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.     

Antagonistic activity of two potential probiotic bacteria from fish intestines and investigation of their effects on growth performance and immune response in rainbow trout (Oncorhynchus mykiss)

The probiotic activity of two bacterial strains, Lactobacillus casei and Lactobacillus plantarum, isolated from common carp intestines was studied using antagonistic tests in vitro against Yersinia ruckeri. Randomly assigned to triplicate groups were 450 rainbow trout (mean weight, 20 ± 3 g) fed three different diets: a commercial feed, or the same feed incorporated into either 5 × 10⁷ CFU g^?1 of L. casei or L. plantarum. After a 30‐day feeding trial, 30 fish in each group were challenged with Yersinia ruckeri by intraperitoneal injection. Growth parameters were significantly increased in both treatment groups. Immune parameters such as lysozyme activity, alternative complement activity and total immunoglobulin level were significantly higher in the L. casei group than in fish fed the control diet, while no significant differences were revealed between the L. plantarum and control groups. Mortality rates of fish fed L. casei and L. plantarum were lower than in fish fed the control diet after challenging with Y. ruckeri.     

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.     

Rapid genotyping assays for the identification and differentiation of Yersinia ruckeri biotype 2 strains

Aims: To establish PCR‐based assays for the rapid identification and differentiation of each of four known biotype 2 (BT2) phenotype‐causing alleles in Yersinia ruckeri strains currently circulating in Europe and the United States. Methods and Results: Novel assays were developed relying on detection of mutant allele‐specific changes in restriction enzyme cleavage sites within targeted PCR products. The developed assays were validated against isolates previously genotyped by DNA sequencing. Conclusions: The described methods were specific, rapid and simple to perform and interpret. Significance and Impact of the Study: The developed genotyping assays provide a valuable tool for identification and differentiation of specific BT2 strains of Y. ruckeri. These assays will be critical for the design and validation of new vaccines or other measures meant to control BT2 strains.     

The inverse autotransporters of Yersinia ruckeri,YrInv and YrIlm,contribute to biofilm formation and virulence

Yersinia ruckeri causes enteric redmouth disease (ERM) that mainly affects salmonid fishes and leads to significant economic losses in the aquaculture industry. An increasing number of outbreaks and the lack of effective vaccines against some serotypes necessitates novel measures to control ERM. Importantly, Y. ruckeri survives in the environment for long periods, presumably by forming biofilms. How the pathogen forms biofilms and which molecular factors are involved in this process, remains unclear. Yersinia ruckeri produces two surface-exposed adhesins, belonging to the inverse autotransporters (IATs), called Y. ruckeri invasin (YrInv) and Y. ruckeri invasin-like molecule (YrIlm). Here, we investigated whether YrInv and YrIlm play a role in biofilm formation and virulence. Functional assays revealed that YrInv and YrIlm promote biofilm formation on different abiotic substrates. Confocal microscopy revealed that they are involved in microcolony interaction and formation, respectively. The effect of both IATs on biofilm formation correlated with the presence of different biopolymers in the biofilm matrix, including extracellular DNA, RNA and proteins. Moreover, YrInv and YrIlm contributed to virulence in the Galleria mellonella infection model. Taken together, we propose that both IATs are possible targets for the development of novel diagnostic and preventative strategies to control ERM.     

Pathogenic activities of live cells and extracellular products of the fish pathogen Pasteurella piscicida.

The pathobiological activities in vivo and in vitro of live cells and extracellular products (ECP) of eleven Pasteurella piscicida strains of different origin were examined. Infectivity trials showed that P. piscicida did not possess strict host specificity since the majority of the isolates were virulent for gilthead seabream, rainbow trout and turbot, with LD50 values ranging between 10(3) and 10(6) live cells. However, none of the strains tested were pathogenic for mice (LD50 > 10(8) cells)). In addition, the ECP were strongly toxic for fish (LD50 ranging from 1.0 to 4.6 micrograms protein per g fish), which clearly demonstrates their important role in the pathogenesis of pasteurellosis. All the ECP samples were cytotoxic for fish and homoiothermic cell lines, possessed notable phospholipase activity and displayed haemolytic activity for sheep, salmon and turbot erythrocytes (but not for trout erythrocytes). However, the production of proteolytic enzymes differed among the P. piscicida strains. Although no strain displayed elastase activity, five isolates (the Japanese and Italian strains) hydrolysed casein and gelatin. All these biological activities in vivo and in vitro were lost after heat treatment (100 degrees C for 10 min). The general enzymic patterns of both live cells and ECP evaluated by the API-ZYM system also revealed some variation among the P. piscicida isolates. Generally, whole cells showed a wider range of enzymic activities than ECP. The results presented here are important for the selection of strains in the development of effective polyvalent pasteurellosis vaccines containing both whole cells and ECP.     

