Uniformity in the biochemical properties of Renibacterium salmoninarum isolates obtained from several sources

Abstract A marked similarity in the biochemical properties of 26 Renibacterium salmoninarum cultures was found. Properties not previously recorded for this bacterium include haemolytic and proteolytic activity, DNase and the presence of glycogen in cells, and negative reactions in tests for nitrates, Tween-80 hydrolysis, amylase, bile solubility, arginine hydrolysis and phosphatase. Selected properties enabled bacteria morphologically similar to R. salmoninarum to be identified.     

Molecular cloning of Renibacterium salmoninarum DNA fragments

A Renibacterium salmoninarum enriched recombinant DNA library was constructed to isolate DNA fragments which could be used as probes to detect gene sequences specific for the causative agent of bacterial kidney disease in salmonid fish. One fragment of 149 base pairs was isolated and its specificity and sequence determined. This probe may prove useful in the design of diagnostic tests for the disease in asymptomatic fish and ova.     

Sequencing, cloning and expression of a β-1,4-mannanase gene, manA, from the extremely thermophilic anaerobic bacterium, Caldicellulosiruptor Rt8B.4

Abstract We report here the isolation of a Renibacterium salmoninarum DNA sequence capable of transforming a non-invasive Escherichia coli strain into a microorganism able to enter the fish cell line, CHSE-214. Immunofluorescence and electron microscopy techniques were used to assess the acquired invasive phenotype by HB101 E. coli cells, upon transformation with pPMV-189. This plasmid carries a 2282-bp R. salmoninarum DNA segment. The invasive phenotype is qonserved upon deletion of approximately 1000 bp at the 3' end of the insert. The remaining segment contains an ORF region encoding a putative protein of about 30 kDa.     

Molecular cloning and sequence analysis of the gene coding for the 57-kDa major soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum

Abstract The complete sequence coding for the 57-kDa major soluble antigen of the salmonid fish pathogen, Renibacterium salmoninarum , was determined. The gene contained an opening reading frame of 1671 nucleotides coding for a protein of 557 amino acids with a calculated M _r value of 57190. The first 26 amino acids constituted a signal peptide. The deduced sequence for amino acid residues 27–61 was in agreement with the 35 N-terminal amino acid residues determined by microsequencing, suggesting the protein in synthesized as a 557-amino acid precursor and processed to produce a mature protein of M _r 54505. Two regions of the protein contained imperfect direct repeats. The first region contained two copies of an 81-residue repeat, the second contained five copies of an unrelated 25-residue repeat. Also, a perfect inverted repeat (including three in-frame UAA stop codons) was observed at the carboxyl-terminus of the gene.     

Immunoelectron microscopic demonstration that Renibacterium salmoninarum is encapsulated

The cell surfaces of four strains of Renibacterium salmoninarum, including the type strain Lea-7-74T, were examined by transmission electron microscopy after immuno-stabilization and staining with ruthenium red. Cells were covered with a layer of capsular material whose thickness varied between 30 to 60 nm. Our results indicate that R. salmoninarum is encapsulated.     

The detection of two antigenic groups among Renibacterium salmoninarum isolates

The analysis of the membrane proteins and their antigenic properties in a group of 14 geographically diverse strains of Renibacterium salmoninarum revealed the existence of antigenic diversity within this species. Eleven isolates, including the type strain ATCC 33209, shared a similar protein profile with a major component of 57 kDa whereas three strains showed a common pattern with a major protein of 30 kDa. The quantitative agglutination tests and Western blotting assays seem to indicate the existence of serological heterogeneity, with two distinct groups being detected.     

