Identification and characterization of a Streptococcus equi ssp. zooepidemicus immunogenic GroEL protein involved in biofilm formation.

Streptococcus equi ssp. zooepidemicus (S. equi spp. zooepidemicus) is an opportunistic pathogen that causes major economic losses in the swine industry in China and is also a threat for human health. Biofilm formation by this bacterium has been previously reported. In this study, we used an immunoproteomic approach to search for immunogenic proteins expressed by biofilm-grown S. equi spp. zooepidemicus. Seventeen immunoreactive proteins were found, of which nine common immunoreactive proteins were identified in planktonic and biofilm-grown bacteria. The immunogenicity and protective efficacy of the S. equi spp. zooepidemicus immunoreactive GroEL chaperone protein was further investigated in mice. The protein was expressed in vivo and elicited high antibody titers following S. equi spp. zooepidemicus infections of mice. An animal challenge experiment with S. equi spp. zooepidemicus showed that 75% of mice immunized with the GroEL protein were protected. Using in vitro biofilm inhibition assays, evidence was obtained that the chaperonin GroEL may represent a promising target for the prevention and treatment of persistent S. equi spp. zooepidemicus biofilm infections. In summary, our results suggest that the recombinant GroEL protein, which is involved in biofilm formation, may efficiently stimulate an immune response, which protects against S. equi spp. zooepidemicus infections. It may therefore be a candidate of interest to be included in vaccines against S. equi spp. zooepidemicus infections.

The effects of upaB deletion and the double/triple deletion of upaB, aatA, and aatB genes on pathogenicity of avian pathogenic Escherichia coli.

Autotransporters (ATs) are associated with pathogenesis of Avian Pathogenic Escherichia coli (APEC). The molecular characterization of APEC ATs can provide insights about their relevance to APEC pathogenesis. Here, we characterized a conventional autotransporter UpaB in APEC DE205B genome. The upaB existed in 41.9 % of 236 APEC isolates and was predominantly associated with ECOR B2 and D. Our studies showed that UpaB mediates the DE205B adhesion in DF-1 cells, and enhances autoaggregation and biofilm formation of fimbria-negative E. coli AAEC189 (MG1655Δfim) in vitro. Deletion of upaB of DE205B attenuates the virulence in duck model and early colonization in the duck lungs during APEC systemic infection. Furthermore, double and triple deletion of upaB, aatA, and aatB genes cumulatively attenuated DE205B adhesion in DF-1 cells, accompanying with decreased 50 % lethal dose (LD50) in duck model and the early colonization in the duck lungs. However, DE205BΔupaB/ΔaatA/ΔaatB might "compensate" the influence of gene deletion by upregulating the expression of fimbrial adhesin genes yqiL, yadN, and vacuolating autotransporter vat during early colonization of APEC. Finally, we demonstrated that vaccination with recombinant UpaB, AatA, and AatB proteins conferred protection against colisepticemia caused by DE205B infection in duck model.

Development of real-time PCR assay for detection of porcine circovirus-like virus P1 in domestic pigs in China.

BACKGROUND: The porcine circovirus-like agent P1 is a newly discovered DNA virus with a single-stranded circular genome that is highly homologous to that of porcine circovirus type 2. P1 infection can cause symptoms resembling postweaning multisystemic wasting syndrome. This study aims to develop a rapid, sensitive and specific method to detect P1. RESULTS: A pair of primers was designed and used to amplify a 119 bp DNA fragment to generate a recombinant plasmid which was served as the standard. A SYBR I qPCR protocol was established using the P1 recombinant plasmid standard and the sensitivity, specificity and stability of this method was analyzed. The results demonstrate a strong correlation with P1 recombinant plasmid titers when virus DNA copy numbers fall in between 10(0) ~ 10(9) copies/µL. This method doesn't detect pseudo rabies, porcine parvovirus or porcine reproductive and respiratory syndrome virus; moreover it can distinguish porcine circovirus type 2 from P1 by melting temperature analysis. Coefficient of variation for each batch of reaction is less than 5%. The serum virus titers of P1 positive in this study were measured by this protocol to be 10(3) to 10(7) copies/mL. CONCLUSIONS: The established qPCR is sensitive, specific, and reliable, which could be a useful tool when applied to quantification of P1 in a variety of samples from infected pigs.

