Role of a protease in the adherence and enterotoxicity ofVibrio mimicus

The role of protease in the intestinal adherence and enterotoxicity ofVibrio mimicus strain E-33 was investigated by usingin vivo ligated rabbit ileal loop model.V. mimicus protease (VMP) was negative in fluid accumulation in the rabbit ileal loop. However, pre-treatment of the loop with purified VMP induced the elevation of fluid accumulation caused by the vibrios with the enhancement of bacterial adherence to the corresponding intestinal mucosa. The augmentation in the fluid accumulation could also be observed in the loop upon supplementation with VMP. In contrast, adherence to the mucosa was reduced by the simultaneous inoculation of VMP. The elevation in the fluid accumulation could also be observed in the loop supplemented or pre-treated with protease from V.cholerae orV. vulnificus. Furthermore, when the vibrios in the loops were accompanied by anti-VMP IgG antibody or inhibitors for VMP, such as ethyleneglycol-bis (\-aminoethylether)-N, N, N’, N’,-tetraacetic acid or tetraethylenepentamine, reductions in the adherence indices with consequent reductions in the fluid accumulations were observed. It is therefore, suggested that the protease produced by the pathogen contributes significantly toward the pathogenicity ofV. mimicus.     

Cellulases ofVibrio agar-liquefaciens isolated from sea mud

Vibrio agar-liquefaciens produced the component enzymes of a cellulase complex (exoglucanase, endoglucanase and -glucosidase). Whilst complete cellulase activity occurred in three media, each containing carboxymethylcellulose as the sole carbon source, different activities of the individual enzymes resulted. The enzymes showed a temporal sequence in their activity and differed in pH and temperature optima. NaCl enhanced the enzyme activities to varying degrees at different substrate concentrations.     

Pressure resistance of aerobic metabolism in eels from different water environments

Eels from different locations were tested comparing their energetic capacities to migrate by studying muscle (red and white) aerobic metabolism. As the migratory activity corresponds to a lengthy swimming activity at depth, their pressure resistance was evaluated by considering fish response to compression, mitochondrial respiration measured under pressure (101 ATA) and cytochrome c oxidase after 3 days under pressure. The results show that only fish from two of the sites have the metabolic capacities to cope with the high pressure encountered during migration.     

Development of a haemolysin gene‐based multiplex PCR for simultaneous detection of Vibrio campbellii,Vibrio harveyi and Vibrio parahaemolyticus

Aim: To develop a haemolysin (hly) gene‐based species‐specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus. Methods and Results: The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species‐specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. Significance and Impact of the Study: Because there is lack of simple, rapid and cost‐effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.     

Analysis of some factors involved in the virulence mechanism of Vibrio cholerae non-01

Abstract A study was carried out to evaluate the potential intestinal toxicity of 188 samples of Vibrio cholerae non-01 isolated from seawater found along the beaches of Rio de Janeiro city. Three different assays were carried out involving: (a) detection of vascular permeability factor (PF) in guinea pigs (together with assessment of two culture media for production of the toxin); (b) intestinal fluid accumulation (FA) in suckling mice; and (c) detection of haemolysin. The results demonstrated that both culture media gave a similar level of performance. In the animal assays, 43% of the samples induced PF in guinea pigs, 28.7% caused intestinal fluid accumulation in suckling mice, and 63.28% contained haemolysin. Only 4.25% of the samples gave positive results in all three tests.     

Distribution of type III secretion systems in Vibrio parahaemolyticus from the northern Gulf of Mexico

