[Comparison between computer aided simulation and dental model orthognathic surgery for the treatment of patients with mandibular excess and facial asymmetries].

Objective: To evaluate the results of the orthognathic surgery with computer aided simulation-three-dimensional(3D) printed surgical guide and dental model surgery in the treatmemt of patients with mandibular excess and facial asymmetries. Methods: The coordinate system was built in ProPlan CMF 2.0 software, and the horizontal plane consisted of PoL, PoR, OrL, middle sagittal plane through nasion point and basion point and the plane perpendicular to the horizontal plane, coronoid plane through nasion point and the plane perpendicular to the horizontal plane and middle sagittal plane. The orientation of maxilla and mandibular distal segment was calculated by each triangle(U1-U6L-U6R, L1-L6L-L6R, Me-M5L-M5R) before and after orthognathic surgery. A total of 60 mandibular excess patients with facial asymmetries were enrolled in this retrospective study. They were divided into two groups, group Ⅰ with computer aided simulation, group Ⅱ with dental model surgery. The difference of maxillary occlusal plane roll and yaw angle, mandibular occlusal plane roll and yaw angle, and mandibular body plane roll and yaw angle were calculated. Statistical analysis was performed with SPSS 17.0 software. Results: The yaw angle of mandibular occlusal plane of the dental model surgery and computer aided simulation was 0.36°± 0.48° and 0.84° ± 0.36° (P=0.043), respectively. The roll angle of mandibular occlusal plane of the dental model surgery and computer aided simulation was 0.26°±0.79° and 0.54°±0.40°(P=0.032), respectively. The yaw angle of mandibular body plane of the dental model surgery and computer aided simulation was 0.60°± 1.04° and 0.23°±0.52°(P=0.008), respectively. The roll angle of mandibular body plane of the dental model surgery and computer aided simulation was 0.82° ± 0.72° and 0.53° ± 0.37° (P=0.028), respectively. The changes in computer aided simulation group were more obvious than that in the dental model surgery group, but the difference was not significant in the yaw angle of maxillary occlusal plane and the roll angle of maxillary occlusal plane between the two groups(P >0.05). Conclusions: It was more effective to correct mandibular asymmetry by computer aided simulation than dental model surgery.

Shake and stew: a non-destructive PCR-ready DNA isolation method from a single preserved fish larva.

A rapid non-destructive alternative to isolate DNA from an individual fish larva is presented, based on the suspension of epithelial cells through vortex forces, and the release of DNA in a heated alkaline solution. DNA from >6056 fish larvae isolated using this protocol has yielded a high PCR amplification success rate (>93%), suggesting its applicability to other taxonomic groups or sources when tissue amount is the limiting factor.

Experimental infection of dogs with H3N2 canine influenza virus from China.

Canine influenza virus (CIV) is an emerging pathogen that causes acute respiratory disease in dogs. The aim of this study was to investigate the pathogenicity of A/canine/Jiangsu/06/2010 (H3N2) virus isolated in China. Nine dogs were inoculated intranasally with 107.95 of 50% egg infectious dose (EID50) of the virus. The onset of clinical signs and virus shedding was observed on day 1 post-infection (p.i.). The peak clinical score occurred between days 4 and 6 p.i. The experimentally infected dogs were found to shed virus not only via the respiratory tract but also via the digestive tract. Viral RNA could be detected in multiple organs including the trachea, lung, liver, spleen, kidney, brain and duodenum. All the sampled organs from infected dogs showed significant lesions and viral antigen staining. The results differed from those reporting using previous CIV strains; the Chinese isolate could induce extrapulmonary infection and cause extensive lesions in dogs.

Associations between anti-microbial resistance phenotypes, anti-microbial resistance genotypes and virulence genes of Escherichia coli isolates from Pakistan and China.

The objective of this study was to determine the association between phenotypic resistance, genotypic resistance and virulence genes of Escherichia coli isolates in Jiangsu province, China and Punjab province Pakistan. A total of 62 E. coli isolates were characterized for phenotypic resistance, genotypic resistance and virulence factor genes. The anti-microbial resistance phenotype and genotypes in relation to virulence factor genes were assessed by statistical analysis. Of 20 tested virulence genes, twelve were found and eight were not found in any isolates. sitA and TspE4C2 were the most prevalent virulence genes. Of the 13 anti-microbial agents tested, resistance to ampicillin, sulphonamide and tetracycline was the most frequent. All isolates were multiresistant, and 74% were resistant to trimethoprim and sulphamethaxazole. Phenotypically, tetracycline-, cefotaxime- and trimethoprim-resistant isolates had increased virulence factors as compared with susceptible isolates. Genotypically, resistant genes Tem, ctx-M, Tet, Sul 1, dhfr1, Cat2 and flo-R showed the association with the virulence genes. Almost all classes of anti-microbial-resistant genes have a high association with virulence. Resistant isolates have more virulent genes than the susceptible isolates.

