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Aeromonas hydrophila isolates from clinical cases (n=43) were tested against 8 antimicrobial agents and typed by outer membrane protein (OMP) pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin (MICs, ≥16 μg mL−1) and sulfamonomethoxine (MICs, ≥64 μg mL−1), but susceptible to norfloxacin (MICs, ≤0.5 μg mL−1). There was a high incidence of resistance to erythromycin (90.70%) and tylosin (93.02%), while a low incidences of resistance to ciprofloxacin (2.33%), enrofloxacin (2.33%) and florfenicol (4.65%). Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.

LuxS/AI-2 quorum sensing (QS) system involves the production of cell signaling molecules vialuxS-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 inStreptococcus 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 aluxSmutant 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 overexpressedluxSwas constructed to comprehensively understand the function of AI-2. OverexpressedluxSwas not able to increase the level ofpfsexpression 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 withluxStranscription.luxSexpression is constitutive, but the transcription ofpfsis perhaps correlated with AI-2 production in SS.

Streptococcus suis is a Gram-positive pathogen that causes serious diseases in pigs. In addition to S. suis serotype 2 (SS2), S. suis serotype 9 (SS9) is another prevalent serotype, which is frequently isolated from the organs of diseased pigs in China. An immunoproteomic-based approach was developed to identify antigens common to SS2 and SS9 for vaccine development. Cell wall proteins extracted from SS2 strain HA9801 were screened by two-dimensional Western blot using anti-SS2 sera, anti-SS9 sera, or pre-immune sera pooled from specific pathogen free (SPF) mice. Protein spots on preparative gels were excised and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, which led to the identification of four shared immunogenic proteins (arginine deiminase, translation elongation factor-Ts, o-acetylserine lyase, and 1-phosphofructokinase). The genes encoding these four proteins from SS9 strain GZ0565 were cloned and their proteins were overexpressed in Escherichia coli BL21. Western blot analysis of these recombinant proteins using the convalescent serum of an SPF mini-pig inoculated with the SS2 strain, anti-SS2 sera, and anti-SS9 sera pooled from SPF mice further confirmed the immunogenicity of these proteins. These immunogenic proteins, which are encoded by genes that are reasonably conserved among SS2 and SS9 strains, could be developed as vaccine candidates.

Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) is a zoonotic pathogen with worldwide distribution. Lacking suitable vaccine and virulent maker is still bottleneck to control this infection. An immunoproteomic approach has been used to screen the membrane-associated and cell wall-associated proteins of S. zooepidemicus isolate in China CY to discover vaccine candidate antigens and therapeutic agents. Finally, 11 membrane-associated proteins, and 13 cell wall-associated proteins were successfully identified. BLAST ( www.sanger.ac.uk ) results also indicated that nucleotide sequences of majority identified proteins shared high homology (>60%) with S. zooepidemicus, except for AC1-3, AC5, AC8, and AC 13. Moreover, genes for 7 of the identified proteins were detected from C Y; compared with ST 171, 3 of them (AMI, AM8 and AC11) were only found in virulent strains (CY). All of the proteins identified in this study remain not to be reported in S. zooepidemicus. Some of the proteins serve a vital role in the immune system and reproduction of host species according to available data, while the functions of the rest were seldom researched.

The invasion pathway of Aeromonas hydrophila in vivo was studied in Crucian carp (Carassius auratus gibelio) with a virulent strain A. hydrophila J-1 transformed with a plasmid encoding green fluorescent protein (pGFPuv) (A. h J-1GFP), which had similar virulence characteristics (haemolysin, extracellular proteases production, toxicity for EPC cells, survival in fish serum and LD50 value) as the parent strain. Fish were divided into four experimental groups: (1) normal fish; (2) bacteria bath challenged, unwounded fish; (3) skin artificially wounded by scalpel, bacterial bath challenged fish; and (4) skin mucus layer partially removed by paper towel, bacterial bath challenged fish. The number of bacteria from blood, gills, kidney, muscle, liver and intestine were detected at 2, 4, 8, 12, and 24 h post-challenge. High bacterial numbers were observed in the muscle of the artificial wound group, in the kidney of the mucus removed group and in the gills of all groups. In conclusion, the gills and damaged skin are likely to be the main routes of entry for A. hydrophila, and GFP can be used as a real-time biomarker to study intimate host–pathogen interaction in fish.