Aims: Two well‐characterized Vibrio parahaemolyticus pathogenicity factors – thermostable direct haemolysin (TDH) and TDH‐related haemolysin – are produced by strains containing the tdh and trh genes, respectively. Most strains of V. parahaemolyticus contain two nonredundant type III secretion systems (T3SS), T3SS1 and T3SS2, both of which contribute to pathogenicity. Furthermore, a recent study has revealed two distinct lineages of the V. parahaemolyticus T3SS2: T3SS2α and T3SS2β. The aim of this study was to determine the incidence of these pathogenicity factors in environmental isolates of V. parahaemolyticus. Methods and Results: We collected 130 V. parahaemolyticus isolates (TCBS agar) containing tdh and/or trh (determined by colony hybridization) from sediment, oyster and water in the northern Gulf of Mexico and screened them and 12 clinical isolates (PCR and agarose gel electrophoresis) for pathogenicity factors tdh, trh, T3SS1, T3SS2α and T3SS2β. The majority of potential pathogens were detected in the sediment, including all tdh^?/trh⁺ isolates. T3SS2α components were detected in all tdh⁺/trh ^? isolates and zero of 109 trh⁺ isolates. One T3SS2α gene, vopB2, was found in all tdh⁺/trh^? clinical strains but not in any of the 130 environmental strains. Fluorescence in situ hybridization adapted for individual gene recognition (RING‐FISH) was used to confirm the presence/absence of vopB2. T3SS2β was found in all tdh^?/trh⁺ isolates and in no tdh⁺/trh^? isolates. Conclusions: The combination of haemolysins found in each isolate consistently corresponded to the presence and type of T3SS detected. The vopB2 gene may represent a novel marker for identifying increased virulence among strains. Significance and Impact of the Study: This is the first study to confirm the presence of T3SS2β genes in V. parahaemolyticus strains isolated from the Gulf of Mexico and one of the few that examines the distribution and co‐existence of tdh, trh, T3SS1, T3SS2α and T3SS2β in a large collection of environmental strains.     

Seasonality and toxigenicity ofVibrio cholerae non-01 isolated from different components of pond ecosystems of Dhaka City,Bangladesh

Diarrhoea due toVibrio cholerae non-01 is common in Bangladesh. Four hundred and eighty samples, including plants, water, phytoplankton and sediment, were collected from five ponds in Dhaka every 15 days for one year.V. cholerae non-01 was isolated from 181 (38%) of the samples. Two peaks were evident: one in April and the other in August/September. Forty-three (23%) of the 181 isolates were examined for toxigenicity and 19% were cytotoxic to Y1 adrenal cells. This study provides evidence of the likely infectious nature of some ponds and may have relevance to the epidemiology of diarrhoea caused byV. cholerae non-01 in Bangladesh.     

Toxicity of the extracellular products ofVibrio damsela isolated from diseased fish

In this work we analyzed the pathogenic in vivo and in vitro activities for both fish and mammals of extracellular products (ECP) of several isolates ofVibrio damsela implicated in disease problems in marine culture. The ECP from all the strains were strongly lethal for fish (LD₅₀ ranging from 0.06 to 3.7 g protein/g fish) and mice (LD₅₀ ranging from 0.02 to 0.43 g protein/g mouse), causing death between 4 and 72 h after inoculation. These ECP samples possessed low proteolytic activity without production of caseinase, gelatinase, or elastase. However, most of them showed remarkable phospholipase and hemolytic activity for sheep, human, and turbot red blood cells. In addition, all the ECP samples were cytotoxic for fish and homoiothermic cell lines. The levels of enzymic and cytotoxic activities were clearly associated with the degree of virulence for fish. Moreover, the enzymic patterns of both live cells and ECP evaluated with the API-ZYM system were very similar among the strains, indicative that most of the activities are associated with exoenzymes.The in vivo and in vitro biological activities were considerably reduced after heat treatment (100°C for 10 min), but not totally lost in the highly virulent strains. Although we have demonstrated that the toxicity of the ECP is not directly associated with their lipopolysaccharides (LPS) content, these compounds could confer some heat-stabilizing effect to the toxic fractions.     

Ion-swimming speed variation ofVibrio cholerae cells

In the present work we report the variation in swimming speed ofVibrio cholerae with respect to the change in concentration of sodium ions in the medium. We have also studied the variation in swimming speed with respect to temperature. We find that the swimming speed initially shows a linear increase with the increase of the sodium ions in the medium and then plateaus. The range within which the swimming speed attains saturation is approximately the same at different temperatures.     

