Impact of water quality parameters on geosmin levels and geosmin producers in European recirculating aquaculture systems.

AIMS: Geosmin is associated with off-flavour problems in recirculating aquaculture systems (RAS) and represents an economic problem for the aquaculture industry. This study aims at investigating factors influencing the composition of the bacterial microbiota, in particular the presence of geosmin producers and the environmental and farming factors favouring geosmin accumulation. METHODS AND RESULTS: Several water quality parameters were correlated to the composition of the microbiota with special emphasis on the presence of geosmin producers within 26 different RAS from four European countries. Three novel groups of geosmin-producing bacteria were quantified to identify potential correlations with geosmin concentration. CONCLUSIONS: The microbiome differed significantly between systems. However, phosphate levels, calcium levels and redox potential correlated to geosmin concentration in the water and the presence of the Actinomycetales geosmin-producers but not with the presence of other groups of geosmin-producing bacteria. Oxygen levels and conductivity were found to negatively correlate with geosmin concentration. A large proportion of the detected geosmin producers represented novel taxonomic groups not previously linked with this activity. SIGNIFICANCE AND IMPACT OF THE STUDY: These results improve our understanding of the diversity of microbiota in RAS and the water quality parameters favouring the populations of geosmin-producing bacteria and the production of geosmin.

Delivering the pain: an overview of the type III secretion system with special consideration for aquatic pathogens.

Gram-negative bacteria are known to subvert eukaryotic cell physiological mechanisms using a wide array of virulence factors, among which the type three-secretion system (T3SS) is often one of the most important. The T3SS constitutes a needle-like apparatus that the bacterium uses to inject a diverse set of effector proteins directly into the cytoplasm of the host cells where they can hamper the host cellular machinery for a variety of purposes. While the structure of the T3SS is somewhat conserved and well described, effector proteins are much more diverse and specific for each pathogen. The T3SS can remodel the cytoskeleton integrity to promote intracellular invasion, as well as silence specific eukaryotic cell signals, notably to hinder or elude the immune response and cause apoptosis. This is also the case in aquatic bacterial pathogens where the T3SS can often play a central role in the establishment of disease, although it remains understudied in several species of important fish pathogens, notably in Yersinia ruckeri. In the present review, we summarise what is known of the T3SS, with a special focus on aquatic pathogens and suggest some possible avenues for research including the potential to target the T3SS for the development of new anti-virulence drugs.

Effects of siRNA silencing on the susceptibility of the fish cell line CHSE-214 to Yersinia ruckeri.

Yersinia ruckeri is a facultative intracellular enterobacterium mostly known as the causative agent of enteric redmouth disease in salmonid fish. In the present study, we applied RNA inhibition to silence twenty pre-selected genes on the genome of a fish cell line (CHSE-214) followed by a gentamicin assay to quantify the effect of silencing on the cells' susceptibility to infection and found that silencing of 18 out of 20 genes significantly reduced the number of Y. ruckeri recovered. These findings improve our understanding of the infection process by Y. ruckeri and of the interactions between this bacterial pathogen and host cells.

Aeromonas spp. suggested as the causative agents of red spot disease in northern Vietnamese grass carp Ctenopharyngodon idella.

In northern Vietnam, a disease called 'red spot disease' has been causing high morbidity and mortality in populations of farmed grass carp Ctenopharyngodon idella for about 2 decades. The name 'red spot disease' refers to a condition characterised by haemorrhagic lesions, reddening and ulceration of the skin. Eight different bacterial isolates, namely Aeromonas hydrophila, A. sobria, Staphylococcus epidermidis, Vibrio alginolyticus, Pseudomonas fluorescens, P. luteola, Citrobacter freundii and P. putida, were isolated from diseased grass carp and used for experimental infection of the same species. Fish were challenged with the different bacterial isolates both by immersion and intramuscular injection. Different concentrations of bacteria were tested to evaluate their pathogenicity. Injection with 1 × 105 CFU of A. hydrophila and A. sobria resulted in clinical signs identical to those of red spot-diseased grass carp in Vietnam. None of the other bacterial isolates tested caused any morbidity or mortality in fish challenged either intramuscularly (1 × 106 CFU) or by bath immersion (1 × 106 or 1 × 108 CFU ml-1).