Summary Objective Our recent study demonstrated that sodium bicarbonate improved cardiac function in macaque models with early-phase endotoxic shock. In the present study, we investigated further the ryanodine receptor/calcium release-channel (RyR) and calcium pump after fluid resuscitation of macaques with early-phase endotoxic shock. Methods Twenty-four anaesthetised macaques were assigned to four groups. Nineteen animals were given an intravenous dose of 2.8 mg kg −1 lipopolysaccharide (LPS). Sixty minutes after the LPS challenge, the animals were given (i) 5 mL kg −1 normal saline (Ns group, n = 6), (ii) 5 mL kg −1 of 5% sodium bicarbonate (Sb group, n = 6) or (iii) 5 mL kg −1 of 3.5% hypertonic sodium chloride (Hs group, n = 7). The control group (Co group, n = 5) received 1 mL kg −1 normal saline and then with 5 mL kg −1 normal saline 60 min later. Results Endotoxin produced a reduction of the density of RyR but did not alter the affinity of RyR. Compared with normal saline, sodium bicarbonate or hypertonic saline induced a restoration of density of RyR but did not influence the affinity of RyR and the calcium pump. Conclusion Up-regulation of RyR performance in myocardium following administration of sodium bicarbonate contributes to the improvement of cardiac function in macaques in the early phase of endotoxic shock.

These studies established a macaque model of early-phase endotoxic shock, and investigated the resuscitation effects of three different solutions. Twenty-four macaques were assigned to four groups. Nineteen animals were given an intravenous dose of 2.8 mg/kg lipopolysaccharide (LPS). At 60 min after LPS challenge, the animals were given (i) 5 mL/kg normal saline (Ns group, n = 6), (ii) 5% of 5 mL/kg sodium bicarbonate (Sb group, n = 6), (iii) hypertonic 3.5% sodium chloride of 5 mL/kg (Hs group, n = 7). The control group (Co group, n = 5) was first injected with 1 mL/kg Ns and with 5 mL/kg Ns 60 min later. Haemodynamic parameters and blood gases were measured during the experiment, and myocardial morphology was examined on termination of the experiment. Administration of LPS caused hypotension and decreases of the left ventricular work index (LVWI). In the Sb group, mean arterial pressure, cardiac index, systemic vascular resistance index, LVWI and right ventricular work index were significantly higher than those of the Ns group. Pathological changes of myocardium were identified in all of the LPS groups. The studies suggest that macaques are suitable models for studying endotoxic shock and potential fluid therapies.

Serratia marcescens jn01 was employed as the host for the isolation of phages from environmental sewage. One strain of phage named SmPjn was purified by picking transparent plaque with 2mm diameter and clear edge on the double-layer agar repeatedly. Electron micrographs indicated that the phage head was icosahedral with head size and tail length of (58 +/- 2.16) x (55 +/- 0.47) nm and (7 +/- 1.25) nm, respectively. On the basis of the morphology, this phage belongs to the family Podoviridae. Host-range determination revealed that the phage was capable of infecting the other two isolates of S. marcescens, P25 and CMCC41002. The optimal multiplicity of infection was 1. A one-step growth curve of SmPjn indicated that the latent period and burst size were estimated at 50 min and 1,125 pfu/cell, respectively . Genomic DNA of SmPjn was above 27kb in size and could be digested by Hind Ill and EcoR I into 11 and 9 visible fragments after electrophoresis, respectively. A novel Podoviridae-phage infecting S. marcescens was firstly reported in China.

