Infectious Diseases In Fish Research Articles

MOLECULAR DIAGNOSIS OF VIRAL DISEASES IN FISHES

The paper provides information about the main viral diseases of fish found in Ukraine and methods of their diagnosis. Rapid diagnosis of fish viruses using polymerase chain reaction is an affordable and relevant way to monitor and prevent outbreaks of viral diseases. The work of the Laboratory of Biotechnology in Aquaculture of the Institute of Fisheries of NAAS is aimed to develop diagnostic kits for infectious diseases of fish and conducting research in accordance with the procedure of surveillance of fish diseases. For developing diagnostic test systems, attention is paid to the trends of cultured fish species and aquafarming as well as to viruses that circulate in Ukraine or can pass into the Ukrainian aquaculture from neighboring countries. An important role is given to the study of genetic variability of fish viruses, characterized by varying types of virulence. Examples of application the techniques in carrying out diagnostics for the purpose of identification of an infectious agent and the reason of an epizootic are resulted.
 A comparative analysis of techniques, components of kits and reagents for optimizing the polymerase chain reaction and its modifications, which can significantly reduce the process of identification of fish viruses, are given. Target genes for oligonucleotide primers selection for PCR diagnosis of viral diseases of fish, namely spring viremia of carp virus (SVCV), cyprinid herpesvirus type 3 (CyHV-3 or KHV), carp edema virus (CEV), infectious pancreatic necrosis virus (IPNV) viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), Piscine orthoreovirus (PRV), Acipenser iridovirus European (AcIV-E).

Polyculture in fish farming as a method of preventing fish diseases

Methods of prevention of fish diseases are aimed at destroying parasites in the host body and in the fish habitat — in the reservoir (chemoprophylaxis, increasing fish resistance), at preventing parasites from entering the reservoir (limiting the import of fish, import of healthy fish, quarantine measures, etc.), as well as preventing their spread and entry into the host body. It is shown that polyculture is not only the most effective method of using the natural forage base of the reservoir and, as a result, increasing fish productivity, but also a means of preventing mass infectious and other fish diseases. To prevent mass infectious diseases of fish and death from them both in safe and unfavorable ponds of carp fish farms, it is advisable to grow other fish species together with carp that do not suffer from diseases peculiar to carp. As a result, so-called sparse species plantings of fish are created in the ponds, while the total biomass remains high. At the same time, the natural food supply of ponds is most fully used and a kind of biological buffer is created that prevents the emergence and spread of contagious diseases. Polyculture can play a particularly important role in pond farms that already have certain problems with rubella, filometroidosis, botryocephalosis, carp pox, etc. Long-term studies of breeding herds in pond farms have shown that most of them have single foci of diseases, which, if there are optimal conditions for the pathogen, are ready to immediately respond with an outbreak of the disease or are generally quarantined for a particular fish disease. When determining the objects of polyculture, it is necessary to reduce the proportion of fish species that have a significant number of common diseases.

Antimicrobial Activity and Chemical Composition of Essential Oils against Pathogenic Microorganisms of Freshwater Fish.

Antimicrobials are widely applied in aquaculture for treatment of infectious diseases in fish. The increased antimicrobial resistance of fish pathogens to conventional antimicrobial treatment highlights the need for research on the antibacterial properties of natural products—in this case, essential oils (EOs). The aim of the present study was to detect the antimicrobial activity of the essential oils on pathogenic microorganisms found in freshwater fish. Freshwater fish isolates of Aerococcus spp., Aeromonas spp., Enterococcus spp., Escherichia spp., Pseudomonas spp., Shewanella spp., Yersinia spp., and Vagococcus spp. were tested for antimicrobial resistance and antimicrobial activity against 14 commercially available essential oils. Antimicrobial resistance was identified in Pseudomonas spp. isolates against cefepime and ciprofloxacin; while all Aeromonas, Enterococcus, and Yersinia isolates were fully susceptible. All tested EOs revealed antimicrobial activity against the tested freshwater fish isolates at different extents. Cinnamomum camphora exhibited strong antimicrobial activity against Aeromonas spp. (3.12 μL/mL), Enterococcus spp. (0.78–1.56 μL/mL), and Pseudomonas spp. with the MIC method. EOs of Gaultheria procumbens and Litsea cubeba showed strong antibacterial activity against Yersinia spp. and Vacococcus spp. (6.25 μL/mL). The study shows the antimicrobial activity of EOs against the most relevant freshwater fish pathogens and indicates the application opportunities in aquaculture.

