains 98HAH33, 05ZYH33. The immunoreactivity was only presented with convalescent sera., Conclusion: The trag was detected from virulent SS isolates but not from avirulent strain, which suggested that this gene may be related to the pathogenicity of SS. The special reactivity was only present with convalescent sera, and it indicated that TRAG might play a role during SS2 invasive course.

A PCR assay was developed to study the distributional characteristics of phage integrase gene in Streptococcus suis serotype 2 (SS2). A 323bp distinct DNA target can be amplified in 25 strains of virulent SS2, while can not be amplified in avirulent strain T15, 5 strains of other serotypes (SS1, SS7, SS9) and strains of group C Streptococcus strains from pigs, which suggested that the phage integrase gene may be related to the pathogenicity of SS2 and can be consider as a detection factor of the virulent gene of SS2. The sequencing and restriction endonuclease analysis of the PCR products were also done. Comparisons between the sequences of phage integrase gene with that of SS2 strain, showed a high homology with SS2 China strains 98HAH33, 05ZYH33 and North American strain 89-1591. Complete cell lysis was observed with SS2 virulent strains but not with avirulent strain T15 after the induction by mitomycin C. Electron microscopy analysis of the lysate from SS2 virulent strains HA9801 and ZY05719 revealed the presence of phage particles. The induced phage, named SS2-HA and SS2-ZY, both have a small isometric nucleocapsid approximately 50 nm in diameter and have no tail and is therefore a member of the Tectiviridae family. The phage integrase gene sequence of phage SS2-HA and SS2-ZY shared high homologue identities with virulent SS2 strains, which suggested that the phage integrase gene of SS2 has high specify. The temperate phage and phage integrase gene can only detected from SS2 virulent strains but not from avirulent strain, and the detection of phage integrase gene was related to the virulence-associate factors of SS2, such as the muramidase-released protein gene (mrp), which suggested that the temperate phage of SS2 may be related to the pathogenicity of SS2.

The bacterial ghost (BG) system is a novel vaccine delivery system endowed with intrinsic adjuvant properties. Bacterial ghosts are nonliving Gram-negative bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology and native surface antigenic structures. They are produced by PhiX174 protein E-mediated lysis of Gram-negative bacteria, and can induce humoral and cellular immune response, including mucosal immune responses. Plasmid pElysis consisting E gene was transformed into AhJ-1. Through shifting the culture temperature from 28 degrees C to 42 degrees C, A. hydrophila J-1 (pElysis) was induced to lyse and the OD600 value of culture media was measured every 15 minutes during the induction. The lysed bacteria were observed by scanning electron microscopy (SEM). The A. hydrophila ghosts (AHG) used as oral vaccine were also investigated. The OD600 value of A. hydrophila J-1(pElysis) began to decline after 30 min of induction, and after 75 min of induction, the OD600 value decline speed become slowly. The efficiency of ghost induction in non-lyophilized A. hydrophila was 99.99%, 16 hours post induced, no live bacteria can be detected in culture. Scanning electron microscopy observation proved that most lysed bacteria were emptied. Fish vaccination experiments shows that the antibody evoked highest degree after 5 weeks by oral administration of bacterial ghost vaccine and the agglutination antibody titer reached 2(7) and continued two weeks, while the agglutination antibody titer of formalin killed vaccine only reached 26 and only maintained one week. After challenged with the parent strain J-1, the survival rate of bacterial ghost vaccinated fish was higher than the control group and formalin killed vaccine group, the relative percent survival (RPS) was 78.95% (16/20), but the RPS of formalin killed vaccine group was 57.9% (12/20). This suggests that the bacterial ghost vaccine has higher potential to induce protective adaptive immunity than normal vaccine.

Streptococcus suis serotype 2 (SS2) is an economically important, zoonotic agent causing death and disease in both human and swine. According to published SS2 European strain P1/7 complete genomic sequence, primers for detection and amplification autolysin were designed. Autolysin gene was detected by PCR with genomic DNA of HA9801 (Jiangsu isolate), ZY05719 (Sichuan isolate), ATCC43765 (reference strain), other SS2 isolates from different regions, and strains of other different serotypes (such as serotype 1, 1/2, 7 and 9). PCR results showed that all 27 SS2 virulent isolates harbored gene autolysin, but not in non-virulent SS2 strain. Among other serotypes, only serotype 7 strain had this gene. The complete autolysin genes of HA9801 and ZY05719 were respectively amplified by PCR and sequenced. Their putative protein sequences were analyzed through online software, results showed both had six repeated domain "GBS&#95;Bsp-like" and one domain "N-acetylmuramoyl-L-alanine amidase". In addition, sequence similarity analysis demonstrated that autolysins of the two strains (HA9801 and ZY05719) showed high homologue to that of European strain P1/7 (99.8%), but obviously differed from Canada strain 89/1589, the latter lacked one domain "GBS Bsp-like". Software DNASTAR was used to analyze autolysin protein sequence and predicted its putative antigenicity. Then partial antigentic segment of autolysin was amplified, cloned and inserted into expression vector pET30a(+), and induced by IPTG to express recombinant autolysin. Its reactivity was analyzed by SDS-PAGE and western blot with swine convalescent sera, blot result showed that the recombinant protein had good reactivity and could be considered as a vaccine candidate.