Biofilm formation of Streptococcus equi ssp. zooepidemicus and comparative proteomic analysis of biofilm and planktonic cells.

Streptococcus equi ssp. zooepidemicus (SEZ) is responsible for a wide variety of infections in many species, including pigs, horses and humans. Biofilm formation is essential for pathogenesis, and the ability to resist antibiotic treatment results in difficult-to-treat and persistent infections. However, the ability of SEZ to form biofilms is unclear. Furthermore, the mechanisms underlying SEZ biofilm formation and their attributes are poorly understood. In this study, scanning electron microscopy (SEM) demonstrated that SEZ strain ATCC35246 formed biofilms comprising a thick, heterogeneous layer with clumps on the coverslips when incubated for 24 h. In addition, we used a two-dimensional gel electrophoresis (2-DE) based approach to characterize differentially expressed protein in SEZ biofilms compared with their planktonic counterparts. The results revealed the existence of 24 protein spots of varying intensities, 13 of which were upregulated and 11 were downregulated in the SEZ biofilm compared with the planktonic controls. Most of proteins expressed during biofilm formation were associated with metabolism, adhesion, and stress conditions. These observations contribute to our understanding of the SEZ biofilm lifestyle, which may lead to more effective measures to control persistent SEZ infections.

Complete Genome Sequence of Aeromonas hydrophila Phage CC2.

Aeromonas hydrophila is one of the major pathogenic bacteria for fish and people. To develop an effective antimicrobial agent, we isolated a bacteriophage from sewage, named CC2, and sequenced its genome. Comparative genome analysis of phage CC2 with its relatives revealed that phage CC2 has higher sequence homology to A. salmonicida phage 65 than to A. hydrophila phage Aeh1. Here, we announce the complete genome sequence of CC2 and report major findings from the genomic analysis.

Complete genome sequence of an H3N2 canine influenza virus from dogs in Jiangsu, China.

A canine influenza virus (CIV) strain of avian origin designated A/Canine/Jiangsu/06/2010 (H3N2) was isolated from dogs exhibiting severe respiratory disease in Jiangsu, China. We announce the complete genome sequence of this viral strain and report major findings from the genomic analysis. This sequence will help us understand the molecular characteristics and evolutionary of H3N2 CIV in China.

Characterization and functional analysis of atl, a novel gene encoding autolysin in Streptococcus suis.

Streptococcus suis serotype 2 (S. suis 2) is an important swine and human pathogen responsible for septicemia and meningitis. A novel gene, designated atl and encoding a major autolysin of S. suis 2 virulent strain HA9801, was identified and characterized in this study. The Atl protein contains 1,025 amino acids with a predicted molecular mass of 113 kDa and has a conserved N-acetylmuramoyl-l-alanine amidase domain. Recombinant Atl was expressed in Escherichia coli, and its bacteriolytic and fibronectin-binding activities were confirmed by zymography and Western affinity blotting. Two bacteriolytic bands were shown in the sodium dodecyl sulfate extracts of HA9801, while both were absent from the atl inactivated mutant. Cell chains of the mutant strain became longer than that of the parental strain. In the autolysis assay, HA9801 decreased to 20% of the initial optical density (OD) value, while the mutant strain had almost no autolytic activity. The biofilm capacity of the atl mutant was reduced ∼30% compared to the parental strain. In the zebrafish infection model, the 50% lethal dose of the mutant strain was increased up to 5-fold. Furthermore, the adherence to HEp-2 cells of the atl mutant was 50% less than that of the parental strain. Based on the functional analysis of the recombinant Atl and observed effects of atl inactivation on HA9801, we conclude that Atl is a major autolysin of HA9801. It takes part in cell autolysis, separation of daughter cells, biofilm formation, fibronectin-binding activity, cell adhesion, and pathogenesis of HA9801.