The nature and consequences of co-infections in tilapia: A review.

Co-infections commonly arise when two or multiple different pathogens infect the same host, either as simultaneous or as secondary concurrent infection. This potentiates their pathogenic effects and leads to serious negative consequences on the exposed host. Numerous studies on the occurrence of the bacterial, parasitic, fungal and viral co-infections were conducted in various tilapia species. Co-infections have been associated with serious negative impacts on susceptible fish because they increase the fish susceptibility to diseases and the likelihood of outbreaks in the affected fish. Co-infections can alter the disease course and increase the severity of disease through synergistic and, more rarely, antagonistic interactions. In this review, reports on the synergistic co-infections and their impacts on the affected tilapia species are highlighted. Additionally, their pathogenic mechanisms are briefly discussed. Tilapia producers should be aware of the possible occurrence of co-infections and their effects on the affected tilapia species and in particular of the clinical signs and course of the disease. To date, there is still limited information regarding the pathogenicity mechanisms and pathogen interactions during these co-infections. This is generally due to low awareness regarding co-infections, and in many cases, a dominant pathogen is perceived to be of vital importance and hence becomes the target of treatment while the treatment of the co-infectious agents is neglected. This review article aimed at raising awareness regarding co-infections and helping researchers and fish health specialists pay greater attention to these natural cases, leading to increased research and more consistent diagnosis of co-infectious outbreaks in order to improve control strategies to protect tilapia when infected with multiple pathogens.

Environmental transformation of n-TiO2 in the aquatic systems and their ecotoxicity in bivalve mollusks: A systematic review.

Over the past decades, titanium dioxide nanoparticles (n-TiO2) have been extensively used in several industrial applications and the manufacture of novel consumer products. Although strict regulations have been put in place to limit their release into the aquatic environment, these nanoparticles can still be found at elevated levels within the environment, which can result in toxic effects on exposed organisms and has possible implications in term of public health. Bivalve mollusks are a unique and ideal group of shellfish for the study and monitoring the aquatic pollution by n-TiO2 because of their filter-feeding behaviour and ability to accumulate toxicants in their tissues. In these animals, exposure to n-TiO2 leads to oxidative stress, immunotoxicity, neurotoxicity, and genotoxicity, as well as behavioral and physiological changes. This review summarizes the uptake, accumulation, and fate of n-TiO2 in aquatic environments and the possible interactions between n-TiO2 and other contaminants such as heavy metals and organic pollutants. Moreover, the toxicological impacts and mechanisms of action are discussed for a wide range of bivalve mollusks. This data underlines the pressing need for additional knowledge and future research plans for the development of control strategies to mitigate the release of n-TiO2 to the aquatic environment to prevent the toxicological impacts on bivalves and protect public health.

Effect of immunostimulatory feed supplements on the development of acquired immunity in rainbow trout (Oncorhynchus mykiss).

Immunostimulatory feed supplements are an increasingly common feature of aquaculture management and their benefit has been confirmed for a wide area of products. However, these investigations have often focused on the benefit of these supplements on the innate immune system. In the current project, we investigated a mixture of two commercial feed supplements (Biotronic® Top 3 and Levabon® Aquagrow E) with a known protective effect against bacterial infections. The effect of the supplemented diet on antibody titters of Oncorhynchus mykiss vaccinated against Yersinia ruckeri was determined by ELISA. Furthermore, an infection trial was performed to confirm the effect of the supplements on the survival of the fish. Finally, their effects on the growth parameters of the fish were also determined. The results from this study found no significant effect on the general antibody titters. However, when considering only the titters of specific anti-Y ruckeri antibodies, the supplemented feed was associated with an improved response to the vaccine, significantly better than in the fish that had received the control feed.

Quorum Quenching Properties and Probiotic Potentials of Intestinal Associated Bacteria in Asian Sea Bass Lates calcarifer.