Identity and virulence properties of Aeromonas isolates from diseased fish, healthy controls and water environment in China.

AIMS: To investigate the species distribution in Aeromonas isolates from diseased fish, healthy controls and water environment in China; to evaluate the frequency of the aerolysin (aer), cytotonic enterotoxin (alt), cytotoxic enterotoxin (act), temperature-sensitive protease (eprCAI) and serine protease (ahp) genes in Aeromonas isolates; and to determine the potential pathogenicity of these isolates. METHODS AND RESULTS: Two hundred and two Aeromonas isolates from diseased fish (n = 42), healthy fish (n = 120) and water environment (n = 40) in China were identified to species levels based on sequencing of the housekeeping gene gyrB, while the distribution of five virulence factors, including aer, alt, act, eprCAI and ahp, was investigated by PCR. Aeromonas veronii (25/42; 60%) and Aeromonas hydrophila (14/42; 33%) were the species most commonly isolated from diseased fish, while Aer. veronii was the most common species in healthy fish (90/120; 75%) and water samples (25/40; 62·5%). All the five virulence genes were present in 9% (19/202), among which 10 strains were from diseased fish and nine were identified as Aer. hydrophila. For the strains carrying five virulence genes, the average 50% lethal doses (LD(50s) ) of strains from diseased fish were lower when compared with the strains from healthy fish and water environment. CONCLUSIONS: Aeromonas veronii is the most common species, but no significant difference exists in the isolates obtained from diseased fish and from healthy fish. However, Aer. hydrophila isolates were significantly more frequent from diseased fish than from healthy fish. aer+alt+act+eprCAI+ ahp+ was more frequent virulence genotype in Aeromonas isolates from diseased fish than from healthy fish and water environment, and the aer+alt+act+eprCAI+ahp+ isolates were more virulent to zebrafish comparing to the other genetic profiles. SIGNIFICANT AND IMPACT OF THE STUDY: Aeromonas species in aquatic environments are various and have considerable virulence potential, and therefore, there is a need for more careful and intensive epidemiology studies.

Detection of three virulence genes alt, ahp and aerA in Aeromonas hydrophila and their relationship with actual virulence to zebrafish.

AIMS: To evaluate the frequency of the aerolysin (aerA), cytotoxic enterotoxin (alt) and serine protease (ahp) genes in Aeromonas hydrophila isolates from different sources, and to determine the relationship between the presence of these genes and virulence of A. hydrophila in zebrafish. METHODS AND RESULTS: Aeromonas hydrophila isolates from clinical cases (n=40), from healthy fish (n=22) and from water environment (n=21) were analysed with respect to the prevalence of aerA, alt and ahp genes by PCR assay. These virulence factors occur among clinical isolates as well as among isolates from healthy fish and water environment. The majority (97·6%) of the strains examined carried one or more virulence genes. The isolates were divided into seven genetic profiles on the basis of PCR result: aerA(+) alt(+) ahp(+) (62·7%), aerA(+) alt(+) ahp(-) (13·3%), aerA(+) alt(-) ahp(+) (10·8%), aerA(-) alt(+) ahp(+) (4·8%), aerA(-) alt(-) ahp(+) (3·6%), aerA(+) alt(-) ahp(-) (2·4%) and aerA(-) alt(-) ahp(-) (2·4%). A higher frequency of genetic group aerA(+) alt(+) ahp(+) was determined in the isolates from diseased animals compared to those from healthy fish or water environments. Virulence properties of 26 representative strains belonging to the seven genetic profiles were further characterized. Results demonstrated that as the present of virulence genes increased, the proteolytic, haemolytic and cytotoxic activities of extracellular products also increased. And the 50% lethal doses (LD(50)s) of aerA(+) alt(+) ahp(+) isolates (<10(5)) in zebrafish were lower when compared with the strains expressing one or combinations of two virulence genes (>10(6)). CONCLUSIONS: Virulence properties of A. hydrophila correlated well with the presence of virulence genes tested. aerA(+) alt(+) ahp(+) was more frequent virulence genotype in A. hydrophila isolates from clinical diseases than from healthy fish and water environment, and the aerA(+) alt(+) ahp(+) isolates were more virulent to zebrafish compared to the other six genetic profiles. SIGNIFICANT AND IMPACT OF THE STUDY: The detection for aerA, alt and ahp can be used for virulence typing of A. hydrophila isolates.