Development of an Aeromonas hydrophila recombinant extracellular protease vaccine.

Aeromonas hydrophila (Ah) exists widely in the aquatic environment and infects a variety of animals. Extracellular protease (EPR) is an important protective antigen that induces a specific antibody response to resist Ah infection. In this study, two genes encoding extracellular protease epr2 and epr3 were linked within the expression vector pET32a to construct a recombinant pET-epr2-3 plasmid. The immunogenicity of the fusion protein epr2-3 was investigated as a subunit vaccine in ICR mice. The recombinant epr2-3 protein induced the production of high antibody titers. The survival rate against homogenous Ah J-1 challenge was significantly higher in the epr2-3 vaccinated group (≥80%) compared with the inactivated Ah vaccinated group and the challenge control group (P < 0.01), thus indicating that the recombinant epr2-3 protein provided significant protection against Ah infection. Therefore, the recombinant epr2-3 is a promising candidate for development as a vaccine against Ah infections.

Complete genome sequence of Streptococcus equi subsp. zooepidemicus strain ATCC 35246.

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.

Molecular cloning and analysis of the UDP-Glucose Pyrophosphorylase in Streptococcus equi subsp. zooepidemicus.

UDP-Glucose Pyrophosphorylase (EC 2.7.7.9, UGPase) plays an important role in Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) cell envelope Hyaluronic acid (HA) biosynthesis and it is also recognized as a virulence determinant in several bacterial species. HA is valuable biopolymer used in the pharmaceutical and cosmetic industry. In addition, encapsulation by HA is considered an important virulence factor in other streptococci. Research UGPase will contribute to the vaccine development of S. zooepidemicus and the production of HA. In this study, The UGPase gene fragment (789 bp) obtained from previous research was amplified using PCR, and located by Genome walking technology (Genebank No.GQ423507). The UGPase was expressed, purified and identified using UGPase antibody. The enzyme kinetic parameters were determined, the temperature and pH of the highest activity for the cloned UGPase were 37°C, pH 7.5. The Km and Kcat value against UTP and G-1-P was 8.5 µM, 69.05 s(-1) and 36.41 µM, 48.81 s(-1), respectively. The homology-modeling was operated. Overexpression of the UGPase in S. zooepidemicus, its virulence was slightly affected, and HA yield reduced. Real-time PCR was carried out to determine the UGPase expression levels of both SEZp and SEZugp in different grow period, the level is high in logarithmic phase and low in Decline phase.

Identification and characterization of inosine 5-monophosphate dehydrogenase in Streptococcus suis type 2.

Streptococcus suis type 2 is a swine pathogen responsible for diverse diseases. Although many virulent factors have been identified and studied, relatively little is known about the pathogenic mechanisms of type 2. The aim of the study was to identify and understand the characterization of Inosine 5-monophosphate dehydrogenase (IMPDH). A 957-bp gene, impdh, was identified in the virulent S. suis serotype 2 (SS2), and analysis of the predicted IMPDH sequence revealed IMP dehydrogenase/GMP reductase domain. The gene encoding for the IMPDH of S. suis was cloned and sequenced. The DNA sequence contained an open reading frame encoding for a 318 amino acid polypeptide exhibiting 23% sequence identity with the IMPDH from Streptococcus pyogenes (YP281355) and Streptococcus pneumoniae (ZP00404150). Using the pET(32) expression plasmid, the impdh gene was inducibly overexpressed in Escherichia coli to produce IMPDH with a hexahistidyl N-terminus to permit its purification. The (His)6 IMPDH protein was found to possess functional IMPDH enzymatic activity after the purification. The impdh-knockout SS2 mutant ( Delta IMPDH) constructed in this study was slower in growth and one pH unit higher than SS2-H after 6 h of culturing, and found to be attenuated in mouse models of infection for 2.5 times and not be capable of causing death in porcine models of infection in contrast with the parent SS2-H.