Quorum quenching (QQ), the enzymatic degradation of N-acyl homoserine lactones (AHLs), has been suggested as a promising strategy to control bacterial diseases. In this study, 10 AHL-degrading bacteria isolated from the intestine of barramundi were identified by 16S rDNA sequencing. They were able to degrade both short and long-chain AHLs associated with several pathogenic Vibrio species (spp.) in fish, including N-[(RS)-3-Hydroxybutyryl]-l-homoserine lactone (3-oh-C4-HSL), N-Hexanoyl-l-homoserine lactone (C6-HSL), N-(ß-Ketocaproyl)-l-homoserine lactone (3-oxo-C6-HSL), N-(3-Oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL), N-(3-Oxotetradecanoyl)-l-homoserine lactone (3-oxo-C14-HSL). Five QQ isolates (QQIs) belonging to the Bacillus and Shewanella genera, showed high capacity to degrade both synthetic AHLs as well as natural AHLs produced by Vibrio harveyi and Vibrio alginolyticus using the well-diffusion method and thin-layer chromatography (TLC). The genes responsible for QQ activity, including aiiA, ytnP, and aaC were also detected. Analysis of the amino acid sequences from the predicted lactonases revealed the presence of the conserved motif HxHxDH. The selected isolates were further characterized in terms of their probiotic potentials in vitro. Based on our scoring system, Bacillus thuringiensis QQ1 and Bacillus cereus QQ2 exhibited suitable probiotic characteristics, including the production of spore and exoenzymes, resistance to bile salts and pH, high potential to adhere on mucus, appropriate growth abilities, safety to barramundi, and sensitivity to antibiotics. These isolates, therefore, constitute new QQ probiotics that could be used to control vibriosis in Lates calcalifer.

Invasion and replication of Yersinia ruckeri in fish cell cultures.

BACKGROUND: Like many members of the Enterobacteriaceae family, Yersinia ruckeri has the ability to invade non professional phagocytic cells. Intracellular location is advantageous for the bacterium because it shields it from the immune system and can help it cross epithelial membranes and gain entry into the host. In the present manuscript, we report on our investigation regarding the mechanisms of Y. ruckeri's invasion of host cells. RESULTS: A gentamycin assay was applied to two isolates, belonging to both the biotype 1 (ATCC 29473) and biotype 2 (A7959-11) and using several cell culture types: Atlantic Salmon Kidney, Salmon Head Kidney and, Chinook salmon embryos cells at both low and high passage numbers. Varying degrees of sensitivity to Y. ruckeri infection were found between the cell types and the biotype 1 strain was found to be more invasive than the non-motile biotype 2 isolate. Furthermore, the effect of six chemical compounds (Cytochalasin D, TAE 226, vinblastine, genistein, colchicine and, N-acetylcysteine), known to interfere with bacterial invasion strategies, were investigated. All of these compounds had a significant impact on the ability of the bacterium to invade host cells. Changes in the concentration of bacterial cells over time were investigated and the results suggested that neither isolate could survive intracellularly for sustained periods. CONCLUSIONS: These results suggest that Y. ruckeri can gain entrance into host cells through several mechanisms, and might take advantage of both the actin and microtubule cytoskeletal systems.

A new age in AquaMedicine: unconventional approach in studying aquatic diseases.

BACKGROUND: Marine and aquaculture industries are important sectors of the food production and global trade. Unfortunately, the fish food industry is challenged with a plethora of infectious pathogens. The freshwater and marine fish communities are rapidly incorporating novel and most up to date techniques for detection, characterization and treatment strategies. Rapid detection of infectious diseases is important in preventing large disease outbreaks. MAIN TEXT: One hundred forty-six articles including reviews papers were analyzed and their conclusions evaluated in the present paper. This allowed us to describe the most recent development research regarding the control of diseases in the aquatic environment as well as promising avenues that may result in beneficial developments. For the characterization of diseases, traditional sequencing and histological based methods have been augmented with transcriptional and proteomic studies. Recent studies have demonstrated that transcriptional based approaches using qPCR are often synergistic to expression based studies that rely on proteomic-based techniques to better understand pathogen-host interactions. Preventative therapies that rely on prophylactics such as vaccination with protein antigens or attenuated viruses are not always feasible and therefore, the development of therapies based on small nucleotide based medicine is on the horizon. Of those, RNAi or CRISPR/Cas- based therapies show great promise in combating various types of diseases caused by viral and parasitic agents that effect aquatic and fish medicine. CONCLUSIONS: In our modern times, when the marine industry has become so vital for feed and economic stability, even the most extreme alternative treatment strategies such as the use of small molecules or even the use of disease to control invasive species populations should be considered.