PH-Controlled Release of Antigens Using Mesoporous Silica Nanoparticles Delivery System for Developing a Fish Oral Vaccine.

The development of effective vaccines and delivery systems in aquaculture is a long-term challenge for controlling emerging and reemerging infections. Cost-efficient and advanced nanoparticle vaccines are of tremendous applicability in prevention of infectious diseases of fish. In this study, dihydrolipoamide dehydrogenase (DLDH) antigens of Vibrio alginolyticus were loaded into mesoporous silica nanoparticles (MSN) to compose the vaccine delivery system. Hydroxypropyl methylcellulose phthalate (HP55) was coated to provide protection of immunogen. The morphology, loading capacity, acid-base triggered release were characterized and the toxicity of nanoparticle vaccine was determined in vitro. Further, the vaccine immune effects were evaluated in large yellow croaker via oral administration. In vitro studies confirmed that the antigen could be stable in enzymes-rich artificial gastric fluid and released under artificial intestinal fluid environment. In vitro cytotoxicity assessment demonstrated the vaccines within 120 μg/ml have good biocompatibility for large yellow croaker kidney cells. Our data confirmed that the nanoparticle vaccine in vivo could elicit innate and adaptive immune response, and provide good protection against Vibrio alginolyticus challenge. The MSN delivery system prepared may be a potential candidate carrier for fish vaccine via oral administration feeding. Further, we provide theoretical basis for developing convenient, high-performance, and cost-efficient vaccine against infectious diseases in aquaculture.

ВПЛИВ ЗМІНИ КЛІМАТУ НА ІНФЕКЦІЙНІ ЗАХВОРЮВАННЯ РИБ (ОГЛЯД)

Purpose. As the climate change impacts freshwater and marine ecosystems, and rising ocean temperatures and acidification continue to this moment, our aim was to analyze the literature and summarize information on the development of fish infectious diseases in the light of global warming. Findings. Even a slight increase in temperature affects the life cycle, physiology, behavior, distribution and structure of populations of aquatic bioresources, especially fish. Recent studies show that some infectious diseases of fish spread much faster with increasing temperature. Climate change contributes to pathogens spread in both marine and freshwater areas. In particular, rising water temperatures can expand the range of diseases. Aquatic bioresources have high cumulative mortality from infectious diseases, and pathogens are rapidly progressing, and these phenomena may be powered by climate change, leading to the geographical spread of virulent pathogens to fisheries and aquaculture facilities, threatening much of global production and food security. The article presents data on the impact of climate change and global warming on aquaculture and fisheries. The list of the main pathogens of fish of various etiology in Ukraine, including viral, bacterial and parasitic diseases is presented. The impact of infectious agents on modern aquaculture is described and the main ideas about the possible long-term consequences of climate change for fish farms are given. Practical Value. The review may be useful for specialists in veterinary medicine, epizootology and ichthyopathology. Key words: climate change, infectious diseases of fish, pathogenesis.

Phytogenics in Aquaculture: A Short Review of Their Effects on Gut Health and Microflora in Fish

Increasing pace in aquaculture development to meet the growing food requirements of the population has greatly compromised the carrying capacity of the culture environment and has placed the aquacultured animals at heightened risk of getting diseases due to pathogens. At present, chemotherapy is widely used as means to prevent or treat infectious diseases in aquaculture; however, the use of these drugs poses multiple negative impacts on fish and human health, as well as the environment. Recently, research initiatives are focused on the use of plant products and their derivatives as a means of controlling diseases in aquaculture. They are regarded as a promising alternative to the use of chemical treatments for infectious diseases in fish. Plant-derived products or phytogenics have been shown to stimulate appetite and promote weight gain in farmed animals, act as immunostimulants, and possess potent anti-pathogenic properties in fish. Their potency is mediated by the presence of bioactive molecules including alkaloids, terpenoids, saponins, and flavonoids, among others. Moreover, nutritional strategies are geared towards the use of these phytogenics in modulating immune and physiological responses, as well as promoting optimum health and microbial community in the gastrointestinal tract of fish. This review synthesizes the current knowledge on the use of phytogenic feed additives in aquaculture by focusing on how these substances act as modulators of health and bacterial community in the gut of fish.

