An alternative efficient procedure for purification of the obligate intracellular fish bacterial pathogen Piscirickettsia salmonis. (1/39)

Piscirickettsia salmonis is an obligate intracellular bacterial pathogen of salmonid fish and the etiological agent of the aggressive disease salmonid rickettsial syndrome. Today, this disease, also known as piscirickettsiosis, is the cause of high mortality in net pen-reared salmonids in southern Chile. Although the bacteria can be grown in tissue culture cells, genetic analysis of the organism has been hindered because of the difficulty in obtaining P. salmonis DNA free from contaminating host cell DNA. In this report, we describe a novel procedure to purify in vitro-grown bacteria with iodixanol as the substrate to run differential centrifugation gradients which, combined with DNase I digestion, yield enough pure bacteria to do DNA analysis. The efficiency of the purification procedure relies on two main issues: semiquantitative synchrony of the P. salmonis-infected Chinook salmon embryo (CHSE-214) tissue culture cells and low osmolarity of iodixanol to better resolve bacteria from the membranous structures of the host cell. This method resulted in the isolation of intact piscirickettsia organisms and removed salmon and mitochondrial DNA effectively, with only 1.0% contamination with the latter.  (+info)

Growth of Piscirickettsia salmonis to high titers in insect tissue culture cells. (2/39)

Piscirickettsia salmonis was grown in established insect, frog, and fish tissue culture cells. The yield of P. salmonis in Sf21 cells was up to 100 times that obtained in CHSE-214 cells, and virulence for Atlantic salmon was retained. The ceiling temperature for growth of P. salmonis in Sf21 cells was 24 degrees C.  (+info)

Isolates of Piscirickettsia salmonis from Scotland and Ireland show evidence of clonal diversity. (3/39)

Salmonid rickettsial septicemia, caused by Piscirickettsia salmonis, causes major mortalities in Chilean salmonid aquaculture and is an increasing problem in Atlantic salmon in Ireland and Scotland. Analysis of 16S-to-23S internal transcribed sequences and 16S ribosomal DNA (rDNA) shows that Irish isolates of P. salmonis form two new groups of the organism while Scottish isolates cluster together with Norwegian and Canadian isolates from Atlantic salmon.  (+info)

Development and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea. (4/39)

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 x 10(3) to 4.4 x 10(9) copies ml(-1) or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 x 10(1) to 2.2 x10(6) copies ml(-1) or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml(-1). The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.  (+info)

CO2-responsive expression and gene organization of three ribulose-1,5-bisphosphate carboxylase/oxygenase enzymes and carboxysomes in Hydrogenovibrio marinus strain MH-110. (5/39)

Hydrogenovibrio marinus strain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, fixes CO2 by the Calvin-Benson-Bassham cycle. Strain MH-110 possesses three different sets of genes for ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO): CbbLS-1 and CbbLS-2, which belong to form I (L8S8), and CbbM, which belongs to form II (Lx). In this paper, we report that the genes for CbbLS-1 (cbbLS-1) and CbbM (cbbM) are both followed by the cbbQO genes and preceded by the cbbR genes encoding LysR-type regulators. In contrast, the gene for CbbLS-2 (cbbLS-2) is followed by genes encoding carboxysome shell peptides. We also characterized the three RubisCOs in vivo by examining their expression profiles in environments with different CO2 availabilities. Immunoblot analyses revealed that when strain MH-110 was cultivated in 15% CO2, only the form II RubisCO, CbbM, was expressed. When strain MH-110 was cultivated in 2% CO2, CbbLS-1 was expressed in addition to CbbM. In the 0.15% CO2 culture, the expression of CbbM decreased and that of CbbLS-1 disappeared, and CbbLS-2 was expressed. In the atmospheric CO2 concentration of approximately 0.03%, all three RubisCOs were expressed. Transcriptional analyses of mRNA by reverse transcription-PCR showed that the regulation was at the transcriptional level. Electron microscopic observation of MH-110 cells revealed the formation of carboxysomes in the 0.15% CO2 concentration. The results obtained here indicate that strain MH-110 adapts well to various CO2 concentrations by using different types of RubisCO enzymes.  (+info)

Microarray analyses identify molecular biomarkers of Atlantic salmon macrophage and hematopoietic kidney response to Piscirickettsia salmonis infection. (6/39)