Production of alternatives to fuel oil from organic waste by the alkane-producing bacterium, Vibrio furnissii M1

AIMS: We investigated the production of alternatives to fuel oil through the bacterial metabolism of organic waste. The availability for this purpose of various sources of organic waste for hydrocarbon production by the alkane-producing bacterium, Vibrio furnissii M1, was examined. METHODS AND RESULTS: We screened 17 authentic compounds which can generally be found in organic waste for their hydrocarbon production. Carbon (3 mmol) in a 50-ml culture with acetic acid, lactic acid, butyric acid, succinic acid, malic acid, pentanoic acid, hexanoic acid glucose, xylose, starch or sucrose yielded 10-27 mg of alkanes or alkenes. The chain length of these alkanes or alkenes varied according to the culture from C14 to C27. Varying the ratio of carbon to nitrogen in the culture had no effect on the hydrocarbon production. Crude blackstrap molasses were also converted into alkanes with a conversion ratio of 20% (half of that in an authentic sucrose medium) of the total carbon consumption. CONCLUSIONS: V. furnissii M1 could produce hydrocarbons corresponding to kerosene or light oil from volatile fatty acids and sugars. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on bacterial hydrocarbon production from organic waste.     

Quantitative changes of CRF-like immunoreactivity in eels treated with reserpine and cortisol

Immunocytochemical techniques were applied to brain and pituitary sections of European eels after experimental manipulation of the pituitary-interrenal activity. A corticotropin-releasing factor (CRF) antiserum allowed the identification of a CRF-like peptide in the preoptic nucleus (PON) and rostral and caudal neurohypophysis (NH). CRF-immunoreactivity (ir) was not affected in solvent-injected eels compared to noninjected eels. Reserpine induced a stimulation of the pituitary interrenal axis, decreased ir-CRF in the rostral NH, but did not affect hypothalamic ir-CRF. Cortisol reduced the immunostaining of hypothalamic CRF-ir perikarya and perikarya cross-sectional area. In the rostral NH, CRF-ir fibers decreased in number and almost disappeared in long-term treated eels. The immunostaining of ACTH cells with ACTH antiserum was greatly reduced. These data suggest that cortisol induces a marked reduction in the activity of the CRF-corticotrop axis. The intensity of the ir-CRF staining observed in the caudal NH, close to the intermediate lobe (IL) was not significantly affected in reserpine-treated eels, and only slightly reduced in long-term cortisol-treated eels. The intensity of ir-CRF staining in the caudal NH did not correlate with melanocorticotropic activity or plasma cortisol level. These data suggest that immunoreactive CRF fibers in the rostral and caudal NH are differently regulated.     

Prolactin,hypercalcemia and corpuscles of stannius in seawater eels

In intact eels in sea water (SW), ovine prolactin (PRL) treatment induces hypercalcemia, but its mechanism of action, which is discussed, remains to be defined. Corpuscles of Stannius (CSt) are modified simultaneously: two cell categories then become evident. The first cell type (type 1) predominates; it has an oval shape and large granules, it shows a nuclear and nucleolar hypertrophy and a mitotic activity, and appears greatly stimulated by PRL; it may elaborate a hypocalcemic factor (hypocalcin) which would compensate for the PRL-induced hypercalcemia. A similar effect, although slightly less intense, is detected in hypophysectomized-PRL treated eels in SW. A second cell type (type 2), is more elongated, smaller in size, and has an oval nucleus and fine granules. Scarcely less active in SW, it is significantly stimulated by PRL despite an increased blood sodium and potassium level. This experiment does not help to clarify its function.     

Intracellular vacuolation induced by culture filtrates of Plesiomonas shigelloides isolated from environmental sources

AIMS: Potential virulence factors produced by culture filtrates of Plesiomonas shigelloides isolated from water were investigated. METHODS AND RESULTS: Culture filtrates of P. shigelloides strains were assayed for cytotoxic activity in CHO (Chinese hamster ovary), Vero (African green monkey kidney), HeLa (human cervix), HT29 (human epithelial intestinal) and SK6 (swine epithelial kidney) cells. Microscopic analyses revealed intensive cytoplasmic vacuolation including cell rounding and swelling, with gradual destruction of the monolayer in filtrate-treated cells. Neutral red assays showed that CHO, HeLa and Vero cells were the most sensitive to the vacuolating activity, which was evident within 30 min of culture filtrate exposure. This activity was inactived by heating at 56 degrees C for 15 min and partially neutralized by antiserum to the cytotoxin of Aeromonas hydrophila. All P. shigelloides strains had a cell-associated haemolysin in the agar plate assay. Three isolates were found to produce a cell-free haemolytic activity at 37 degrees C. In the suckling mouse test, two P. shigelloides culture supernatants were positive for enterotoxic activity. CONCLUSIONS: P. shigelloides culture filtrates isolated from aquatic environment cause intracellular vacuolation on mammalian cells, and produce haemolytic and enterotoxic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Plesiomonas strains.     