Specifi c composition of microfl ora circulating at the FARM “Quant System” and its resistance to antibacterial medicines

Accumulation of opportunistic pathogenic bacteria in the aquatic environment may lead to the changes in the structure of the surface cover microbiocenosis as well as the fish intestinal tract. In its turn, it causes the growth of pathological processes in the fish organism, reduces the barrier functions of tissues and mucus and, as a consequence, may induce the spread of bacterial infections. Thus, assessing the state of fish organism, it is necessary to take into account the analysis of the epizootic situation in water (i.e. presence of infectious and invasive diseases of fish, death of fish) as well as condition of the skin, gills and gastrointestinal tract. The results of bacteriological studies of washes from the skin, gills and intestines showed that in the conditions of farming among rainbow trout of different age groups, E. coli, low fermented E. coli, Enterobacter spp., Klebsiella pneumoniae, Staphylococcus spp., Flavibacterium spp., Enterococcus spp. and Citrobacter spp. For the purposes of determining the sensitivity of isolated cultures to antibacterial drugs, it was found that Enterobacteriaceae are sensitive to enrofloxacin, cotrimoxazole, doxacillin, chloramphenicol, gentamicin, ceftriaxone, cefoperazone, ceftazidime and cefpiromycin and resistant to oxytetracycline, amoxicillin and ampicillin sulbactam. E. coli also demonstrated resistance to amoxicillin and cefpirome and sensitivity to enrofloxacin, oxytetracycline, cotrimoxazole, doxacillin, chloramphenicol, ampicillin sulbactam, gentamicin, ceftriaxone, cefoperazone, ceftazidime. Klebsiella pneumoniae was resistant to enrofloxacin, amoxicillin and ampicillin sulbactam, while Flavobacteriaceae were resistant to amoxicillin and ampicillin sulbactam and sensitive to all other antibacterial drugs. Key words: rainbow trout, microflora, skin, gills, intestines, antibacterial agents, resistance, sensitivity.

Presence of viral haemorrhagic septicaemia virus (VHSV) in the environment of virus-contaminated fish farms and processing plants.

After the first outbreak of viral haemorrhagic septicaemia virus (VHSV) in Finnish brackish water rainbow trout Oncorhynchus mykiss farms, infection spread rapidly between the farms. The infrastructure of fish farming did not take into account spreading of infectious fish diseases. To show the presence of VHSV in the environment, we tested seawater, sediment and wild blue mussels Mytilus edulis from VHSV-infected fish farms, and liquid waste from a processing plant that handled infected rainbow trout. Additionally, blue mussels were bath-challenged with VHSV (exposed to cultivated virus or naturally infected rainbow trout). To detect VHSV, virus isolation in cell culture and real-time reverse transcriptase polymerase chain reaction (qRT-PCR) were used. The virus or viral RNA was detected in sea water and in liquid waste from processing plants during wintertime when water temperature is close to 0°C and sunlight is sparse. VHSV did not appear to replicate in blue mussels in our study. Therefore, blue mussels were not considered relevant carriers of VHSV. However, traces of viral RNA were detected up to 29 d post challenge in mussels. Contact with water from processing plants handling VHSV-infected fish populations increases the risk of the disease spreading to susceptible fish populations, especially during cold and dark times of the year.

Effect of extracts of luffa cylindrica, xanthosoma sagittifolium and momordica charantia on saprolegnia spp. Mycelial growth

Among the major infectious diseases in fish, those caused by zoosporic organisms are important, especially saprolegniasis. These diseases can affect few animals or even the entire population of the production environment. The use of alternative treatments for infectious diseases in aquatic animals has proven to be effective. It prevents the mortality of part of the crop, as well as the contamination of water by chemical components. Within this scenario, the use of plants stands out, mainly unconventional food plants (UFPs). The objective of this work was to evaluate the effect of the aqueous extracts of Luffa cylindrica, Xanthosoma sagittifolium and Momordica charantia on the mycelial growth of Saprolegnia spp. The extracts obtained from the maceration of dry and ground leaves were incorporated into the potato agar dextrose (PDA), at concentrations of 0, 5, 15, 25 and 50%. Later, filter paper disks containing the mycelium of Saprolegnia spp. were applied on the agar and incubated at 25°C. Evaluations were performed every 24 hours, for 10 days. In the study, all concentrations of M. charantia extract had an inhibitory action on the mycelial growth in the order of 75% of Saprolegnia spp. and can be considered a promising alternative for controlling this pathogen.