Invasion of Aeromonas hydrophila strain Ah J-1 isolated from diseased fish to cultured HEp-2cells monolayer was evaluated by the recovery of gentamincin-resistant (Gm') bacteria from Triton X-100 cell lysates. The invasive efficiency (IE) could reach to 0.2% at 37 degrees C. In addition, potential signal transduction pathways that precede bacterial internalization were studied by using signal transduction inhibitors. Bacterial internalization of Ah J-1 involved microfilaments and protein tyrosine kinase since cytochalasin D (an inhibitor of microfilament polymerization) and genistein (an inhibitor of protein tyrosine kinase) prevented internalization. Staurosporine (a protein kinase C inhibitor) and Sodium orthovanadate (a protein tyrosine phosphatase inhibitor) accelerated internalization of Ah J-1 into HEp-2 cells.

Aeromonas hydrophila (Ah) biofilm (BF) model in vitro was established and antibiotic effects of 11 antimicrobials on BF and free-cell(FC) bacteria were tested. After the Ah J-1 on the surfaces of the silicon pieces was incubated for 7 days in TSB, the intact BF was formed by detection of AgNO3 solution staining. The FC bacteria were resistant to Penicillin but susceptible to Enrofloxacin and Fulgram. Their minimum bactericidal concentrations (MBCs) were 256 microg/mL, 0.03 microg/mL and 0.25 microg/mL respectively. Florfenicol had a strong antibacterial effect on BF bacteria and the ratio of MBC between BF and FC bacteria was 2 to 1, but more than 32 to 1 occurred for Kanamycin, Penicillin and Neomycin. Morphology and structure of BFs with/without the treatment of Enrofloxacin were observed under scanning electron microscope, while the bactericidal curve was detected. Enrofloxacin could eradicate the FC bacteria completely but not the BF bacteria at a concentration of 4 fold MBC. The 32 fold MBC Enrofloxacin could entirely kill the FC bacteria in 4h, but in 24h for BF bacteria. The result suggested that BF enabled Ah to have strong resistance to antimicrobials and the potential influence of BF should be highly considered.

The haemolysin of Streptococcus suis type 2 Jiangsu isolate was purified. Effects of protein modification reagents on the haemolysin activity were detected. The haemolysin was not affected by PCMB, succinic anhydride (SA), EDC, and N-AI modification, and it indicated that sulfhudryl groups, amino groups, carboxyl groups and tyrosine residues were not essential to haemolysin activity. The haemolysin activity was significantly decreased after NBS, DEPC, 2, 3-Diacetyl, H2O2 modification and was greatly increased after DTT modification. The results indicated that tryptophane residues, histidine residues, arginine residues and disulfides groups seemed to be essential to the haemolysin activity.

The pathogenic role of muramidase released protein (MRP), a virulence factor of Streptococcus suis type 2 (SS2) is poorly understood. The purified MRP was co-incubated with HEp-2 cells to determine the effect of MRP on epithelial cells. Under light microscope, two principal morphologic changes were observed. Firstly, the cells were fused to form syncytia and a apoptosis followed. Secondly, single cell was also induced to apoptosis at high level as 18%, which was verified by transmission electron microscopy and flow cytometry. It showed that MRP alone was capable of a virulence factor of SS2.

The genes encoding precursor heat-stabile (pro-ST) and mature peptide of B unit of heat-labile (LT) enterotoxins of Escherichia coli from piglet were amplified by polymerase chain reaction (PCR). The 3' terminus of gene encoding pro-ST was genetically fused to the 5' terminus of the LTB subunit gene in nest-PCR. The fusion genes encoding pro-ST and LTB were cloned into pGEM-T vector and subcloned into the pQE30. The recombinant plasmid was expressed in E. coli by IPTG induction. The fusion protein possessed both ST and LTB antigenicity, as well as it had lost the biological toxicity of ST or LT toxin.

Virulence-related proteins, muraminidase-released protein (MRP) and extracellular factor (EF) of Streptococcus suis type 2, were extracted from Jiangsu Isolate HA9801 and were used as antigens for preparation of antibodies. Bacterium cell envelope proteins and extracellular proteins of swine Streptococcus strains including 17 Chinese isolates, 1 German strain and 1 human isolate of Streptococcus suis type 2, were analyzed by SDS-PAGE and immunoblotting using the above antibodies, 11 strains produced MRP and 10 strains possessed EF or EF. They exist four phenotypes: MRP+ EF+ (8/19), MRP+ EF (1/19), MRP+ EF- (1/19), MRP- EF- (10/19).