Leaves from banana (Musa nana) and maize (Zea mays) have no phyto-prophylactic effects on the susceptibility of grass carp (Ctenopharyngodon idella) to Aeromonas hydrophila infection.

BACKGROUND: The ubiquitous and opportunistic bacterial pathogen Aeromonas hydrophila has been associated with ulcerative dermatitis in fish, especially under stressful conditions. It can cause severe losses in fresh water aquaculture and is particularly prevalent in tropical and subtropical regions. Fresh leaves from maize and bananas have been used as feed supplement by fish farmers in Vietnam and it has been reported that they may have phyto-prophylactic benefits. In the present study, a feeding trial was conducted to investigate the benefits of providing maize and banana leaves as feed supplement: to determine if they were taken up and digested by grass carp (Ctenopharyngodon idella), if this uptake resulted in improved growth performance, and if leaf supplementation protected fish when challenged with A. hydrophila by intramuscular injection. RESULTS: All fish were fed an identical ratio of commercial pelleted feed relative to biomass. However, in 12/18 tanks, this diet was supplemented with either fresh banana leaves or fresh maize leaves; offered ad libitum. Addition of leaves increased the overall feed conversion ratio (FCR) significantly. However, if only the pellet were taken into account, then no difference was found between treatments. Changes to the isotopic composition of the fish showed leaf nutrient uptake occurred. No prophylactic effects of feeding banana or maize leaves were detected against infection with A. hydrophila, and the diet did not induce changes in the fish haematocrit. However, addition of the maize leaves was associated with significantly reduced severity of the skin lesions, which could improve the market value of the fish. CONCLUSIONS: Addition of the leaf supplement did not result in significantly improved growth performance. Similarly, the effect of the supplement on the fish survival to infection was not significant.

The impact of co-infections on fish: a review.

Co-infections are very common in nature and occur when hosts are infected by two or more different pathogens either by simultaneous or secondary infections so that two or more infectious agents are active together in the same host. Co-infections have a fundamental effect and can alter the course and the severity of different fish diseases. However, co-infection effect has still received limited scrutiny in aquatic animals like fish and available data on this subject is still scarce. The susceptibility of fish to different pathogens could be changed during mixed infections causing the appearance of sudden fish outbreaks. In this review, we focus on the synergistic and antagonistic interactions occurring during co-infections by homologous or heterologous pathogens. We present a concise summary about the present knowledge regarding co-infections in fish. More research is needed to better understand the immune response of fish during mixed infections as these could have an important impact on the development of new strategies for disease control programs and vaccination in fish.

Shotgun proteomic analysis of Yersinia ruckeri strains under normal and iron-limited conditions.

Yersinia ruckeri is the causative agent of enteric redmouth disease of fish that causes significant economic losses, particularly in salmonids. Bacterial pathogens differentially express proteins in the host during the infection process, and under certain environmental conditions. Iron is an essential nutrient for many cellular processes and is involved in host sensing and virulence regulation in many bacteria. Little is known about proteomics expression of Y. ruckeri in response to iron-limited conditions. Here, we present whole cell protein identification and quantification for two motile and two non-motile strains of Y. ruckeri cultured in vitro under iron-sufficient and iron-limited conditions, using a shotgun proteomic approach. Label-free, gel-free quantification was performed using a nanoLC-ESI and high resolution mass spectrometry. SWATH technology was used to distinguish between different strains and their responses to iron limitation. Sixty-one differentially expressed proteins were identified in four Y. ruckeri strains. These proteins were involved in processes including iron ion capture and transport, and enzymatic metabolism. The proteins were confirmed to be differentially expressed at the transcriptional level using quantitative real time PCR. Our study provides the first detailed proteome analysis of Y. ruckeri strains, which contributes to our understanding of virulence mechanisms of Y. ruckeri, and informs development of novel control methods for enteric redmouth disease.