Phylogenetic characteristics, virulence properties and antibiogram profile of motile Aeromonas spp. isolated from ornamental guppy (Poecilia reticulata).

Aeromonas spp. are opportunistic pathogenic bacteria related to an assembly of infectious diseases in ornamental fish. In the present study, virulence properties and antibiotic susceptibility of 52 guppy-borne Aeromonas spp. were investigated. The isolates were identified as A. veronii (n = 34), A. dhakensis (n = 10), A. hydrophila (n = 3), A. caviae (n = 3) and A. enteropelogenes (n = 2) by gyrB gene sequencing. The gyrB sequence deviation within and among the species ranged from 0 to 2.6% and 2.7-9.2%. Each species formed a distinct group in the unrooted neighbor-joining phylogenetic tree. The phenotypic virulence factors such as β-hemolysis, slime, caseinase, DNase, gelatinase and lipase production were observed in 28 (53.9%), 33 (63.5%), 28 (53.9%), 42 (80.8%), 37 (71.2%) and 42 (80.8%) isolates, respectively. The virulence genes were detected by PCR assay in the following proportions- act (84.6%), hly (80.8%), aer (73.1%), lip (73.1%), gcaT (73.1%), ascV (53.8%), ahyB (53.8%) fla (51.9%), alt (48.1%), ast (36.5%) and ser (34.6%), respectively. The amoxicillin, ampicillin, imipenem, nalidixic acid, oxytetracycline and rifampicin were resistant to more than 70.0% of the isolates in antibiotic susceptibility test. Our study suggests that the ornamental guppy can be a potential reservoir of virulent and multi-drug resistant Aeromonas spp.

Infectious diseases in aquarium ornamental pet fish

Aquarium ornamental pet fish constitute a major segment in the pet industry, with the United States, Europe, and Japan dominating the market. There are approximately 1,500 marine fish species and over 4,500 freshwater fish species commercialized as aquarium ornamental pet fish. Fish are the fourth most common pet present in Brazilian homes. In Brazil, aquarium ornamental pet fish can be marketed and distributed from different parts of the Brazilian territory and the world. Commercialization and circulation of living animals without the use of adequate prophylactic management procedures enables dissemination of a number of agents responsible for infectious diseases. Aquarium pet fish can also carry pathogenic agents, of bacterial, viral, fungal, or parasitic etiology, that may have a zoonotic feature endangering the persons handling the animals. This review presents the main pathogenic infectious agents of bacterial, viral, andfungal etiology that affect aquarium pet fish, as well as the prevention and control measures to ensure sanitary excellence in this segment.

Saprolegnia parasitica impairs branchial phosphoryl transfer network in naturally infected grass carp (Ctenopharyngodon idella): prejudice on bioenergetic homeostasis