[Lysogenic infection of a Shiga toxin 2-converting bacteriophage changes host gene expression, enhances host acid resistance and motility].

Shiga toxin 2 (Stx2)-converting bacteriophages can infect and lysogenize other bacteria in vivo and in vitro, and, thus, contribute to a genotypic heterogeneity of infected host. However, the global transcription patterns accompanying the lysogenic infection of E. coli host have not been clearly resolved. In this study, gene expression profiles of Stx2 phage phi Min27(delta stx::cat) converted and native E. coli MG1655 hosts were compared using microarray assay. The phi Min27(delta stx::cat) conversion had a direct effect on the global expression of bacterial host genes as 166 genes were found to be differentially expressed (104 up-regulated and 62 downregulated). These genes were predominantly responsible for bacterial central metabolism, transport and transcription. It was shown that in addition to the down-regulation of genes involved in synthesis of thiamine and protein transporters, expression of genes associated with bacterial energy production (e.g., fadABDEHIJL, aceK, and acnA) was also suppressed. Conversely, most up-regulated genes were transport genes, flagellar synthesis genes (fliDESTZ), and acid resistance genes (e.g., gadEW, hdeABD, and adiY). Futhermore, conversion of phi Min27(delta stx::cat) was shown to change physiological properties of the host cell. In comparison with the uninfected cells the converted bacteria host had increased acid tolerance and promoted swimming motility on a semisolid agar surface.

High-resolution melting analysis (HRMA): a highly sensitive inexpensive genotyping alternative for population studies.

High-resolution melting analysis (HRMA) is a highly sensitive closed-tube genotyping method used primarily in clinical studies. As the method is rapid, inexpensive and amenable to high throughput, we decided to investigate its applicability to population studies. Small amplicons and unlabelled probes were used to genotype the nuclear genes, lactate dehydrogenase-A (ldh-A), myosin light chain-2 (mlc-2), acidic ribosomal phosphoprotein P0 (ARP) and calmodulin (CaM) in populations of swordfish, Xiphias gladius. Results indicate that HRMA is a powerful genotyping tool to study wild populations.

Effect of oral immunization with Aeromonas hydrophila ghosts on protection against experimental fish infection.

AIMS: To investigate whether oral immunization with Aeromonas hydrophila ghosts (AHG) vaccine can elicit mucosal and systemic immune responses of Carp (Carassius auratus gibelio) compared to conventional formalin-killed bacteria (FKC). METHODS AND RESULTS: Fish were fed diets coated with AHG, FKC or phosphate buffered saline (PBS) alone, after immunization, more antigen-specific antibody was significantly detected in serum and intestinal mucus in AHG group than FKC group and PBS group. In addition, after challenged with the parent strain J-1, the survival of bacterial ghost-vaccinated fish was higher than PBS group and FKC group, the relative per cent survival (RPS) being 76.8%, 58.9%, respectively. CONCLUSIONS: Oral immunization with A. hydrophila ghosts can elicit systemic and mucosal adaptive immune responses and has higher potential to induce protective adaptive immunity than normal vaccine. SIGNIFICANCE AND IMPACT OF THE STUDY: Oral immunization with bacterial ghosts is a promising new solution with potential application to prevent diseases in fish.

Genetic characterization of H1N1 swine influenza A viruses isolated in eastern China.

Three influenza H1N1 viruses were isolated in 2005 from pigs with respiratory disease on a farm in eastern China. The three isolates were characterized to determine their probable origin. Each of the eight genes of the isolates was most closely related to the corresponding gene from the classical swine H1N1 virus. Also, phylogenetic analysis further confirmed that each of the eight genes of the isolates was closely related to the classical swine H1N1 viruses, especially those isolated in China. The HA1 proteins of the three isolates were identical to that of A/Swine/Guangdong/ 1/01, a virus isolated in 2001 in China, even though three nucleotide differences were observed. These results further support the concept that swine can serve as a reservoir of genetically stable influenza viruses.

Analysis of putative recombination hot sites in the S gene of canine coronaviruses.