Aeromonas salmonicida: updates on an old acquaintance.

Aeromonas salmonicida is the oldest known infectious agent to be linked to fish disease and constitutes a major bacterial pathogen of fish, in particular of salmonids. This bacterium can be found almost worldwide in both marine and freshwater environments and has been divided into several sub-species. In this review, we present the most recent developments concerning our understanding of this pathogen, including how the characterization of new isolates from non-salmonid hosts suggests a more nuanced picture of the importance of the so­called 'atypical isolates'. We also describe the clinical presentation regarding the infection across several fish species and discuss what is known about the virulence of A. salmonicida and, in particular, the role that the type 3 secretion system might play in suppressing the immune response of its hosts. Finally, isolates have displayed varied levels of antibiotic resistance. Hence, we review a number of solutions that have been developed both to prevent outbreaks and to treat them once they occur, including the application of pre- and probiotic supplements.

Yersinia ruckeri, the causative agent of enteric redmouth disease in fish.

Enteric redmouth disease (ERM) is a serious septicemic bacterial disease of salmonid fish species. It is caused by Yersinia ruckeri, a Gram-negative rod-shaped enterobacterium. It has a wide host range, broad geographical distribution, and causes significant economic losses in the fish aquaculture industry. The disease gets its name from the subcutaneous hemorrhages, it can cause at the corners of the mouth and in gums and tongue. Other clinical signs include exophthalmia, darkening of the skin, splenomegaly and inflammation of the lower intestine with accumulation of thick yellow fluid. The bacterium enters the fish via the secondary gill lamellae and from there it spreads to the blood and internal organs. Y. ruckeri can be detected by conventional biochemical, serological and molecular methods. Its genome is 3.7 Mb with 3406-3530 coding sequences. Several important virulence factors of Y. ruckeri have been discovered, including haemolyin YhlA and metalloprotease Yrp1. Both non-specific and specific immune responses of fish during the course of Y. ruckeri infection have been well characterized. Several methods of vaccination have been developed for controlling both biotype 1 and biotype 2 Y. ruckeri strains in fish. This review summarizes the current state of knowledge regarding enteric redmouth disease and Y. ruckeri: diagnosis, genome, virulence factors, interaction with the host immune responses, and the development of vaccines against this pathogen.

Tetracapsuloides bryosalmonae infection affects the expression of genes involved in cellular signal transduction and iron metabolism in the kidney of the brown trout Salmo trutta.

Tetracapsuloides bryosalmonae is an enigmatic endoparasite which causes proliferative kidney disease in various species of salmonids in Europe and North America. The life cycle of the European strain of T. bryosalmonae generally completes in an invertebrate host freshwater bryozoan and vertebrate host brown trout (Salmo trutta) Linnaeus, 1758. Little is known about the gene expression in the kidney of brown trout during the developmental stages of T. bryosalmonae. In the present study, quantitative real-time PCR was applied to quantify the target genes of interest in the kidney of brown trout at different time points of T. bryosalmonae development. PCR primers specific for target genes were designed and optimized, and their gene expression levels were quantified in the cDNA kidney samples using SYBR Green Supermix. Expression of Rab GDP dissociation inhibitor beta, integral membrane protein 2B, NADH dehydrogenase 1 beta subcomplex subunit 6, and 26S protease regulatory subunit S10B were upregulated significantly in infected brown trout, while the expression of the ferritin M middle subunit was downregulated significantly. These results suggest that host genes involved in cellular signal transduction, proteasomal activities, including membrane transporters and cellular iron storage, are differentially upregulated or downregulated in the kidney of brown trout during parasite development. The gene expression pattern of infected renal tissue may support the development of intraluminal sporogonic stages of T. bryosalmonae in the renal tubular lumen of brown trout which may facilitate the release of viable parasite spores to transmit to the invertebrate host bryozoan.

Protein expression and transcription profiles of three strains of Aeromonas salmonicida ssp. salmonicida under normal and iron-limited culture conditions.