Precise coupling of spatially separated intracellular adenosine triphosphate (ATP) producing and ATP-consuming exerts a key role in bioenergetic balance, and the phosphoryl transfer network, catalyzed by creatine kinase (CK), adenylate kinase (AK), and pyruvate kinase (PK), is fundamental in energetic homeostasis of tissues with high-energy requirements, as the branchial tissue. This whole system is very sensitive, and our hypothesis is that it can be altered in cases of infectious diseases in fish, such as that caused by the oomycete Saprolegnia parasitica. The effects of S. parasitica infection on gills remain poorly understood and limited only to histopathological studies. Thus, the aim of this study was to evaluate whether natural infection by S. parasitica impairs the enzymes of the phosphoryl transfer network in gills of grass carp (Ctenopharyngodon idella), as well as the pathways involved in this inhibition. In this study, we used sick carp and compared to healthy carp, fish of similar age and receiving the same feed, but allocated in different tanks. Branchial CK (cytosolic and mitochondrial) activity and ATP levels decreased in infected fish compared to uninfected on day 7 post-infection (PI), while no significant difference was observed between groups regarding branchial AK and PK activities. Branchial sodium-potassium ion pump (Na+, K+-ATPase) activity decreased in infected carp compared to uninfected on day 7 PI, while reactive oxygen species (ROS) and thiobarbituric acid reactive substance (TBARS) levels were higher. Gill histopathology revealed massive necrosis, loss of branchial epithelium, and detachment of the epithelium interlayer with structural loss of secondary lamellae. Based on these data, the impairment of CK activity elicited by S. parasitica caused an impairment in branchial energetic homeostasis, reducing the ATP availability in the gills and provoking an impairment on Na+, K+-ATPase activity. Moreover, the inhibition on CK activity appears to be mediated by ROS overproduction and lipid peroxidation, which contribute to disease pathogenesis linked to branchial tissue.

Use of STF-1/56 preparation for prevention and treatment of infectious diseases in fish

Carps exposed to infection were investigated objects. The body of aquatic organisms is not a natural reproduction environment of Salmonella, however, as a result of cross-infection, Salmonella bacillus may appear in fish. The use of probiotic additives from living bacteria has become an alternative to the use of antibiotics, probiotics have antagonistic abilities against pathogens. A group of carp yearlings was selected for research in an aquarium installation. The average weight of animal specimen was 900 grams. During the experiment, the main hydrochemical parameters were monitored. Oxygen disturbances were not allowed by using aerators. The temperature of the water was kept at 19–20 ° C. The diet was standard for raising carp and herbivorous fish. The presence of Salmonella bacteria in the control group was revealed in all fish three days after infection. Experimentally established high efficiency of the drug "STF-1/56" in fish farming, as a method of combating Salmonella infection without disrupting the microbial balance of microflora in the intestinal cavity.

An agent-based approach for predicting patterns of pathogen transmission between aquaculture sites in the Norwegian fjords

The aquaculture industry is a main industry in Norway, and it must be sustainable, i.e. experience long-term growth and development: It is necessary to build an environmentally sustainable aquaculture industry that minimizes risks to the marine environment and biological diversity, – including the transmission of fish diseases. The process of fish disease transmission in aquaculture systems is influenced by many factors, including individual (fish and pathogen) conditions, movement behavior and environmental conditions. Fish disease dynamics originates from a complex system, and the transmission of viruses is an unstable process, making it difficult to predict and analyze. In preparation for this paper, we built an agent-based model to predict patterns of pathogen transmission with the purpose of identifying risks and hazards in the space and time domains. The model presented explores the potential effects of different factors, such as the conditions of agents, movement behavior and environmental conditions, on the simulated spread of a fish disease.We applied the model developed to different case studies in the Norwegian fjords. The results demonstrated how the infection risk at any point around the infected site is dependent on both the pathogen and the fish density at that point, and the infection risk increases when the pathogen or fish densities increase. The pathogen density decreases exponentially as a function of an increase in the water temperature, and the pathogen density increases with the velocity of the current or the fish density at the infected site. The pathogens are moved faster by higher current velocity, so this will slow the infection process at the local infected site. Nevertheless, the current will carry the pathogens to nearby places faster. The direction of the current is very important since the pathogens are predominantly moved by the currents.The agent-based method helps us advance our understanding of pathogen transmission and builds risk maps to help us reduce the spread of infectious fish diseases. By using this method, we may study the spatial and dynamic aspects of the spread of infections and address the stochastic nature of the infection process.