Piscirickettsia salmonis is the intracellular bacterium that causes salmonid rickettsial septicemia, an infectious disease that kills millions of farmed fish each year. The mechanisms used by P. salmonis to survive and replicate within host cells are not known. Piscirickettsiosis causes severe necrosis of hematopoietic kidney. Microarray-based experiments with QPCR validation were used to identify Atlantic salmon macrophage and hematopoietic kidney genes differentially transcribed in response to P. salmonis infection. Infections were confirmed by microscopy and RT-PCR with pathogen-specific primers. In infected salmon macrophages, 71 different transcripts were upregulated and 31 different transcripts were downregulated. In infected hematopoietic kidney, 30 different transcripts were upregulated and 39 different transcripts were downregulated. Ten antioxidant genes, including glutathione S-transferase, glutathione reductase, glutathione peroxidase, and cytochrome b558 alpha- and beta-subunits, were upregulated in infected macrophages but not in infected hematopoietic kidney. Changes in redox status of infected macrophages may allow these cells to tolerate P. salmonis infection, raising the possibility that treatment with antioxidants may reduce hematopoietic tissue damage caused by this rickettsial infection. The downregulation of transcripts involved in adaptive immune responses (e.g., T cell receptor alpha-chain and C-C chemokine receptor 7) in infected hematopoietic kidney but not in infected macrophages may contribute to infection-induced kidney tissue damage. Molecular biomarkers of P. salmonis infection, characterized by immune-relevant functional annotations and high fold differences in expression between infected and noninfected samples, may aid in the development of anti-piscirickettsial vaccines and therapeutics.  (+info)

Infectivity of a Scottish isolate of Piscirickettsia salmonis for Atlantic salmon Salmo salar and immune response of salmon to this agent. (7/39)

A Scottish isolate of Piscirickettsia salmonis (SCO-95A), previously shown by intraperitoneal injection to have a lethal dose (LD50) of < 2 x 10(3) infectious rickettsial units, was tested for virulence by bath challenge, surface application to the skin, or dorsal median sinus injection. Atlantic salmon Salmo salar post-smolts were used in all experiments, and exposure to 1 x 10(5) tissue culture infective doses (TCID) of P. salmonis ml(-1) for 1 h in a bath challenge resulted in only 1 mortality, 18 d later, in 10 exposed fish. Application of 2.5 x 10(6) TCID of P. salmonis SCO-95A to paper discs on the skin failed to induce any mortalities within 42 d. Intraperitoneally, fish were administered vaccines containing 10(9) heat-inactivated (100 degrees C, 30 min) or 10(9) formalin-inactivated P. salmonis SCO-95A in adjuvant, with a control group receiving phosphate-buffered saline (PBS) in adjuvant. After an induction period of over 6 mo fish were challenged by injection of P. salmonis into the dorsal median sinus. Mortalities in the control group reached 81.8% and the heat-inactivated and formalin-inactivated vaccines gave significant protection from P. salmonis, with relative percentage survivals of 70.7 and 49.6%, respectively. The nature of the protective antigen is unknown, but could be lipopolysaccharide or a heat-stable outer membrane protein. Fish that survived a dorsal median sinus challenge of P. salmonis or were cohabitants showed a strong immune response to P. salmonis.  (+info)

Sulfuricurvum kujiense gen. nov., sp. nov., a facultatively anaerobic, chemolithoautotrophic, sulfur-oxidizing bacterium isolated from an underground crude-oil storage cavity. (8/39)

A facultatively anaerobic, chemolithoautotrophic, sulfur-oxidizing bacterium, strain YK-1(T), was isolated from an underground crude-oil storage cavity at Kuji in Iwate, Japan. The cells were motile, curved rods and had a single polar flagellum. Optimum growth occurred in a low-strength salt medium at pH 7.0 and 25 degrees C. It utilized sulfide, elemental sulfur, thiosulfate and hydrogen as the electron donors and nitrate as the electron acceptor under anaerobic conditions, but it did not use nitrite. Oxygen also served as the electron acceptor under the microaerobic condition (O(2) in the head space 1 %). It did not grow on sugars, organic acids or hydrocarbons as carbon and energy sources. The DNA G+C content of strain YK-1(T) was 45 mol%. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that its closest relative was Thiomicrospira denitrificans in the 'Epsilonproteobacteria', albeit with low homology (90 %). On the basis of physiological and phylogenetic data, strain YK-1(T) should be classified into a novel genus and species, for which the name Sulfuricurvum kujiense gen. nov., sp. nov. is proposed. The type strain is YK-1(T) (=JCM 11577(T)=MBIC 06352(T)=ATCC BAA-921(T)).  (+info)

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