Influence of the growth conditions on the hydrophobicity of Renibacterium salmoninarum evaluated by different methods

The influence of medium and salinity on the cell surface hydrophobicity of Renibacterium salmoninarum was investigated using three different methods: bacterial adherence to hydrocarbons (BATH), salt agglutination test (SAT), and binding to nitrocellulose filters (NCF). The possible relationship among hydrophobicity, haemagglutination and adherence to cell lines was also evaluated. R. salmoninarum showed to be highly hydrophobic regardless of the growth conditions or technique employed. Nevertheless, slight differences can be detected depending on the method used. In the SAT and NCF assays very uniform values were obtained within the strains. All the R. salmoninarum isolates agglutinated in (NH4)2SO4 in a range of 0.05-0.2 M and displayed a 77-100% of adherence to nitrocellulose filters. However, more variable results were observed in the BATH method depending on the hydrocarbon, buffer and strain employed. Although all of the isolates produced haemagglutinins for homeotherm erythrocytes, the majority of them failed to agglutinate poikilothermic red blood cells and were unable to adhere to fish cell lines. Therefore, a general correlation among hydrophobicity, agglutinating capacity for fish erythrocytes and adherence to fish cells can not be established for R. salmoninarum.     

Investigation of factors influencing the ability ofRenibacterium salmoninarumto stimulate rainbow trout macrophage respiratory burst activity

The early stages of contact betweenRenibacterium salmoninarumand rainbow trout macrophages were investigated. LiveR. salmoninarumelicited a respiratory burst, which was enhanced by heat-killing the microorganism. This phenomenon, though, was not observed using UV killed bacteria. Both responses were enhanced when a combination of LPS and TNFαwas used to activate the macrophages prior to contact withR. salmoninarum. Further studies demonstrated that both compounds synergised to enhance O₂⁻production, and that this was correlated with the ability to kill the pathogen. Opsonisation ofR. salmoninarumwith serum factors also increased the respiratory burst, but no difference was found between normal serum and heat-inactivated serum.     

Cloning, functional expression and partial characterization of the glucose kinase from Renibacterium salmoninarum

In recent years, different molecular techniques have led to an important progress in the characterisation of Colletotrichum species, but there are no available methods which permit the easy identification of Colletotrichum strains and their assignation to classical species. In the present work, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene, were used to identify a total of 80 strains of Colletotrichum, the majority of them isolated from strawberry. One of the most interesting results derived from this study was the easy and reliable distinction, using the endonuclease MvnI, between Colletotrichum fragariae and Colletotrichum gloeosporoides, both responsible of anthracnose on strawberry and phenotypically indistinguishable. Moreover, we propose the restriction fragments generated by the endonucleases MvnI, PvuII and ScrFI as a rapid method to differentiate species of the Colletotrichum genus.     

A new value for mol percent guanine + cytosine of DNA for the salmonid fish pathogen Renibacterium salmoninarum

The mol% G + C of DNA extracted from seven different isolates of Renibacterium salmoninarum was determined. Organisms studied were from selected geographical areas (U.S.A., Canada, England and France) and were isolated from five different species of salmonid fish. The mol% G + C was determined to be 55.5, higher than the currently reported value of 53.     

应用聚合酶链反应检测口蹄疫病毒的实验研究

聚合酶链反应用于直接检测口蹄疫病毒(FMDV),可快速、灵敏地检出乳鼠组织或细胞繁殖的病毒的核酸。其灵敏度可达0.062pg,整个检测过程可缩短至4～5个小时内完成,比其它检测方法都敏感和快速。本文还探讨了直接用PCR扩增合成生物素化核酸探针检测口蹄疫病毒核酸的存在。     

Phylogenetic relationship of the fish pathogenic Renibacterium salmoninarum to Arthrobacter, Micrococcus and related taxa

Abstract The phylogenetic position of the obligate fish pathogenic Gram-positive eubacterium Renibacterium salmoninarum was determined by the 16S ribosomal RNA cataloguing approach. Comparison of the catalogue to those of more than 165 Gram-positive organisms from 50 genera revealed that R. salmoninarum is a member of the actinomycetes subdivision. As derived from similarity coefficients (S_AB values), this organism is related to a subgroup harbouring morphologically and chemotaxonomically rather heterogeneous taxa, including Anthrobacter, Micrococcus, Cellulomonas, Jonesia, Promicromonospora, Stomatococcus and Brevibacterium . Thus, the inclusion of organisms characterized by regular rods increases the phenotypic variability of this phylogenetically coherent group.     