To explore development of a vaccine against PCV2 infections, the gene of capsid protein (Cap) was inserted into the swinepox virus (SPV) genome by homologous recombination. The recombinant swinepox virus expressing capsid protein (rSPV-cap) was verified by PCR, western blot and immunofluorescence assays. To evaluate the immunogenicity of rSPV-cap, twenty-four PCV2 seronegative minipigs were immunized with rSPV-cap, wild type SPV (wtSPV; negative control), or PBS (challenge control). After inoculation with PCV2, pigs in the rSPV-cap immunized group showed significantly higher average daily weight gain (ADG) and shorter fever duration compared with the wtSPV treated group (P

Streptococcus equi subsp. zooepidemicus is an opportunistic pathogen. It has caused a very large economic loss in the swine industry of China and has become a threat to human health. We announce the complete genome sequence of S. equi subsp. zooepidemicus strain ATCC 35246, which provides opportunities to understand its pathogenesis mechanism and genetic basis.

Aeromonas hydrophila is a motile bacterium present in numerous freshwater habitats worldwide and is frequently the cause of infections in fish and numerous terrestrial vertebrates including humans. Because A. hydrophila is also a component of the normal intestinal flora of healthy fish, virulence mechanisms are not well understood. Considering that fish models used for the examination of A. hydrophila genes associated with virulence have not been well defined, we established an infection model using the free-living, ciliate protozoa Tetrahymena thermophila. The expression of A. hydrophila virulence genes following infection of T. thermophila was assessed by reverse transcription-PCR and demonstrated that the aerolysin (aerA) and Ahe2 serine protease (ahe2) genes (not present in the avirulent A. hydrophila NJ-4 strain) in the virulent J-1 strain were upregulated 4-h postinfection. Furthermore, the presence of intact A. hydrophila J-1 within T. thermophila suggested that these bacteria could interfere with phagocytosis, resulting in the death of the infected protozoan 48-h postinfection. Conversely, A. hydrophila NJ-4-infected T. thermophila survived the infection. This study established a novel T. thermophila infection model that will provide a novel means of examining virulence mechanisms of A. hydrophila.

The ibeA gene is located on a genomic island, GimA, which is involved in the pathogenesis of neonatal meningitis Escherichia coli (NMEC) and avian pathogenic E. coli (APEC). The prevalence of ibeA in the APEC collection in China was investigated, and 20 of 467 strains (4.3%) were positive. In addition, analysis of the association of the E. coli reference (ECOR) groups with positive strains revealed that ibeA was linked to group B2. The ibeA gene in DE205B was analyzed and compared to those of APEC and NMEC, which indicated that the specificity of ibeA was not consistent along pathotypes. The invasion of chicken embryo fibroblast DF-1 cells by APEC DE205B and RS218 was observed, which suggested that DF-1 cells could be a model to study the mechanism of APEC invasion. The inactivation of ibeA in APEC DE205B led to the reduced capacity to invade DF-1 cells, defective virulence in vivo , and decreased biofilm formation compared to the wild-type strain. In addition, strain AAEC189 expressing ibeA exhibited enhanced invasion capacity and biofilm formation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) analysis and animal system infection experiments indicated that the loss of ibeA decreased the colonization and proliferation capacities of APEC in the brain during system infection.

