Inhibitory activity of antibiotic-producing marine bacteria against fish pathogens

The activity of antibiotic-producing marine bacteria was assayed against bacterial fish pathogens belonging to the genera Vibrio, Aeromonas, Pasteurella, Edwardsiella, Yersinia and Pseudomonas with the aim of evaluating the possible use of these marine strains for controlling epizootics in aquaculture. Inhibition tests on solid medium showed that, in general, the majority of fish bacteria were strongly sensitive to the marine bacteria. Only two strains ( Edwardsiella tarda and Pseudomonas aeruginosa ), were resistant to all the antibiotic-producing strains. The results of antagonism assays in sea water, however, varied according to the fish pathogens examined. Experiments conducted using cell-free supernatant fluids of marine bacteria demonstrated the involvement of antibiotic substances in the inhibition of fish pathogens.     

Chromosome-mediated iron uptake system in pathogenic strains of Vibrio anguillarum.

We describe in this work a new iron uptake system encoded by chromosomal genes in pathogenic strains of Vibrio anguillarum. This iron uptake system differs from the plasmid-encoded anguibactin-mediated system present in certain strains of V. anguillarum in several properties. The siderophore anguibactin is not utilized as an external siderophore, and although characteristic outer membrane proteins are synthesized under iron-limiting conditions, these are not related to the plasmid-mediated outer membrane protein OM2 associated with ferric anguibactin transport. Furthermore, the siderophore produced by the plasmidless strains may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants, although its behavior under various chemical treatments suggested major differences from that siderophore. Hybridization experiments suggested that the V. anguillarum chromosome-mediated iron uptake system is unrelated genetically to either the anguibactin or enterobactin-associated iron assimilation systems.     

Plasmids mediating iron uptake in Vibrio anguillarum strains isolated from turbot in Spain

Vibrio strains isolated from diseased turbot in an experimental fish farm on the Atlantic coast of northwest Spain were identified as Vibrio anguillarum. The isolates shared many biochemical characteristics with V. anguillarum strains obtained from other sources, and harboured a plasmid species that showed extensive homology with plasmid pJM1, carried by V. anguillarum strain 775 isolated from an epizootic in North America. Restriction endonuclease analysis showed that the two plasmids were very similar albeit not identical. The presence of the plasmid in the turbot isolates was associated with their ability to cause disease in fish. Plasmid-carrying bacteria could also grow under conditions of iron limitation. Two outer membrane proteins, of 86 and 79 kDal, were induced, and a similar siderophore activity to that produced by V. anguillarum 775 was also detected under these conditions. The 86 kDal outer membrane protein cross-reacted immunologically with antiserum raised against the outer membrane protein OM2 produced by strain 775. Nonvirulent plasmidless derivatives were unable to grow under iron-limiting conditions, and were also unable to produce either siderophore activity or the 86 kDal outer membrane protein, suggesting the plasmid-mediated nature of these components.     

Surface and virulence properties of environmental Vibrio cholerae non-O1 from Albufera Lake (Valencia, Spain).

A total of 140 environmental Vibrio cholerae non-O1 isolates, together with several culture collection strains from both environmental and clinical sources, were studied in relation to hemagglutination, surface hydrophobicity, and the enzymatic, hemolytic, cytotoxic, and enterotoxic activities of their extracellular products. A total of 78 and 62% of the strains produced hemagglutinins and exohemagglutinins, respectively. Four different hemagglutinating and two exohemagglutinating activities were found by using eight sugars in the inhibition assays. Cell-bound mannose-sensitive hemagglutination was detected mainly in chicken blood, whereas fucose-sensitive hemagglutination was recorded only in human blood. Cell-bound hemagglutinin resistant to all sugars tested was the only one related to surface hydrophobicity. The surface properties varied along the growth curves. The non-O1 strains displayed strong enzymatic and hemolytic activities, except for esculin hydrolysis. Of 26 non-O1 isolates selected for cytotoxin and enterotoxin production, 23 showed a wide spectrum of cytotoxic effects on cell lines of poikilothermic and homoiothermic species, but they were weakly enterotoxigenic in the infant mouse test. All extracellular products of cytotoxic strains were proteolytic, lipolytic, and hemolytic, and a high percentage produced hemagglutination of chicken blood. The cytotoxic factors in the non-O1 strains analyzed were not R plasmid mediated.     

Iron uptake by Pasteurella piscicida and its role in pathogenicity for fish.