用PCR技术检测活检组织和鼻咽上皮细胞内EB病毒基因

选用Epstin-Barr病毒(EBV)基因组内部重复序列1(IR1)片断作为多聚酶链反应(Polymerase Chain Reaction,PCR)扩增引物,用于检测了31例不同病例活检组织和4例新鲜鼻咽组织经体外培养6周以上的新生上皮细胞内EBV基因,其中检出EBVDNA:高分化鼻咽癌5/5,低分化鼻咽癌4/4,何杰金氏病5/5,非何杰金氏病0/2,头颈其他肿瘤1/6,鼻咽慢性炎症0/5,正常鼻咽组织0/4;新生上皮细胞DNA抽提物;低分化鼻咽癌2/2,炎症0/1,正常人胚鼻咽上皮0/1;携带EBV基因组细胞系(Raji,B_(95-8)各1)2/2,致淋巴细胞转化之B_(95-8)病毒为10~(-4),PCR检测10~(-4)～10~(-6)均阳性,10~(-7)未检出。结果表明EBV与鼻咽癌与何杰金氏病有关,常规石蜡包埋切片仅8μm×0.1mm~2,贮存时间至三年仍可用于PCR检测EBV DNA,证实PCR是一种快速、灵敏和特异测捡EBV基因组的方法,可作为肿瘤和疚病病毒病因回顾性调直研究的有力手段。     

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.     

Identification of specific gene sequences in preimplantation embryos by genomic amplification: detection of a transgene

Endogenous and foreign DNA sequences can be detected in an extremely small number of cells via sequence amplification in vitro. The polymerase chain reaction (PCR) technique applied in multiple cycles allows the amplification of specific short regions of the genome to levels that can be detected by DNA blotting techniques. Cow and mouse blastocysts were analyzed by PCR for the presence of an endogenous singlecopy gene or an integrated foreign gene. The endogenous single-copy gene encoding the beta chain of bovine luteinizing hormone was detectable in cow blastocysts and in purified bovine genomic DNA representing as few as 25 cells. To determine whether exogenous genes (transgenes) can be detected in preimplantation embryos, transgenic male mice hemizygous for the prokaryotic gene encoding neomycin resistance were bred to nontransgenic females, and the resulting blastocysts were analyzed. The neo gene was detected in approximately half of the embryos. The capability to identify specific gene sequences in a limited number of embryonic cells affords investigators the opportunity to study genetics in early development.     

Evaluation of non-culture diagnosis of invasive meningococcal disease by polymerase chain reaction (PCR)

Antibiotic treatment prior to transport or admission to hospital has reduced the proportion of cases of invasive meningococcal disease (IMD) from which Neisseria meningitidis can be isolated by standard microbiological techniques. Identification of meningococci by polymerase chain reaction (PCR) was assessed in relation to microbiological diagnosis for cases over a 4-year period between 1998 and 2001. A screening assay for the IS1106 gene was used to detect meningococcal DNA and five additional assays for siaD and orf-2 genes were performed to determine the serogroup. PCR results were compared with results of bacteriological culture, other laboratory test results and clinical data. The sensitivity of the PCR assay for culture-confirmed cases was 98.5%. The specificity of the assay was 96% based on test results for patients from whom other bacteria were isolated, children with viral meningitis and afebrile negative controls. The siaD B/C/W-135 and Y as well as the orf-2 gene for serogroup A PCR assays were able to determine the serogroup for 75.2% of cases that were positive by PCR screening assay. When isolates from patients with IMD were tested by both agglutination and PCR, the results agreed in all cases. PCR is a useful tool for diagnosis of IMD when Gram stain and culture tests are negative due to antibiotic treatment prior to collection of samples for microbiological analyses.