To construct a gene knock-out mutant of response regulator named RevS in Streptococcus suis serotype 2 virulent strain 05ZYH33, and to investigate the effects of its deletion on the biological characters of this pathogen and the pathogenesis to mice and piglets.Recombinant gene knock-out vector consisting of Spc(r) cassette was constructed and flanking was constructed consisting of Spc(r) cassette with flanking homology regions to the RevS genes while the isogenic RevS-deficient mutant was screened by allelic replacement. The effects of RevS deletion on the basic biological characters of 05ZYH33 including growth stability, colonial morphology, haemolysis, Gram staining, growth curve and protein expression were examined in vitro. The mice and piglets were infected with 10(8) CFU wild virulent and mutant isolates.PCR analysis confirmed that the coding genes of RevS were replaced completely by Spc(r) cassette and the basic biological characters of 05ZYH33 did not undergo any apparent change. Balb/c mice infection assay indicated that RevS play a role in the pathogenesis of Streptococcus suis infections, while no remarkable difference was observed in the piglets' pathogenesis infection rates between mutant isolates deltaA05ZYH33 and wild-type isolates 05ZYH33.The mutant of Streptococcus suis 05ZYH33 response regulator was successfully constructed, while the mutation did not obviously affect the bacterial biological characters, while the knock-out mutant of RevS was shown to be attenuated in pathogenesis to mice and piglets.

By using suppression subtractive hybridization (SSH) and cDNA microarrays, we studied the differentially expressed genes in haemocytes of the crayfish (Procambarus clarkii) infected with white spot syndrome virus (WSSV). Thirty three differentially expressed genes were detected in which 31 were up-regulated and 2 were down-regulated. The up-regulated genes include serine protease inhibitors, chaperonin, synaptasome-associated protein of 25 kD(SNAP25), tubulin, zinc-finger protein, intracellular fatty acid binding protein, extracellular superoxide dismutase precursor, arginine kinase, 70 kD heat shock like protein and Bax inhibitor-1. A lot of genes including the 2 down-regulated genes are still unknown. All these immuno-related genes responding to the virus infection provide a new insight for further study in the shrimp innate immunity.

An immunogenic protein, named HM3, of Streptococcus suis type 2 HA9801 was identified by using immunoproteomic assay. Some characters of this protein were analyzed by several online bioinformatical tools, including BLAST, SinglP, HMMTOP and PSORTb. The most homologous sequence of HM3 was extracellular solute-binding protein (gi/69246104) of Enterococcus faecium. The predictions results showed that HM3 contained Signal peptide and one transmembrane region, and Non-Cytoplasmic Localization were also predicted. Partial gene of this protein were amplified from the genome of HA9801 by PCR and inserted into expression plasmid pET-32a (+) after double digested by BamH I and Sal I, then transformed into BL21 (DE3) where they were induced to express by IPTG. After induced, there was specific proteins band of approximately 45kDa on the SDS-PAGE gel. Western-blotting showed that recombinant protein could react with immune serum of HA9801 of SPF (Specefic pathogen Free) mini-swine. This protein could be taken as vaccine candidate of SS2.

According to the conservative sequence in the epitaxial variable region of Thailand strain of WSSV published in GenBank,a pair of primers were designed to amplify the variable region genes of 5 local WSSV strains (ZHSH, ZHJ, HN, QD1, QD2) by PCR and then cloned. In accordance with the CN, the results indicated that the number of nucleotide of 5 strains were deleted more than 591bp of the TW and TH strains. The ZHSH and HN strains that deleted 591bp at the 3' end, and 454 bp at the 5' end of variable gene was highly homologous with CN strain about 99.3%. 229bp of ZHJ strain at the 5' end was homologous with CN about 99.3%, and deleted of 816bp at the 3' end. 97bp at the 5' end and 171bp at the 3' end of QD1 and QD2 strains were homologous with CN strain about 99.3%, and about 777bp were absent in the middle. The above data showed that the variable region genes of WSSV had mutated more in China. The variable region gene of QD2 strain was coincidence with that of QD1 after propagating 10 generations in crayfish. The results indicated that the crayfish inoculation did not result in mutation of variable region genes of WSSV.