Antigenic and cross-protection studies of biotype 1 and biotype 2 isolates of Yersinia ruckeri in rainbow trout, Oncorhynchus mykiss (Walbaum)

Aims: The study investigated antigen characteristics of biotype (bt) 1 and bt 2 isolates of Yersinia ruckeri. Methods and Results: The cell surface characteristics of Y. ruckeri were compared for their antigenic characteristics using polyclonal antibodies that revealed that both biotypes had a homogenous whole‐cell protein antigenic profile. Notable differences in the antigenic properties were observed in the lipopolysaccharide profile of both biotypes. Two iron‐regulated outer membrane proteins (IROMP) of c. 90 and 100 kDa were shown to be major specific antigens. The results demonstrate for the first time differences in antigens between bt 1 and bt 2 isolates of serotype O1 isolates of Y. ruckeri. The protection induced in rainbow trout by a commercial monovalent, and bivalent inactivated vaccine was tested with the outcome that the ability of isolates to cause mortality in vaccinated fish varied with geographical location. In this context, vaccination studies suggested that the O antigen was the dominant immunogenic molecule involved in protection against the disease. Conclusions: The O antigen of Y. ruckeri was the dominant immunogenic molecule involved in the protection of rainbow trout against enteric redmouth disease. Significance and Impact of the Study: There are distinct phenotypic and antigenic differences in Y. ruckeri bt 1 and bt 2 with O antigen recognized as the dominant immunogenic molecule. The data have significance in explaining the lack of success of the earlier monovalent vaccine and demonstrate the effectiveness of the newer bivalent vaccine.     

Identification of Flagellar Motility Genes in Yersinia ruckeri by Transposon Mutagenesis

Here we demonstrate that flagellar secretion is required for production of secreted lipase activity in the fish pathogen Yersinia ruckeri and that neither of these activities is necessary for virulence in rainbow trout. Our results suggest a possible mechanism for the emergence of nonmotile biotype 2 Y. ruckeri through the mutational loss of flagellar secretion.Yersinia ruckeri is the etiologic agent of enteric redmouth disease, a disease of salmonid fish species that is found worldwide in areas where salmonid fish species are farmed (3, 6, 18, 20). Vaccines for enteric redmouth disease have been used successfully for nearly 3 decades and consist of immersion-applied, killed whole-cell preparations of motile serovar 1 Y. ruckeri strains (22). Recently though, outbreaks have been reported in vaccinated fish at trout farms in the United Kingdom (2), Spain (9), and the United States (1). The Y. ruckeri strains isolated from these outbreaks are uniformly atypical serovar 1 isolates lacking both flagellar motility and secreted lipase activity. These variants have been classified as Y. ruckeri biotype 2 (BT2) and are believed to have a reduced sensitivity to immersion vaccination (2). The objective of this study was to obtain a better understanding of the emergence of BT2 Y. ruckeri by identifying genetic elements necessary for expression of the Y. ruckeri flagellum and determining the role that the flagellum plays in virulence by using a rainbow trout infection model.     

Occurrence and Phenotypic Characterization of Yersinia ruckeri Strains with Biofilm-Forming Capacity in a Rainbow Trout Farm

The presence of Yersinia ruckeri in a French fish farm was investigated. Y. ruckeri was isolated mainly from algae and sediment samples rather than from water. Twenty-two Y. ruckeri isolates were obtained, and three strains were distinguished by enterobacterial repetitive intergenic consensus PCR amplification. These strains were able to adhere to solid supports. This characteristic was correlated with flagellum-mediated motility. Killing experiments showed that sessile cells were more resistant to oxolinic acid than their planktonic counterparts. Our results demonstrate that surface colonization of fish farm tanks by Y. ruckeri biofilms is a potential source of recurrent infection for extended periods of time.     

