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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_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.

A total of 36 streptococcal strains, including seven S. equi ssp.zooepidemicus, two S. suis type 1 (SS1), 24 SS2, two SS9, and one SS7, were tested for glyceraldehyde-3-phosphate dehydrogenase gene (gapdh). Except from non-virulent SS2 strain T1 5, all strains harboured gapdh. The gapdh of Chinese Sichuan SS2 isolate ZY05719 and Jiangsu SS2 isolate HA9801 were sequenced and then compared with published sequences in the GenBank. The comparison revealed a 99.9 % and 99.8 % similarity of ZY05719 and HA9801, respectively, with the published sequence. Adherence assay data demonstrated a significant ((p<0.05)) reduction in adhesion of SS2 in HEp-2 cells pre-incubated with purified GAPDH compared to non pre-incubated controls, suggesting the GAPDH mediates SS2 bacterial adhesion to host cells.

An RT-nPCR assay was used for testing fecal samples of dogs, foxes, raccoon dogs and minks for the presence of canine coronavirus (CCV). The animals were raised in homes, dog schools or farms. Seventy out of 81 healthy dog feces from three cities and 21 out of 48 diarrhea feces from pet dogs were positive for type II CCV. From a total of 61 healthy fox feces, 43 were positive for type II and 29 for type I CCV, out of which 25 were simultaneously positive for the two different genotypes. Among 24 raccoon dogs samples, 22 were CCV type II-positive, and from those 16 were additionally type I positive. No CCVs was detected from healthy mink feces. Sequence analysis found that ten type II CCVs fragments of M gene shared a high similarity with reference strain CCV 1-71 (96.5-99.5%), and four type I CCVs shared a high similarity (96.7%-98.1%) with a reported FCV-like CCV strain. The sequence of one particular M gene fragment was found to cluster between the type I and type II CCV branches in phylogenetic analysis, suggesting the existence of a novel strain. Our study confirmed that type II CCVs infection is very common in domestic dog, fox, and raccoon dog populations in China. This is also the first report on the co-existence of two CCV genotypes in healthy foxes and raccoon dogs.

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).