Identification of differentially expressed genes in haemocytes of the crayfish (Procambarus clarkii) infected with white spot syndrome virus by suppression subtractive hybridization and cDNA microarrays.

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.

Identification and distribution of putative virulent genes in strains of Streptococcus suis serotype 2.

In order to identify gene sequences unique to the virulent strains, suppression subtractive hybridization (SSH) was conducted using virulent Streptococcus suis type 2 (SS2) strain HA9801 and avirulent S. suis type 2 strain T15. Thirty genomic regions were absent in T15, and the DNA sequences of these regions in HA9801 were determined. These DNA fragments, containing putative virulence genes, encoded 28 proteins that were homologous to proteins involved in various aspects of cellular surface structure, molecular synthesis, energy metabolism, regulation, transport systems and others of unknown function. According to the published SS2 genomic sequence of the Chinese strain 98HAH33, PCR primers for 14 significant DNA fragments were designed and used for detection of the distribution of these fragments in S. suis strains from different sources, serotypes, regions, groups and times. The results showed that these 14 DNA fragments were widely distributed in 37 detected SS2 strains, yet were absent among the avirulent strain T15. Moreover, these fragments could be detected in other serotypes of S. suis, but each serotype had a different distribution of the fragments.

Genetic characterization of phenicol-resistant Escherichia coli and role of wild-type repressor/regulator gene (acrR) on phenicol resistance.

The genetic basis for phenicol resistance was examined in 38 phenicol-resistant clinical Escherichia coli isolates from poultry. Out of 62 isolates, 38 showed resistance for chloramphenicol and nine for florfenicol, respectively. Each strain also demonstrated resistance to a variety of other antibiotics. Molecular detection revealed that the incidence rates of the cat1, cat2, flo, flo-R, cmlA, and cmlB were 32, 29, 18, 13, 0, and 0%, respectively. Nineteen strains were tolerant to organic solvents. PCR amplification of the complete acrR (regulator/repressor) gene of five isolates revealed the amino acid changes in four isolates. DNA sequencing showed the non-synonymous mutations which change the amino acid, silent mutation, and nucleotide deletion in four isolates. MY09C10 showed neither deletion nor mutation in nucleotide. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in these strains. Complementation with a plasmid-borne wild-type acrR gene reduced the expression level of AcrA protein in the mutants and partially restored antibiotic susceptibility one- to fourfold. This study shows that mutations in acrR are an additional genetic basis for phenicol resistance.

[Cloning, expression and characterization analysis of the arginine deiminase of Streptococcus suis of China isolates].

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.

[Development and characterization of monoclonal Antibody against M-like protein of Streptococcus equi subsp. zooepidemicus from pig].

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.

[Identification and characterization of a novel antigenic protein of Streptococcus suis type 2].

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.

Effect of fluid resuscitation on ryanodine receptor in macaques with endotoxic shock.

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 mgkg(-1) lipopolysaccharide (LPS). Sixty minutes after the LPS challenge, the animals were given (i) 5 mLkg(-1) normal saline (Ns group, n = 6), (ii) 5 mLkg(-1) of 5% sodium bicarbonate (Sb group, n = 6) or (iii) 5 mLkg(-1) of 3.5% hypertonic sodium chloride (Hs group, n = 7). The control group (Co group, n = 5) received 1 mLkg(-1) normal saline and then with 5 mLkg(-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.

[Cloning and characterization of the gene encoding IMPDH of Streptococcus suis serotype 2].

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.

Endotoxic shock model with fluid resuscitation in Macaca mulatta.

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.