Highly sensitive detection and quantification of the pathogen Yersinia ruckeri in fish tissues by using real-time PCR

Yersinia ruckeri is the causative agent of enteric redmouth diseases (ERM) and one of the major bacterial pathogens causing losses in salmonid aquaculture. Since recent ERM vaccine breakdowns have been described mostly attributed to emergence of Y. ruckeri biotype 2 strains, rapid, reproducible, and sensitive methods for detection are needed. In this study, a real-time polymerase chain reaction (PCR) primer/probe set based on recombination protein A (recA) gene was designed and optimized to improve the detection of Y. ruckeri. The primer/probe set proved to have a 100 % analytical specificity and a sensitivity of 1.8 ag μl^?1, equivalent to 1.7 colony-forming units (CFU)?ml^?1, for purified DNA, 3.4 CFU g^?1 for seeded liver, kidney, and spleen tissues, and 0.34 CFU/100 μl^?1 for seeded blood, respectively. The assay was highly reproducible with low variation coefficient values for intra- and inter-run experiments (2.9 % and 9.5 %, respectively). Following optimization, the assay was used to detect changes in the bacterial load during experimental infection. Rainbow trout (Onchorhynchus mykiss) were exposed to two strains of Y. ruckeri (biotype 1 and biotype 2) by intraperitoneal inoculation. Internal organs (liver, kidney, spleen) and blood were biopsied from dead fish daily for 15 days to quantify copies of pathogen DNA per gram of tissue. The findings showed the efficacy of this real-time PCR assay to quantify Y. ruckeri cells in the fish tissues and also confirmed this assay as a non-lethal method for the detection of this pathogen in blood samples.     

Characterization of probiotic properties of lactic acid bacteria isolated from an estuarine environment for application in rainbow trout (Oncorhynchus mykiss, Walbaum) farming

Bacterial attachment to fish surfaces and the capacity to compete with pathogens for adhesion sites are essential characteristics in order to select a candidate probiotic for aquaculture. Twelve lactic acid bacteria (LAB) isolated from fish and sediments from Bahía Blanca Estuary, Argentina, were examined for in vitro adhesion to rainbow trout mucus, cell surface properties and competitive exclusion against two salmonid pathogens, Yersinia ruckeri and Aeromonas salmonicida. In order to assess their survival through the digestive tract, pH and rainbow trout bile tolerance were evaluated. All LAB strains survived for 1.5 h incubation in 10% rainbow trout bile. Most of the strains survived 1.5 h at pH 3.0 and three of them showed a reduction of viable counts lower than 2 logarithms, with respect to control (pH 6.5). Only a few strains showed tolerate pH 2.0. All the strains were able to attach to rainbow trout skin mucus (10⁴–10⁶ cells/cm²), to glass (10⁴–10⁵ cells/cm²) and to stainless steel (10³–10⁴ cells/cm²). Sixty percent of LAB strains were capable of competing with and successfully excluding Y. ruckeri and all strains were able to displace it. Against A. salmonicida, 75% of LAB strains competed successfully, 50% were capable of displacing and 60% excluded this pathogen. Our data suggest the potential of these strains as anti-infective agents for use in rainbow trout culture. This study is the first report on the probiotic potential of LAB strains isolated from an estuarine environment from Argentina.     

Adhesive Properties and Hydrolytic Enzymes of Oral <Emphasis Type="Italic">Candida albicans</Emphasis> Strains

Several virulence factors in Candida albicans strains such as production of hydrolytic enzymes and biofilm formation on surfaces and cells can contribute to their pathogenicity. For this, control of this opportunistic yeast is one of the factors reducing the nosocomial infection. The aim of this study was to investigate biofilm formation on polystyrene and polymethylmethacrylate and the production of hydrolytic enzymes in Candida albicans strains isolated from the oral cavity of patients suffering from denture stomatitis. All strains were identified by macroscopic, microscopic analysis and the ID 32 C system. Our results showed that 50% of the total strains produced phospholipase. Furthermore, protease activity was detected in seven (35%) strains. All Candida albicans strains were beta haemolytic. All C. albicans strains adhered to polystyrene 96-well microtiter plate at different degrees, and the metabolic activity of C. albicans biofilm formed on polymethylmethacrylate did not differ between tested strains. The atomic force micrographs demonstrated that biofilm of Candida albicans strains was organized in small colonies with budding cells.     

Toxicity of Crude Extracellular Products of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> on Rohu, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.)

In the present study the haemolytic and proteolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila (CAHH14 strain) were studied with respect to temperature and different time of incubation as well as its lethal toxicity on rohu, Labeo rohita. The strain was isolated from Catla catla (showing abdominal dropsy symptom) collected from the pond of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India and was characterized on the basis of biochemical tests. The highest production of haemolysin was achieved when the bacteria was grown at 35°C for 30 h. The proteolytic activity was found to be highest when the bacterium was grown at 30°C for 36 h. The haemolytic and proteolytic toxin produced by Aeromonas hydrophila was found to be lethal to rohu (LD₅₀ 1.7 × 10⁴ cfu/ml). The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min. This indicates that protease activity and haemolytic activity of A. hydrophila ECP was temperature dependant.     

