Iron uptake mechanisms as key virulence factors in bacterial fish pathogens

This review summarizes the current knowledge about iron uptake systems in bacterial fish pathogens and their involvement in the infective process. Like most animal pathogens, fish pathogens have evolved sophisticated iron uptake mechanisms some of which are key virulence factors for colonization of the host. Among these systems, siderophore production and heme uptake systems are the best studied in fish pathogenic bacteria. Siderophores like anguibactin or piscibactin, have been described in Vibrio and Photobacterium pathogens as key virulence factors to cause disease in fish. In many other bacterial fish pathogens production of siderophores was demonstrated but the compounds were not yet chemically characterized and their role in virulence was not determined. The role of heme uptake in virulence was not yet clearly elucidated in fish pathogens although there exist evidence that these systems are expressed in fish tissues during infection. The relationship of other systems, like Fe(II) transporters or the use of citrate as iron carrier, with virulence is also unclear. Future trends of research on all these iron uptake mechanisms in bacterial fish pathogens are also discussed.     

Antimicrobial activity in serum of Atlantic cod Gadus morhua L.: comparison between cultured and wild fish

The present investigation determined the antibacterial activities in the sera of both cultured and wild Atlantic cod Gadus morhua . Serum samples from both groups of fish were quantified for total protein, and their effects against fish pathogens Listonella anguillarum , Aeromonas salmonicida and Yersinia ruckeri were determined using co-incubation assay. Total serum protein concentrations were not significantly different between farmed and wild Atlantic cod. Sera of cultured Atlantic cod significantly decreased growth of L. anguillarum by at least two-log₁₀ reductions in bacterial count, while those of the wild Atlantic cod were able to inhibit the growth of all three fish bacterial pathogens. The present study showed that sera from wild fish possess broader antibacterial activities than cultured Atlantic cod and that these could provide an explanation for the differences regarding their immunity to bacterial infections as well as their health status.     

Inhibitory activity of antibiotic-producing marine bacteria against fish pathogens

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

Inhibitory activity of antibiotic-producing marine bacteria against fish pathogens

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

Comparison of antimicrobial activity in the epidermal mucus extracts of fish

The mucus layer on the surface of fish consists of several antimicrobial agents that provide a first line of defense against invading pathogens. To date, little is known about the antimicrobial properties of the mucus of Arctic char (Salvelinus alpinus), brook trout (S. fontinalis), koi carp (Cyprinus carpio sub sp. koi), striped bass (Morone saxatilis), haddock (Melanogrammus aeglefinus) and hagfish (Myxine glutinosa). The epidermal mucus samples from these fish were extracted with acidic, organic and aqueous solvents to identify potential antimicrobial agents including basic peptides, secondary metabolites, aqueous and acid soluble compounds. Initial screening of the mucus extracts against a susceptible strain of Salmonella enterica C610, showed a significant variation in antimicrobial activity among the fish species examined. The acidic mucus extracts of brook trout, haddock and hagfish exhibited bactericidal activity. The organic mucus extracts of brook trout, striped bass and koi carp showed bacteriostatic activity. There was no detectable activity in the aqueous mucus extracts. Further investigations of the activity of the acidic mucus extracts of brook trout, haddock and hagfish showed that these fish species had specific activity for fish and human pathogens, demonstrating the role of fish mucus in antimicrobial protection. In comparison to brook trout and haddock, the minimum bactericidal concentrations of hagfish acidic mucus extracts were found to be approximately 1.5 to 3.0 times lower against fish pathogens and approximately 1.6 to 6.6 folds lower for human pathogens. This preliminary information suggests that the mucus from these fish species may be a source of novel antimicrobial agents for fish and human health related applications.     

Applications of monoclonal antibodies in aquaculture

Monoclonal antibodies (MAbs) currently are being applied to the study of fish immunology and fish infectious diseases. MAbs to fish immunoglobulins (Igs) have helped isolate fish Igs, identify heavy and light chain variants in fish Ig, study the ontogeny of B lymphocytes, and improve techniques for the measurement of fish Ig and specific antibodies (Abs). MAbs have been obtained against several leucocyte surface antigens and are being used as markers for different subsets of fish leukocytes: neutrophils, non-specific cytotoxic cells and cells responsible for the mixed leucocyte reaction. The sensitivity and specificity of many immunoassays for identifying fish pathogens have been improved by the use of MAbs. Further improvement of these tests is currently being attempted by the use of MAbs together with the polymerase chain reaction (PCR). Epizootiological studies of fish diseases are beginning to emerge from the use of these reagents and techniques. In the near future these new methods should detect low levels of pathogens in adult carriers and perhaps in eggs, thus helping to prevent the dissemination of fish diseases. MAbs to fish pathogens are also being used for passive immunization studies as well as for conformational probes in the development of genetically engineered vaccines.     

Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment

The accelerated growth of finfish aquaculture has resulted in a series of developments detrimental to the environment and human health. The latter is illustrated by the widespread and unrestricted use of prophylactic antibiotics in this industry, especially in developing countries, to forestall bacterial infections resulting from sanitary shortcomings in fish rearing. The use of a wide variety of antibiotics in large amounts, including non-biodegradable antibiotics useful in human medicine, ensures that they remain in the aquatic environment, exerting their selective pressure for long periods of time. This process has resulted in the emergence of antibiotic-resistant bacteria in aquaculture environments, in the increase of antibiotic resistance in fish pathogens, in the transfer of these resistance determinants to bacteria of land animals and to human pathogens, and in alterations of the bacterial flora both in sediments and in the water column. The use of large amounts of antibiotics that have to be mixed with fish food also creates problems for industrial health and increases the opportunities for the presence of residual antibiotics in fish meat and fish products. Thus, it appears that global efforts are needed to promote more judicious use of prophylactic antibiotics in aquaculture as accumulating evidence indicates that unrestricted use is detrimental to fish, terrestrial animals, and human health and the environment.     

Interaction of fishes with pathogenic micro-organisms and application of phages for their control: a review

The present condition of aquacultural industry is influenced by the economical losses due to various aquacultural diseases causing fish mortality. The present review provides benchmark information related to application of bacteriophages in aquacultural industries over available traditional treatment procedures like antibiotics and chemotherapy. The traditional methods are mostly less advantageous due to development of resistance, non-specific targeting of bacteria including intestinal microflora, etc. In short here we discuss the interaction between fish, bacteria and their phages in order to have an alternate treatment method for the pathogens responsible for aquacultural diseases.     

Molecular identification and phylogenetic analysis of fungal pathogens isolated from diseased fish in Xinjiang,China

The outbreaks of fungal diseases in cultured fish have been severe in recent years, which is harmful to the healthy and sustainable development of fish farming. In this study, an investigation was conducted for significant fungal infections of 12 species of fish in four regions in Xinjiang, China, to understand the distribution of local fish fungal pathogens. Twenty-six fungal strains with pathogenicity were isolated, and the challenge experiment showed that eight strains from Changji area had high infection rate to fish eggs. Based on internal transcribed spacer sequence data and molecular analysis, the 26 strains were classified into nine different species of six fungal genera. Phylogenetic analysis showed that all strains were divided into two clades, namely Cluster 1 (contains only the genus Mucor) and Cluster 2 (consists of five small branches), and the distribution of strains from the same region was scattered in two clusters. There is no strict host selectivity for these fungi to infect fish. Mucor sp. are the main fungal pathogen of fish in these four regions, whereas Hypophthalmichthys molitrix and Carassius auratus are two types of fish that were susceptible to pathogen. In addition, the environmental adaptability experiments showed that eight highly pathogenic strains have different adaptability to the environment, and their optimum temperature and pH were 25°C and 7.0, respectively, whereas the concentration of NaCl was negatively correlated with the growth of strains. Therefore, these results indicated that the coinfection of multiple fungal pathogens in a culture region should be considered in the future study.     

Pathogenesis of gram-negative bacterial infections in warmwater fish

Knowledge concerning the pathogenesis of many bacterial diseases in fish is limited, especially in those diseases that occur in warmwater species. This limited knowledge base is due to the relative recent emergence of warmwater fish culture as a major industry in many parts of the world, and to the previous economic insignificance of warmwater aquaculture and the bacterial pathogens affecting warmwater species. This article is an overview of the important gram-negative pathogens of warmwater fish, including members of the genera Aeromonas, Edwardsiella, Pasteurella, Pseudomonas, and Vibrio. The current knowledge of the pathogenesis of these organisms is emphasized, including: the source of the pathogen, its preferred site and method for attaching to and penetrating the host, its adaptations for surviving the host immune system, and its strategies for obtaining nutrients required for proliferation and growth. Although information for many of these pathogens is limited, the intent of this article is to provide a baseline for the development of future research projects. Increases in worldwide aquaculture production will result in a demand for knowledge about the pathogenesis of bacterial pathogens in warmwater fish, because of its importance in making health management decisions, in deciding on treatment regimens, and in the development of vaccines.     