BACKGROUND: Aeromonas salmonicida is an important fish pathogen that produces a wide and varied array of virulence factors. Here we used iron deprivation by addition of the chelator 2'2-dipyridyl to induce the expression of several such virulence factors in three isolates of Aeromonas salmonicida (one avirulent and two virulent). By using SDS-PAGE followed by mass spectrometry, we identified proteins that appeared differentially expressed under these conditions. The differential transcription of the identified gene products were subsequently measured by reverse transcription quantitative real-time PCR (RT-qPCR). RESULTS: Our initial screening using SDS-PAGE identified five proteins that appeared differentially expressed in virulent and avirulent isolates or, within the same isolates, between bacteria cultivated under iron-rich or iron-deprived conditions. The transcription of the genes coding for these proteins were subsequently quantified by RT-qPCR. Results of this analysis demonstrated that the gene coding for alkyl hydroperoxide reductase (AhpC), a protein involved in oxidative stress response, was transcribed at a higher rate in the virulent strain as compared to the avirulent strain. Additionally, it was observed that addition of an iron chelator to the culture medium lead to a reduction of the transcription levels of the regulatory histone-like nucleoid structuring protein (H-NS). This was consistent in all three isolates. On the other hand, the transcription levels of the virulence array protein (VapA) and the protein ATP-synthetase F (ATPF) displayed only limited changes, despite being the dominant component of a protein fraction that displayed changes during the preliminary SDS-PAGE screening. This was true regardless of the culture conditions and of the isolates considered. Finally, transcription of the enzyme enolase was upregulated in the iron-deprived broths in all isolates. CONCLUSIONS: We identified several genes differentially expressed under culture conditions known to lead to the overexpression of virulence factors. In addition, we identified alkyl hydroperoxide as being overexpressed in the virulent isolates compared to the avirulent isolates. The results from this study will contribute to enhance our understanding of the virulence of A. salmonicida and may suggest new directions for further research.

Use of in vivo induced antigen technology to identify genes from Aeromonas salmonicida subsp. salmonicida that are specifically expressed during infection of the rainbow trout Oncorhynchus mykiss.

BACKGROUND: Aeromonas salmonicida is a major fish pathogen associated with mass mortalities in salmonid fish. In the present study, we applied In Vivo Induced Antigen Technology (IVIAT), a technique that relies on antibodies adsorbed against in vitro cultures of the pathogen, to a clinical isolate of A. salmonicida subsp. salmonicida. RESULTS: The results from IVIAT allowed identification of four proteins that were upregulated in the fish samples: A UDP-3-O-acyl-N-acetylglucosamine deacetylase, an RNA polymerase sigma factor D as well as TonB and a hypothetical protein. Subsequent investigations were performed using real-time PCR and cDNA synthesised from infected spleen, liver and anterior kidneys. These confirmed that the transcription level of each of these genes was significantly upregulated during the infection process compared to bacteria in vitro. CONCLUSIONS: The present studied identified four genes that were upregulated during the infectious process and are likely to play a role in the virulence of A. salmonicida. Because these are antigenic they might constitute potential targets for the development of new vaccine as well as therapeutic agents.

Non-structural protein pORF 12 of cyprinid herpesvirus 3 is recognized by the immune system of the common carp Cyprinus carpio.

Cyprinid herpesvirus 3 is an important pathogen and the causative agent of koi herpesvirus disease, which has been associated with mass mortalities in koi and common carp Cyprinus carpio. Currently, the only available commercial vaccine is an attenuated version of the virus. This has led to concerns about its risk to reversion to virulence. Furthermore, the vaccine is currently only available in Israel and the United States. In order to investigate the antigenic profile of the virus, western blot was performed using infected cell culture supernatant and sera from carp that had survived exposure to the virus. Only one antigen could be detected, and mass spectrometry analysis identified the corresponding protein as ORF 12, a putative secreted tumour necrosis factor receptor homologue. In other herpesviruses, such proteins have been associated with the viral infectious process in a number of ways, including the entry into the host cell and the inhibition of apoptosis in infected cells. The reason why only one antigen could be detected during this study is unknown.