The repeat structure of two paralogous genes,Yersinia ruckeri invasin (yrInv) and a “Y. ruckeri invasin-like molecule”, (yrIlm) sheds light on the evolution of adhesive capacities of a fish pathogen

Inverse autotransporters comprise the recently identified type Ve secretion system and are exemplified by intimin from enterohaemorrhagic Escherichia coli and invasin from enteropathogenic Yersiniae. These proteins share a common domain architecture and promote bacterial adhesion to host cells. Here, we identified and characterized two putative inverse autotransporter genes in the fish pathogen Yersinia ruckeri NVH_3758, namely yrInv (for Y. ruckeri invasin) and yrIlm (for Y. ruckeri invasin-like molecule). When trying to clone the highly repetitive genes for structural and functional studies, we experienced problems in obtaining PCR products. PCR failures and the highly repetitive nature of inverse autotransporters prompted us to sequence the genome of Y. ruckeri NVH_3758 using PacBio sequencing, which produces some of the longest average read lengths available in the industry at this moment. According to our sequencing data, YrIlm is composed of 2603 amino acids (7812 bp) and has a molecular mass of 256.4 kDa. Based on the new genome information, we performed PCR analysis on four non-sequenced Y. ruckeri strains as well as the sequenced. Y. ruckeri type strain. We found that the genes are variably present in the strains, and that the length of yrIlm, when present, also varies. In addition, the length of the gene product for all strains, including the type strain, was much longer than expected based on deposited sequences. The internal repeats of the yrInv gene product are highly diverged, but represent the same bacterial immunoglobulin-like domains as in yrIlm. Using qRT-PCR, we found that yrIlm and yrInv are differentially expressed under conditions relevant for pathogenesis. In addition, we compared the genomic context of both genes in the newly sequenced Y. ruckeri strain to all available PacBio-sequenced Y. ruckeri genomes, and found indications of recent events of horizontal gene transfer. Taken together, this study demonstrates and highlights the power of Single Molecule Real-Time technology for sequencing highly repetitive proteins, and sheds light on the genetic events that gave rise to these highly repetitive genes in a commercially important fish pathogen.     

The UvrY response regulator of the BarA–UvrY two-component system contributes to Yersinia ruckeri infection of rainbow trout (Oncorhynchus mykiss)

To identify virulence-associated genes of a fish pathogen Yersinia ruckeri, we screened a total of 1056 mini-Tn5-Km2 signature-tagged mutants in rainbow trout by immersion challenge. Of 1056, 25 mutants were found survival-defective as they could not be re-isolated from fish kidney 7 days after infection. Mutated gene in F2-4 mutant, one of the 25 mutants, was homologous to uvrY that encodes UvrY response regulator of BarA–UvrY two-component system (TCS). Mutant F2-4 was significantly more sensitive (P < 0.05) to H₂O₂-mediated killing and was less able to infect Epithelioma papulosum cyprini cells. However, UvrY mutation did not affect survival of F2-4 mutant in the presence of non-immune fish serum and its ability to grow under iron starvation. In a time-course co-infection, mutant F2-4 had lower bacterial loads on day 1 itself, and by day 5 there was nearly a 1,000-fold difference in infection levels of the parent and mutant strains. The barA homolog of Y. ruckeri was PCR-amplified and sequence analyses identified four domains that were characteristic of hybrid histidine kinases. To conclude, the BarA–UvrY TCS contributes to the pathogenesis of Y. ruckeri in its natural host rainbow trout, possibly by regulating invasion of epithelial cells and sensitivity to oxidative stress induced by immune cells.     