We evaluated the iron uptake mechanisms in Pasteurella piscicida strains as well as the effect of iron overload on the virulence of these strains for fish. With this aim, the capacity of the strains to obtain iron from transferrin and heme compounds as well as their ability to overcome the inhibitory activity of fish serum was analyzed. All the P. piscicida strains grew in the presence of the iron chelator ethylene-diamine-di (O-hydroxyphenyl acetic acid) or of human transferrin, which was used by a siderophore-mediated mechanism. The chemical tests and cross-feeding assays showed that P. piscicida produced a siderophore which was neither a phenolate nor a hydroxamate. Cross-feeding assays as well as preliminary chromatographic analysis suggest that this siderophore may be chemically related to multocidin. All the P. piscicida isolates utilized hemin and hemoglobin as an iron source, since the virulence of the strains increased when the fish were preinoculated with these compounds. This effect was stronger in the avirulent strains (50% lethal dose was reduced by 4 logs when fish were pretreated with hemin or hemoglobin). Only the pathogenic P. piscicida isolates were resistant to the bactericidal action of the fresh fish serum. The nonpathogenic strains grew in fish serum only when it was heat-inactivated or when it was supplemented with ferric ammonium citrate, hemin, or hemoglobin. In all the strains, at least three iron-regulated outer membrane proteins (IROMPs) (105, 118, and 145 kDa) were increased when the strains were cultured in iron-restricted medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibility of turbot (Scophthalmus maximus), coho salmon (Oncorhynchus kisutch, and rainbow trout of. my kiss) to strains of Vibrio anguillarum and their exotoxins

The susceptibility of turbot, coho salmon, and rainbow trout to strains of Vibrio anguillarum of serotypes 01 and 02 and their extracellular products (ECP) was investigated in order to clarify the role of exotoxins in the mechanism of virulence of both serotypes. All V. anguillarum isolates were virulent for trout, salmon, and turbot. Despite the origin of the strains tested, rainbow trout was the most susceptible fish species to experimentally induced vibriosis. Coho salmon and turbot did not differ significantly in their susceptibility to V. anguillarum live cells. In contrast, the ECP from Vibrio strains of serotypes 01 and 02 exhibited similar lethal dose for turbot, salmon, and trout (ranging from 4.52 to 7.32 μg protein/g fish). Therefore, differences in susceptibility to vibriosis are not completely due to a differential sensitivity of fish to the extracellular products of Vibrio strains. The ECP from 7 of 10 V. anguillarum strains possessed vascular permeability factors, and all the extracts displayed proteolytic, hemolytic and cytotoxic activities. All the biological activities of ECP were lost after heat treatment at 80° C/10 min.     

RS‐1 enhances CRISPR‐mediated targeted knock‐in in bovine embryos

Targeted knock‐in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)‐assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR‐assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS‐1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single‐stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS‐1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS‐1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS‐1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.     

Comparison of PCR protocols for detecting Histoplasma capsulatum DNA through a multicenter study

BackgroundA multicenter study was conducted. A panel containing DNA from Histoplasma capsulatum, as well as negative and cross-reaction controls, was sent to five different laboratories, members of the MICOMOL network from CYTED Program.AimsThe objective was to assess the accuracy of different PCR protocols to detect H. capsulatum DNA.MethodsSeven different PCR protocols were tested. They were based on PCR techniques and used unicopy and multicopy targets.ResultsMost of these protocols (4/7) were able to detect the smallest amounts of fungal DNA (10² fg/μl). Overall sensitivity was 86% and specificity was 100%. The protocol based on a unicopy target (SCAR₂₂₀) presented lower sensitivity (43%) but 100% specificity. The real-time protocols tested were highly reproducible, sensitive, and specific. Neither false positives nor cross-reactions were detected in any protocol.ConclusionsAll laboratories were able to amplify H. capsulatum DNA, and real-time PCR seems to be a promising tool to efficiently detect this pathogen in clinical samples.     

Mycosis Due to Tropicoporus tropicalis (= Inonotus tropicalis) in a Domestic Dog

Mycopathologia - Mycelial basidiomycetes rarely produce mycoses in animals including humans. We report a case of a 9-year-old female mongrel dog with lesions in the prescapular lymph nodes. The...     

Phenotypic characterization and description of two major O-serotypes in Tenacibaculum maritimum strains from marine fishes

Tenacibaculum maritimum is the etiological agent of marine flexibacteriosis disease, with the potential to cause severe mortalities in various cultured marine fishes. The development of effective preventive measures (i.e. vaccination) requires biochemical, serological and genetic knowledge of the pathogen. With this aim, the biochemical and antigenic characteristics of T. maritimum strains isolated from sole, turbot and gilthead sea bream were analysed. Rabbit antisera were prepared against sole and turbot strains to examine the antigenic relationships between the 29 isolates and 3 reference strains. The results of the slide agglutination test, dot-blot assay and immunoblotting of lipopolysaccharides (LPS) and membrane proteins were evaluated. All bacteria studied were biochemically identical to the T. maritimum reference strains. The slide agglutination assays using O-antigens revealed cross-reaction for all strains regardless of the host species and serum employed. However, when the dot-blot assays were performed, the existence of antigenic heterogeneity was demonstrated. This heterogeneity was supported by immunoblot analysis of the LPS, which clearly revealed 2 major serological groups that were distinguishable without the use of absorbed antiserum: Serotypes O1 and O2. These 2 serotypes seem to be host-specfic. In addition, 2 sole isolates and the Japanese reference strains displayed cross-reaction with both sera in all serological assays, and are considered to constitute a minor serotype, O1/O2. Analysis of total and outer membrane proteins revealed that all strains share a considerable number of common bands that are antigenically related.