The S gene sequence of Canine coronavirus strain 1-71 (CCoV 1-71) was cloned, sequenced, and compared to those of other CCoVs, Transmissible gastroenteritis virus (TGEV), and Feline coronavirus (FCoV). The sequence analysis showed that CCoV 1-71 displayed a 98.8-99.8% identity with CCoVs strains V1, K378, and GP. Four putative recombination sites were found at the 5'-end of the S gene, namely at nt 53, 75, 425, 991. Both sequences flanking each site were significantly different. Three recombination hot regions were found on the S gene, namely at nt 337-437, 1545-3405, and 4203-4356, which shared a common recombination signal with Group 2 coronaviruses. The G/CTAAAAA/GT sequence downstream of the recombination site may represent a specific recombination signal in CCoVs. The CCoV 1-71 S protein sequence was found to be similar to those of other CCoVs except for several N-glycosylation sites at the N-terminus of the S protein, which could be related to the differences in virulence and cell tropism in individual CCoVs. This study indicated that the similarity of CCoVs in virulence and tropism was mostly acquired by the homologous RNA recombination and not only by simple mutation and selection.

Purified recombinant phage lysin LySMP: an extensive spectrum of lytic activity for swine streptococci.

Bacteriophage lysin has attracted considerable attentions as possible antimicrobial agents for solution of antibiotic resistance. SMP was a Streptococcus suis serotype 2 bacteriophage isolated from nasal swabs of healthy Bama minipigs. The putative SMP bacteriophage lysin, designated LySMP, was recombinantly expressed in Escherichia coli BL21, and chromatographically purified. Treated with 0.8% of beta-mercaptoethanol, LySMP exhibited an extensive lysin spectrum than those of whole phage against bacteria investigated. S. suis serotype 2, S. suis serotype 7 and S. suis serotype 9 strains were recovered from diseased pigs between 1998 and 2005 in China. Fifteen of seventeen strains of S. suis serotype 2 could be lysed, as well as S. suis serotype 7 and 9, Streptococcus equi ssp. zooepidemicus and Staphylococcus aureus. But E. coli and Salmonella enterica were not affected. Purified LySMP showed high degrading efficiency against PMSF or lysozyme treated cells comparing to PBS washed cells. Optimum pH and temperature conditions for the lysin were investigated by turbidity reduction assay. The lysin exerted efficient lysis activity at 37 degrees C, pH 5.2. The turbidity of bacterium investigated was observed to decrease by 1.2-68% in 30 min. Result indicated that putative LySMP could be a candidate antimicrobial agent in controlling S. suis infection.

Detection of autoinducer-2 and analysis of the profile of luxS and pfs transcription in Streptococcus suis serotype 2.

The only quorum sensing (QS) system shared by Gram-positive and Gram-negative bacteria involves the production of autoinducer-2 (AI-2). However, there have been no reports concerning the AI-2 in Streptococcus suis serotype 2 (SS2). In this study, we investigated AI-2 production and analyzed the relationship between the transcription level of luxS and pfs and AI-2 production. A homologue of luxS, the gene required for AI-2 synthesis in Vibrio harveyi, was isolated from the SS2 genome. V. harveyi BB170 bioassay demonstrated luxS functionality in SS2 and its ability to complement the luxS-negative phenotype of Escherichia coli DH5alpha. Further studies showed that AI-2 activity peaked in the late exponential phase, and sodium chloride or glucose increased the production of AI-2. Real-time PCR showed that the level of transcription of pfs is highly correlated with the level of AI-2 production, while the level of transcription of luxS does not correlate with the level of AI-2 production. The results presented here demonstrate that SS2 can secrete AI-2 and that the profile of pfs transcription is highly correlated with the level of AI-2 production.

Isolation and identification of a bacteriophage capable of infecting Streptococcus suis type 2 strains.

Streptococcus suis (S. suis) type 2 infection is considered to be a major problem worldwide in the swine industry. Studying phages of S. suis type 2 would be crucial for understanding the ecology and evolution of the Gram-positive bacteria. However, at the present, very little is known about them. An S. suis type 2 bacteriophage, named SMP, was isolated from nasal swabs of healthy Bama minipigs and was characterized at the microbiological and molecular levels. Phage SMP had an isometric head of 50 nm, a noncontractile tail of approximately 135 nm, and a linear double-stranded DNA genome. The host range of phage SMP was limited to 2 of 24 S. suis type 2 strains tested. The genome of phage SMP contained 36,216 bp with an average G+C content of 41.6%.

Immunoproteomic assay of membrane-associated proteins of Streptococcus suis type 2 China vaccine strain HA9801.

Immunoproteomic approaches were undertaken to study the immunogenicity of the membrane-associated proteins of the Streptococcus suis type 2 (SS2) China vaccine strain HA9801. The membrane-associated proteins were enriched using the Triton X-114 extraction protocol and were analysed by two-dimensional gel electrophoresis (2-DE) and subsequent immunoblotting using the hyperimmune serum of SS2-HA9801-immunized specific pathogen free (SPF) minipigs. A total of 11 proteins were recognized, and the corresponding spots on a duplicate gel were excised and identified by MALDI-TOF MS.