PCR analysis demonstrated the presence of the ad gene in all 29 S. suis strains tested, but none of the seven S. equi subsp. zooepidemicus strains. The fragment of ad gene of virulent isolate SS2-HA9801 was later cloned into pBAD/Myc-HisC vector via restriction endonuclease and then transformed into host strain TOP10. A recombinant protein of 47000Da was highly expressed after induced by L-ararose and purified by Ni-nitrilotriacetic acid affinity chromatography. Western blotting demonstrated that the recombinant protein can reacted to the polyclonal antibody raised against whole-cell protein of SS2-HA9801, which suggested that it possessed some immunogenicity and may be important for further research. Enzymatic assay revealed that the optimum temperature for its activity is 37 degrees C and pH is 6.5. Studies with class-specific inhibitors supported the assignment of a sulfhydryl enzyme with some metallo class characteristics.

Streptococcus suis type 2 (SS2) is a porcine zoonotic pathogen with worldwide distribution, and lacking suitable vaccine and virulent maker were bottleneck to control this infection. An immunoproteomic assay was used to identify antigenic proteins from the total extracellular proteins of the virulent Chinese SS2 strain ZY05719. The convalescent serum of a specific pathogen free (SPF) mini-pig recognized nine protein spots on PVDF membrane. Antigenic proteins on a duplicate gel, as well as those with a similar placement of extracellular proteins from another virulent strain (HA9801) and an avirulent strain (T15) on 2-D gels, were excised and identified by MALDI-TOF-MS. PMF of the protein spots were performed using the MASCOT server. Two proteins were found in all three strains. Comparative proteomic analysis between the two virulent strains and the avirulent strain revealed nine differential proteins, eight of which were successfully identified. Genes for six of the differentially expressed proteins were found in both virulent strains, and of those were present in the avirulent stain.

Streptococcus equi subsp. zooepidemicus belongs to lancefield group C streptococcus, which can cause disease both in animals and humans. It has been associated with a wide variety of serious infections, including meningitis, pneumonia, septic arthritis and mastitis. The M like proteins on the surface of S. equi subsp. zooepidemicus have an antiphagocytic role analogous to that of group A streptococcal M proteins that are essential in establishing infection. In the present study, the M-like gene without partial signal peptide sequence was amplified from genomic DNA of S. equi subsp. zooepidemicus ATCC35246 strain isolated from pig by polymerase chain reaction (PCR). Then the amplified fragment was cloned in the proper orientation into the site between EcoR I and Xho I of pET32-a(+) via restriction endonuclease EcoR I and Xho I. The recombinant plasmid was verified by restriction endonuclease analysis and nucleotide sequencing, then transformed into E. coli BL21. An fusion protein was expressed in BL21 after induced by IPTG, SDS-PAGE analysis showed that the recombinant protein had a molecular weight of 60 kD, Western blotting showed a positive reaction with the antiserum against ATCC35246. To prepare the monoclonal antibodies (McAbs) against the M-like protein, 6-8 weeks old BABL/c mice were immunized endermicly with purified recombinant M-like protein by Ni-nitrilotriacetic acid affinity chromatography. Splenocytes from the immuniszed mice were fused with SP2/0 and indirect ELISA was used to screen hybridoma cells. 12 hybridoma cell lines secreting McAbs against M-like protein of Streptococcus equi subsp. zooepidemicus were generated, and indirect ELISA confirmed that these McAbs only reacted with M-like protein, but not reacted with other bacteria such as group A Streptococci, Streptococcus suis type 2, Streptococcus equi. The indirect ELISA titers of these 12 ascites McAbs were about 2.56 x 10(4) to 1.01 x 10(5) , and the subtype of these McAbs belong to IgG2b, IgG1, IgM. The results of adhersion inhibition showed McAbs 2C8 could inhibit the adhersion of M-like protein to HEp-2 cell.