Characterization of Virulence Determinants And Multiple Antimicrobial Resistance Profiles in Motile Aeromonas Spp. Isolated from Ornamental Goldfish (Carassius Auratus)

As opportunistic pathogens, Aeromonas spp. can cause a multitude of infectious diseases in ornamental fish. In the present study, virulence properties and multiple antimicrobial resistance in 65 goldfish-borne Aeromonas spp. were investigated. The isolates were identified as A. hydrophila (n = 30), A. veronii biovar veronii (n = 32), and A. caviae (n = 3) by gyrB gene sequencing, and each of the species formed distinct clusters in the neighbor-joining phylogenetic tree. The virulence-related genes were detected by Polymerase chain reaction (PCR) assay in the following proportions: aer (87.7%), ahyB (86.2%), fla (83.1%), ascV (67.7%), ser (67.7%), act (66.2%), ast (61.5%), alt (56.9%), gcaT (53.8%), and lip (49.2%). According to the in vivo challenge test with A. hydrophila and A. veronii biovar veronii, the strains (AH1 and AV1) harboring the highest number of genes displayed LD50 values 2.2 × 106 and 9.8 × 106 CFU fish−1 whereas the strains (AH30 and AV32) harboring the lowest number of genes showed LD50 values 1.2 × 107 and 4.6 × 107 CFU fish−1, respectively. A negative relationship (r = −0.85) was found between the frequency of virulence genes and LD50 values. In antimicrobial disk diffusion test, each of the isolates was resistant to at least 4 antimicrobials. The multiple antimicrobial resistance index values ranged from 0.22 to 0.56. Our results suggest that ornamental goldfish can be the reservoir of virulent and multidrug resistant motile aeromonads.

Combination of solar disinfection (SODIS) with H2O2 for enhanced disinfection of marine aquaculture effluents

Technologically advanced recirculation aquaculture systems (RAS), developed for intensive farming, allows to recycle about 90–99% of water. As a result, accumulation of pathogenic bacteria in water often occurs and possesses threat of infectious fish diseases. The goal of this study was to evaluate feasibility of solar disinfection in combination with hydrogen peroxide for seawater and real marine aquaculture effluent. Specific roles of solar disinfection (SODIS), hydrogen peroxide and highly reactive oxidizing species (experiments conducted in presence of tert-butyl alcohol), generated in combined SODIS/H2O2 process were investigated. Natural consortia of Vibrio spp., known as highly resistant pathogen, present in seawater and real marine aquaculture effluent were chosen as target microorganisms. The SODIS/H2O2 process significantly enhances inactivation of natural Vibrio consortia (Vibrio harveyi, Vibrio owensii and Vibrio alfacsensis) in seawater in comparison with individual SODIS and H2O2 treatment. The best results in terms of Vibrio spp. inactivation efficiency was obtained with SODIS/H2O2 conducted using open channel raceway reactor. The efficiency of Vibrio spp. inactivation by SODIS/H2O2 in raceway and compound parabolic collector (CPC) reactors was found to be similar, requiring the dose of 28–29 Wh m−2 for complete inactivation of Vibrio spp. present in seawater. Absence of toxic effect of residual H2O2 for Nannochloropsis gaditana, as well as absence of Vibrio spp. regrowth (48 h) indicate suitability of SODIS/H2O2 process for inactivation of pathogenic bacteria in aquaculture effluent and following nutrients removal by microalgae. Moreover, feasibility of SODIS/H2O2 performed in raceway reactor for disinfection of real aquaculture effluent, containing more than 5⋅103 CFU mL−1 of Vibrio harveyi, was demonstrated requiring the dose of 34.4 Wh m−2 for complete Vibrio spp. inactivation. The annual operation and maintenance cost of aquaculture effluent disinfection using CPC and raceway reactors were evaluated.

Applications of Electronic-Nose Technologies for Noninvasive Early Detection of Plant, Animal and Human Diseases

The development of electronic-nose (e-nose) technologies for disease diagnostics was initiated in the biomedical field for detection of biotic (microbial) causes of human diseases during the mid-1980s. The use of e-nose devices for disease-diagnostic applications subsequently was extended to plant and animal hosts through the invention of new gas-sensing instrument types and disease-detection methods with sensor arrays developed and adapted for additional host types and chemical classes of volatile organic compounds (VOCs) closely associated with individual diseases. Considerable progress in animal disease detection using e-noses in combination with metabolomics has been accomplished in the field of veterinary medicine with new important discoveries of biomarker metabolites and aroma profiles for major infectious diseases of livestock, wildlife, and fish from both terrestrial and aquaculture pathology research. Progress in the discovery of new e-nose technologies developed for biomedical applications has exploded with new information and methods for diagnostic sampling and disease detection, identification of key chemical disease biomarkers, improvements in sensor designs, algorithms for discriminant analysis, and greater, more widespread testing of efficacy in clinical trials. This review summarizes progressive advancements in utilizing these specialized gas-sensing devices for numerous diagnostic applications involving noninvasive early detections of plant, animal, and human diseases.