Comparison of four infectious bursal disease viruses isolated from different bird species.

Four isolates of infectious bursal disease virus (IBDV), isolated from chicken, duck, goose and sparrow in Jiangsu province of China in 2002, were compared. The viruses were stable to the treatments of 60 degrees C for 1 h, pH 2.0 and lipid solvents. Their antigenic relatedness values (R) were from 0.76 to 0.78. Chickens infected with the chicken isolate showed severe clinical symptoms of IBD and the mortality rate was 33.3% (2/6). Chickens infected with the other three viruses survived but their bursas were damaged and the bursa/body-weight ratios were lower than those of the uninfected control (p< 0.01). The titers of anti-IBDV antibody in infected chicken sera reached up to 1600 by virus neutralization and 6400 by ELISA at 10 days post infection. The sequences of the variable region of VP2 were aligned and compared, showing nucleotide variations ranging from 1.5 to 6.7% and deduced aminoacid variations from 0.8 to 2.2%. All had the same heptapeptide, S-W-S-A-S-G-S, Asp279, and Ala284. The four viruses clustered on a phylogenetic tree and were distant from the STC strain. These findings suggested that different bird species naturally infected with IBDV could serve as carriers or reservoirs in IBDV transmission and might play a role in the emergence of variant IBDV.

Contribution of AhyR to virulence of Aeromonas hydrophila J-1.

Aeromonas hydrophila is a broad-host-range pathogen and its pathogenesis is multifactorial. A regulatory mechanism known as quorum sensing has been found to be involved in the regulation of virulence in many bacteria. In A. hydrophila the ahyR gene encodes LuxR-type response regulator. Here we describe the inactivation of the ahyR gene of A. hydrophila J-1 by the insertion of a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of A. hydrophila J-1 by means of the suicide plasmid pJP5603. Cytotoxic effects on EPC cells assay and LD(50) determinations in fish demonstrated that the ahyR mutant was highly attenuated relative to the wild-type strain. Compared with the parent strain, some characteristics, such as biochemical characters and outer membrane protein profiles, had changed. Some main virulent determinants could not be detected, including proteases, amylase, Dnase, hemolysin and S layer. This article confirmed the important function of AhyR in the pathogenesis of A. hydrophila J-1.

Genetic characterization of novel reassortant H1N2 influenza A viruses isolated from pigs in southeastern China.

In December 2004, three influenza H1N2 viruses were isolated from lung samples of pigs that had died from respiratory disease on a farm in southeastern China. To determine the genetic characterization and probable origin, one of the three isolates, A/Swine/Zhejiang/1/2004 (Sw/ZJ/1/2004), was genetically analyzed. Sw/ZJ/1/2004 was a reassortant with an NA gene most closely related to the corresponding gene from a human-like H3N2 virus circulating in 1995. The remaining seven genes were most closely related to those from the classical swine H1N1 virus. Sw/ZJ/1/2004 appeared to be a novel reassortant H1N2 virus that was genetically distinguishable from other H1N2 viruses found in pigs worldwide. The isolation of Sw/ZJ/1/2004 provided further evidence for pigs serving as a "mixing vessel" for the generation of new reassortant genotypes of influenza viruses and emphasizes the importance of reinforcing influenza virus surveillance in pigs in China.

Selection of immunodominant mimics of IROMP-99 of rabbit Pasteurella multocida from a random 12-peptide library.

A random 12-peptide library was used to screen immunodominant mimics of 99kDa iron-regulated outer membrane protein (IROMP-99) of rabbit Pasteurella multocida. In the present study, expression of IROMPs of rabbit P. multocida strain C51-12 were analyzed by SDS-PAGE, and Western blot to determine the specificity of rat antiserum antibodies against IROMP-99. Only IROMP-99 whose expression was induced under iron-restricted conditions was detected on nitrocellulose paper. The phage display library was screened with rat normal and IROMP-99-specific antiserum. The positive phage clones were identified using enzyme-linked immunoadsorbent assay (ELISA) and inhibition assays for their reactivity to the antiserum. Out of the 18 randomly selected positive clones that showed higher reactivity to rat antiserum, only ten clones efficaciously inhibited binding of rat antisera to IROMPs and their displayed peptides were determined. Alignment using DNAStar-MegAlign software, results showed that motif WHxTxP was highly conserved among nine clones, only clone A7 had no obvious linear homology with either. Our findings suggest that the motif WHxTxP could be an immunodominant mimic epitope of IROMP-99 of rabbit P. multocida strain C51-12.