Given the lack of effective vaccines to control Streptococcus suis infection and the lack of a rapid and reliable molecular diagnostic assay to detect its infection, S. suis serotype 2 was sequenced partly in an effort to identify important virulence factors. Two new open reading frames were found located between orf2 and mrp. One of new open reading frame (2738 - 3694) that encoded a polypeptide of 319 amino acid residues with a calculated molecular mass of 33.5kDa was identified by Western blot. GenBank database search revealed that the derived amino acid sequence shared low homology with sequences of known function from other genes. Second structure was analyzed by InterPro, PHD, DNAstar software, the deduced protein had functional domains typical of IMP dehydrogenase (IMPDH). The PCR product of the open reading frame was transformed into E. coli BL21 and the fusion protein of 48kDa was expressed. The recombinant protein was reactive with serum from pigs experimentally infected with virulent strains of S. suis type 2, suggesting that the protein is immunogenic. IMPDH activity staining confirmed that the protein has IMPDH function and can catalyze the rate-limiting reaction of GTP biosynthesis, the NAD-dependent reduction of IMP into XMP. Flow cytometry (FCM) revealed that the protein had apparent effect on HEp-2 cell cycle.

Background Our previous study has established a macaque model with early-phase endotoxic shock. The present study further investigated myocardial and blood vessel injury in Macaques by examining the subsequent expression of ACP, selectins, iNOS, and cTnI in response to LPS treatment. Methods In an experiment with anaesthetised, instrumental macaques, eleven animals were randomised into: an En group (n = 6), receiving a dose of 2.8 mg kg−1 lipopolysaccharides (LPS) by i.v.; and a Co group (n = 5), injected with normal saline of 1 ml kg−1. Cytochemistry of acid phosphatase (ACPase) in heart was performed by electron microscope at 120 min following endotoxin injection. Three immunochemical stains, namely, L-selectin, P-selectin and iNOS protein in heart, were studied. In addition, cardiac troponin I (cTnI), L-selectin and P-selectin in plasma were detected. Results In the early phase of endotoxic shock, LPS caused myocardial lysosome damage. The data of immunochemical staining showed the thrombus formation in vessels and the increase of iNOS, L-Selectin and P-Selectin expression in heart, but LPS challenge did not change L-selectin, P-selectin and cTnI in plasma. Conclusion The increase of iNOS, L-selectin and P-selectin protein expression following endotoxin administration may have caused vessel injury and myocardial damage in macaques.

To rapidly and sensitively detect the four virulence-associated factors of Streptococcus suis, a multiplex PCR was developed.In the process of this reaction, four distinct DNA targets were amplified. One target was based on the serotype 2 (and 1/2) specific cps gene and the others were based on Streptococcus suis mrp, epf (epf*) and sly gene, encoding the MRP, EF(EF*) and Sly proteins of Streptococcus suis. 72 isolates, which including 48 strains of Streptococcus suis and 24 strains of negative control, and 49 clinical specimens were detected by the multiplex PCR assay.All PCR products were detected by electrophoresis on 1.2% agarose gels. With the 48 Streptococcus suis strains, the positive detection rates of cps2+, mrp+, epf+, epf*+ and sly+ were 16/48, 14/48, 12/48, 3/48 and 26/48,respectively. The results were confirmed by bacteriological examination. There were no specific amplification products including 49 clinical specimens and 24 negative control strains.The results demonstrated that multiplex PCR was a highly specific and sensitive diagnostic tool for the detection of virulence-associated factors of streptococcus suis.

Bacterial 16S rDNA library of Siberian tiger was developed and 15 different clones were obtained using EcoR I and Hind III in restriction fragment length polymorphism analysis. DNA sequencing and similarity analysis showed that 10 clones matched corresponding Clostridium sequences, of which 6 sequences had over 99% similarity with Clostridium novyi type A, and 4 sequences had 97% similarity with Swine manure bacterium RT-18B, which identified as Peptostreptococcus spp. The other five 16S rDNA sequences had 94% - 95% similarity with Clostridium pascui, Clostridium tetani E88, Clostridium sp. 14505 Clostridium perfringens and Carnobacterium sp. R-7279 respectively.