The two-component regulatory system CpxA/R is required for the pathogenesis of Aeromonas hydrophila.

Aeromonas hydrophila is a causative pathogen of many infectious diseases in fish and human. The two-component regulatory system enables bacteria to respond to a wide range of stimuli, growth conditions and environments. The two-component system CpxA/R is prevalent in microorganisms and involved in the pathogenesis of a number of pathogens. In this study, we generated a ΔcpxA/R mutant of A. hydrophila ZYAH72 to explore the biological functions mediated by the CpxA/R in this organism. Compared with the wild type strain, the knockout mutant strain ΔcpxA/R exhibited a significant impairment in adherence to human epithelial cells Caco-2 and resistance against host killing in fish blood. However, the mutant strain and the wild type strain showed no difference in the cytotoxicity assay, which revealed that the absence of CpxA/R did not influence the cytopathic effect of this pathogen in vitro. Furthermore, the virulence of ΔcpxA/R was attenuated in zebrafish (Danio rerio) and grass carp (Ctenopharyngodon idella) infections, with reduced mortality or delayed death time. These findings suggest that CpxA/R is required for the virulence of A. hydrophila.

Effects of florfenicol and oxytetracycline on the tropical cladoceran Ceriodaphnia silvestrii: A mixture toxicity approach to predict the potential risks of antimicrobials for zooplankton

Antimicrobials are commonly used in aquaculture to treat infectious diseases in fish. The overuse of these chemicals, however, has made them a contamination source for the aquatic environments. In this study, single and combined effects of florfenicol (FLO) and oxytetracycline (OTC), two antimicrobials widely used in the fish farming, were evaluated in acute and chronic toxicity tests using the tropical cladoceran Ceriodaphnia silvestrii as a model species. Also, a preliminary risk characterization of FLO and OTC for zooplankton was carried out, taking into account different exposure scenarios. The results obtained revealed that FLO and OTC have adverse effects on the mobility, reproduction and population growth rate of C. silvestrii in single exposures. In addition, mixture effects on the C. silvestrii were more severe than predicted effects based on the Concentration Addition model, showing a synergistic deviation for the mobility and a dose-level dependent deviation for the reproduction (synergism at higher levels than EC60). In relation to the risk characterization, risk quotients (RQs) exceeded 1 for chronic toxicity data obtained in both OTC and mixture exposures, indicating that the concentrations of these chemicals in Brazilian freshwater bodies could potentially present risks for the reproduction of zooplankton species in tropical regions. The RQs obtained for the mixtures were higher than those obtained for each chemical separately. Therefore, it is highly recommended that RQs are derived from single and mixture exposure data in order to obtain a more accurate risk characterization.

The stress hormone norepinephrine increases the growth and virulence of Aeromonas hydrophila.

Stress is an important contributing factor in the outbreak of infectious fish diseases. To comprehensively understand the impact of catecholamine stress hormone norepinephrine (NE) on the pathogenicity of Aeromonas hydrophila, we assessed variations in bacterial growth, virulence‐related genes expression and virulence factors activity after NE addition in serum‐SAPI medium. Further, we assessed the effects of NE on A. hydrophila virulence in vivo by challenging fish with pathogenic strain AH196 and following with or without NE injection. The NE‐associated stimulation of A. hydrophila strain growth was not linear‐dose‐dependent, and only 100 μM, or higher concentrations, could stimulate growth. Real‐time PCR analyses revealed that NE notably changed 13 out of the 16 virulence‐associated genes (e.g. ompW, ahp, aha, ela, ahyR, ompA, and fur) expression, which were all significantly upregulated in A. hydrophila AH196 (p < 0.01). NE could enhance the protease activity, but not affect the lipase activity, hemolysis, and motility. Further, the mortality of crucian carp challenged with A. hydrophila AH196 was significantly higher in the group treated with NE (p < 0.01). Collectively, our results showed that NE enhanced the growth and virulence of pathogenic bacterium A. hydrophila.