Comparison of outer membrane protein profiles of Vibrio vulnificus biotypes 1 and 2

Abstract The outer membrane proteins of 17 Vibrio vulnificus biotype 2 strains from Japanese and European cels, and 12 biotype 1 strains from clinical and environmental sources have been compared. The overall profile in both biotypes was similar, and a major protein band of molecular mass 36 kDa was detected in the majority of the strain. Differences in the minor bands allowed differentiation of strains from different origins, suggesting that outer membrane protein profiles could be useful as epidemiological markers in the species V. vulnificus . Immunoblotting with antisera to whole cells of selected strains of biotypes 1 and 2 showed a strong antigenic response to outer membrane proteins 66, 60, 48, 46 and 44 kDa; these were common to all strains examined, independent of their biotypes and origins. These results demonstrate the presence of antigenically related outer membrane proteins in both biotypes of V. vulnificus .     

Isolation and characterization of a visibly luminous variant ofVibrio fischeri strain ES114 from the sepiolid squidEuprymna scolopes

Vibrio fischeri strains isolated from light organs of the sepiolid squid Euprymna scolpes are non-visibly luminous and fast growing in laboratory culture, whereas in the symbiosis they are visibly luminous and slow growing. A spontaneous, visibly luminous, slow-growing variant was isolated from a laboratory culture of the squid-symbiotic V. fischeri strain ES114. Taxonomic and DNA-homology analyses demonstrated that the variant was V. fischeri and was very similar to the original form. However, the variant grew at one-fourth the rate of the original form, produced 30,000-fold more luminescence, induced luminescence at a lower cell density, and produced a higher level of V. fischeri luminescence autoinducer. Regulation of luminescence, nonetheless, was similar in the two forms and typical of V. fischeri with respect to responses to autoinducer, glucose, the iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid), and 3′:5′-cyclic AMP. Compared to the original form, cells of the variant were smaller, exhibited from zero to two polar, sheathed flagella instead of a tuft of three to eight flagella, produced a deeper yellow-orange pigment, did not acidify media containing glycerol, and produced a more distinct pellicle. The two forms also differed in the levels of several outer membrane and soluble proteins. These results establish a distinctive physiological, morphological, and biochemical dimorphism in V. fischeri ES114 in which the variant exhibits several traits similar to V. fischeri cells in the symbiotic state. The variant and its conversion from the original form in laboratory culture may provide insight into the properties of V. fischeri cells in the symbiosis and may serve as a model for elucidating the mechanism for their pleiotropic conversion upon colonization of the squid. Received: 10 January 1995 / Accepted: 24 May 1995     

鱼源溶藻弧菌胞外产物的特性研究

溶藻弧菌经胰胨大豆胨琼脂培养基(TSA 2%NaCl)培养48h后,培养物用PBS洗脱、离心,再经孔径0.22μm微孔滤膜过滤,制得胞外产物(Extracellular product,ECP)。经检测,该ECP具有淀粉酶、酪蛋白酶、卵磷脂酶和脂肪酶等4种酶活性,其中淀粉酶的活性最高,酪蛋白酶、脂肪酶活性次之,卵磷脂酶活性较低,未检测出明胶酶及脲酶活性;能溶解断斑石鲈(Pomadasys hasta)、眼斑拟石首鱼(Sciaenops ocellatus)和赤点石斑鱼(Epinephelus akaara)的红细胞,而不能溶解鸡、羊及O型人红细胞;对小白鼠和石斑鱼的LD50分别为0.08mg/g和0.17mg/g,95%可信限分别为0.06—0.12mg/g和0.07—0.28mg/g;50mmol/L EDTA和100mmol/L苯甲基磺酰氟能使胞外蛋白酶(Extracellular pro-teinase,ECPase)的活性分别下降83.40%和51.32%;ECPase对热稳定性较差,碱性条件下活性较高,其最适温度为50℃,最适pH为8.0。     