Fusion expression plasmid pFBPS, pFBPS (7 - 82), pFBPS (7 - 165) and pFBPS3 (87 - 320) of Streptococcus suis type strain HA9801 were generated by cloning different fragments of fbps amplified from pMD-T-FBPS by PCR into plasmid pET-32a(+). It has been confirmed that the recombinant proteins, FBPS, FBPS(87 -320), and FBPS(7 - 167), which are expressed by recombinant plasmid pFBPS, pFBPS(87 - 320) and pFBPS(7 - 165), respectively, bound human fibronectin by ligand affinity Western blot assay. The results indicate the primary fibronectin-binding domain of FBPS lies within 87 - 165 amino acid residues region.

A number of 65 Streptococcus species isolated from diseased pigs were surveyed for in vitro minimal inhibitory concentration (MIC) to 32 drugs by microdilution test. Clinical categorization of isolates as susceptible or resistant was based on MIC interpretative standards of the National Committee for Clinical Laboratory Standards(NCCLS). None of the strains were susceptible to all antimicrobial compounds tested and most strains (96.6%) were multidrug-resistant. High level resistance to streptomycin was also detected. Resistance to sulfonamides (92.3% - 98.3%), aminoglycosides (70.8% - 78.5%), lincosamides (66.2% - 64.6%), tetracyclines (72.3%) and macrolides (53.8% - 67.7%) were most frequent, then beta-lactam (18.5% - 56.9%) and quinolones (36.9% - 78.5%) resistance, while all strains were susceptible to florfenicol, 78.5% strains were susceptible to Taimulin. All resistant strains to beta-lactam were tested for beta-lactamase production and the results were negative.

61 fecal samples from healthy foxes and 24 from healthy raccoon dogs were examined by RT-nested PCR assays for the presence and genotypic identification of Canine coronaviruses (CCVs). 77.0% fox samples were recognized as CCV positive, 43 of which belonged to type II and 29 to type I, as well as both genotypes were simultaneously detected in 25 samples. Out of the total 24 fecal samples from raccoon dogs, 22 were CCV positive for type II and 16 for type I. M gene fragments of 8 samples were sequenced, 4 of which were confirmed as CCV type I and the other 4 as CCV type II. Sequence analysis showed that the M gene of CCV type I had a high similarity of 96.7% - 98.1% between the fox-and raccoon dog strains and the reported Italian strain from diarrhea dogs. The two genotypes, with an identity of 88.3% - 89.7%, formed two separate branches in phylogenetic tree. Interestingly, the sequence at several nucleic acid sites of CCV type II differed between foxes and raccoon dogs. The co-existence and popularity of the two CCV genotypes in healthy foxes and raccoon dogs were first confirmed in this article.

Cell conjugation was carried out between the donor E. coli CG120 with pAM120 (Tc(r)/Tn916) and the recipient Aeromonas hydrophila J-1 by filter mating. 3800 positive clones were gained according to the ability of growth on LB medium with 10 g/mL tetracycline (Tc) and 100 g/mL cephazolin (cfz). Conjugation efficiency was 3 x 10(-5) per donor. When 16S rDNA PCR amplification was performed with special primers, positive results were gained in all the 38 conjugants. To demonstrate the insertion of Tn916 into the genomes of conjugants, tetracycline gene (tet) PCR amplification was performed. A special band could be gained in the conjugants with Tc(r). Compared with the parent J-1 strain, some genes of main virulence determinants, such as proteases, hemolysins, DNase and amylases, could not be expressed in the conjugants. The pathogenicity capability of conjugants was greatly decreased and the 50% lethal dose for mice was more than 10(9) CFU. By serially passaged for 10 times, the above virulent characters had not been regained despite the disappearance of Tc(r). The results showed that by the insertion of Tn916, stable and avirulent A. hydrophila mutants could be gained. The mechanisms, by which Tn916 induced the changes of virulence characters in A. hydrophila, are not definite. It is suspected that there might exist hot spots of Tn916 or pathogenicity island on the chronosome of A. hydrophila.