Potential of ochratoxin A production by Aspergillus carbonarius strains isolated from grapes at different ecological factors

Aspergillus carbonarius is known to colonize and produce ochratoxin A (OTA) on grapes and its derived products which is harmful to humans. We screened and tested A. carbonarius strains which isolated from grapes for production of OTA and selected three high OTA producing strains (ACSP1, ACSP2, ACSP3) for this study. These strains were further tested for their ability to produce OTA at different ecological factors [temperature 15, 25, 30, 35°C; water activity (a_w) 0.98, 0.95, 0.90, 0.88; and pH 4.0, 7.0, 9.0, 10.0]. Out of the three strains tested, A. carbonarius ACSP3 produced high levels of OTA than ACSP2 and ACSP1 in all the ecological factors. At 30°C A. carbonarius strains produced the highest OTA compared with other temperature regimes. With reference to water activity, a_w 0.98 favoured mycelial growth and accumulation of more OTA with all the three A. carbonarius strains. Further, pH 4.0 was encouraged the greatest production of OTA in all the strains. No growth was observed at a_w 0.88 and pH 10.0 in all the three strains except the strain ACSP3 at high pH. Our work demonstrated that temperature 30°C, a_w 0.98 and pH 4.0 is optimum for growth and production of OTA by A. carbonarius strains. Maximum amounts of OTA were found at earlier growth stages (7–9 days of incubation) in all the strains of A. carbonarius. The present study revealed that different ecological factors had great impact on OTA production by A. carbonarius which is useful for understanding OTA contamination and to develop proper management practices in future research programmes.     

The effects ofPerkinsus marinusextracellular products and purified proteases on oyster defence parametersin vitro

The effects of extracellular products (ECP) and purified proteases from the protozoan parasitePerkinsus marinuson three host defence parameters (haemocyte motility, lysozyme and haemagglutinin) of the eastern oyster,Crassostrea virginica, were investigated. ECP with high proteolytic activities, as well as purified proteases, significantly decreased the random migration of haemocytes through micro-porous filters in Boyden chambers. Stimulation of haemocyte migration byP. marinuscells orP. marinuscell lysate was also dramatically reduced by ECP and purified proteases. Incubation of oyster plasma with ECP and purified proteases caused a significant decrease in lysozyme activity and also appeared to reduce haemagglutinin titres. These data suggest thatP. marinusECP, as well as the proteolytic fraction of the ECP, can modulate some defence parameters of oystersin vitro.     

Safety and probiotic functionality of isolated goat milk lactic acid bacteria

Purpose

Lactic acid bacteria (LAB) are traditionally employed in the food industry. LAB strains from goat milk may also present probiotic potential, and it is fundamental to study the safety and functionality aspects which are desirable for their use in food. The objective of this study was to verify the probiotic potential of lactic bacteria isolated from goat milk.

Methods

The presence of safety-related virulence factors (hemolytic activity, gelatinase production, coagulase, and sensitivity to antibiotics) as well as functionality (exopolysaccharide (EPS) production, proteolytic activity, autoaggregation, gas production, survival in the gastrointestinal tract, and antimicrobial activity against bacteria that impair oral health) were determined.

Result

The selected LAB strains are safe against the evaluated parameters and have characteristics of possible probiotic candidates. Especially L. plantarum (DF60Mi) and Lactococcus lactis (DF04Mi) have potential to be added to foods because they have better resistance to simulated gastrointestinal conditions. In addition, they are isolated with already proven antimicrobial activity against Listeria monocytogenes, an important food-borne pathogen. DF60Mi was able to produce EPS (exopolysaccharides). LS2 and DF4Mi strains, both Lactococcus lactis subsp. lactis, demonstrated antimicrobial activity against S. mutans ATCC 25175, a recurrent microorganism in oral pathologies, mainly caries.

Conclusion

This study provides subsidies for future exploration of the potentialities of these LAB strains for both the development of new functional foods and for application in oral health.

     

Isolation and characterization of OmpF-like porin from <Emphasis Type="Italic">Yersinia ruckeri</Emphasis>

The polypeptide profile of the porin protein fraction of Yersinia ruckeri, a Gram-negative bacterium causing yersiniosis in fish, has been shown to depend on cultivation temperature. OmpF-like porins are expressed mainly in the outer membrane (OM) of the “cold” variant (4°C) of the microorganism and OmpC-like proteins are expressed in the OM of the “warm” variant (37°C). Both types of porins are present in the OM of Y. ruckeri at room temperature. The OmpF-like porin of the “cold” variant was isolated and characterized. The molecular weight and primary structure of the protein were determined. The methods of optical spectroscopy (circular dichroism and intrinsic protein fluorescence) have shown that the protein has a spatial structure typical of β-structured porins from the OM of Gram-negative bacteria. The functional activity of isolated protein was characterized by the bilayer lipid membrane (BLM) technique. The most probable level of channel conductivity was 320 ± 60 pS, corresponding to the channel conductivity of OmpF porins of the genus Yersinia. The distinctive feature of OmpF porin from Y. ruckeri is high thermostability of its functionally active conformation: the protein forms stable pores in the BLM even after heating to 85°C.