Changes in the gene expression profiles of the brains of male European eels (Anguilla anguilla) during sexual maturation

Background

The vertebrate brain plays a critical role in the regulation of sexual maturation and reproduction by integrating environmental information with developmental and endocrine status. The European eel Anguilla anguilla is an important species in which to better understand the neuroendocrine factors that control reproduction because it is an endangered species, has a complex life cycle that includes two extreme long distance migrations with both freshwater and seawater stages and because it occupies a key position within the teleost phylogeny. At present, mature eels have never been caught in the wild and little is known about most aspects of reproduction in A. anguilla. The goal of this study was to identify genes that may be involved in sexual maturation in experimentally matured eels. For this, we used microarrays to compare the gene expression profiles of sexually mature to immature males.

Results

Using a false discovery rate of 0.05, a total of 1,497 differentially expressed genes were identified. Of this set, 991 were expressed at higher levels in brains (forebrain and midbrain) of mature males while 506 were expressed at lower levels relative to brains of immature males. The set of up-regulated genes includes genes involved in neuroendocrine processes, cell-cell signaling, neurogenesis and development. Interestingly, while genes involved in immune system function were down-regulated in the brains of mature males, changes in the expression levels of several receptors and channels were observed suggesting that some rewiring is occurring in the brain at sexual maturity.

Conclusions

This study shows that the brains of eels undergo major changes at the molecular level at sexual maturity that may include re-organization at the cellular level. Here, we have defined a set of genes that help to understand the molecular mechanisms controlling reproduction in eels. Some of these genes have previously described functions while many others have roles that have yet to be characterized in a reproductive context. Since most of the genes examined here have orthologs in other vertebrates, the results of this study will contribute to the body of knowledge concerning reproduction in vertebrates as well as to an improved understanding of eel biology.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-799) contains supplementary material, which is available to authorized users.     

Study on the relationship of protease production and luminescence in Vibrio harveyi

AIMS: To demonstrate that Vibrio harveyi produces various types of toxins and how the production of those toxins is related with luminescence. METHODS AND RESULTS: Luminescence and toxicity of eight V. harveyi were evaluated. We demonstrated that all V. harveyi emitting luminescence were isolated from marine organisms and also showed that they were highly pathogenic when compared with culture collection V. harveyi based on cytotoxic assay test. On the contrary, V. harveyi isolated from shrimp farm showed no luminescence but showed high pathogenicity based on toxicity test. The effect of protease inhibitors on pathogenicity and luminescence was also investigated. We demonstrated that light emission of pathogenic V. harveyi remarkably decreased after addition of protease inhibitor. Furthermore, extracellular proteins from cell-free culture supernatant of luminescent and nonluminescent V. harveyi were compared using SDS-PAGE analysis. Results showed that there were differences in molecular weight and amount of proteins. CONCLUSIONS: Vibrio harveyi parasiting marine organisms have both luminescence and pathogenicity. Based on this study, luminescence and protease toxin activity in V. harveyi are related. Moreover, this paper clarified that V. harveyi produces various types of toxins. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study demonstrated that V. harveyi produces two kinds of toxins, haemolysin and protease toxin. It may be clear roots of V. harveyi toxin.     

Pathogenicity of live bacteria and extracellular products of motile Aeromonas isolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD₅₀ 10^5·4-10^7·6 cfu fish^-1) but no Aer. sobria , Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 10^8·4 cfu fish^-1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD⁵⁰ 1·0–3·2 μg (g fish)^-1) reproducing the symptoms associated with natural disease. ECP from non-pathogenic species were inactive on fish cell lines as well as being poorly lethal for eels (LD⁵⁰ > 9·2 μg (g fish)^-1). All these biological activities of Aeromonas ECP were lost after heat treatment. These findings indicate differences between pathogenic and non-pathogenic Aeromonas species with respect to the expression of virulence factors, and show that elastases, haemolysins and exotoxins play a leading role in the pathogenicity of motile Aeromonas for eels.