Recommendations for control of pathogens and infectious diseases in fish research facilities

Concerns about infectious diseases in fish used for research have risen along with the dramatic increase in the use of fish as models in biomedical research. In addition to acute diseases causing severe morbidity and mortality, underlying chronic conditions that cause low-grade or subclinical infections may confound research results. Here we present recommendations and strategies to avoid or minimize the impacts of infectious agents in fishes maintained in the research setting. There are distinct differences in strategies for control of pathogens in fish used for research compared to fishes reared as pets or in aquaculture. Also, much can be learned from strategies and protocols for control of diseases in rodents used in research, but there are differences. This is due, in part, the unique aquatic environment that is modified by the source and quality of the water provided and the design of facilities. The process of control of pathogens and infectious diseases in fish research facilities is relatively new, and will be an evolving process over time. Nevertheless, the goal of documenting, detecting, and excluding pathogens in fish is just as important as in mammalian research models.     

The role of the diagnostic laboratory in fish disease control

The diagnostic laboratory is becoming increasingly important to the productivity and profitability of hatcheries, fish farms, and aquaculture stations. The basic function of the laboratory personnel is to isolate and identify viral, bacterial, protozoan, and other fish pathogens present in cultured and feral fish stocks. New, rapid and accurate methods for the detection and identification of fish disease agents based on immunological, biochemical, and physiological assays are becoming commonly used. Nearly every North American state or province and many foreign countries have fish health regulations that require inspection of stocks for certain disease agents before the fish are shipped into their areas. Decisions from the diagnostic laboratory on identification, treatment, guaranteed isolation, immunization, and disposal of fish populations affect administrative directives, hatchery placement, and national and international transportation of fish and fish products. This paper reviews concepts and describes the equipment, supplies, biologics, and media needed for the basic diagnostic laboratory. Information management, including training of staff, certification procedures, and quality control are also discussed.     

Histopathological survey of lesions and infections affecting sick ornamental fish in pet shops in New South Wales, Australia

The objective of this study was to describe the frequency of histopathological lesions and categorize histopathologically evident infections in sick ornamental fish from pet shops in New South Wales, Australia. We examined 108 fish that had evidence of morbidity or mortality, including 67 cyprinids, 25 osphronemids, 11 poeciliids, 4 characids and 1 cichlid, sourced from 24 retail outlets. Conditions frequently observed in the study population included branchitis (62/86, 72.1%), visceral granulomas (41/108, 38.0%), dermatitis (17/55, 30.9%), wasting (31/108, 28.7%), and intestinal coccidiosis (18/104, 17.4 %). Branchitis and dermatitis were usually due to monogenean flukes, or flagellate or ciliate protozoa. Intralesional Microsporidia (16/41, 39.0%), mycobacteria (7/41, 17.%), or Myxosporidia (5/41, 12.2%) were identified in the majority of fish with visceral granulomas; however, special stains were critical in their identification. The proportion of histologically evident infections was remarkably high (77/108, 71.3%), and parasitic infections predominated. Many pathogens identified in the study have low host specificity and/or direct life cycles which would facilitate transmission to exposed naive fish populations, potentially posing a threat to native and commercial fish populations. Those caring for sick ornamental fish should take appropriate steps to investigate infectious disease and should take precautions that prevent the spread of pathogens.     

Bacterial resistance to antimicrobial agents used in fish farming: A critical evaluation of method and meaning

The use of antimicrobial agents in aquaculture has resulted in the increase in the frequency of strains resistant to these agents. Potentially these resistant strains can have an impact on the therapy of fish diseases, the therapy of human diseases, or the environment of the fish farms. Analysis of the extent of these impacts is hindered by the limited information available and the variation in methods that have been used. There is, for example, considerable variation in the methods used to measure the sensitivity of strains and in the criteria used to determine the clinical significance of these laboratory data. It is important that some standardisation of sensitivity testing methods is attempted. The available data on the frequency of resistance in fish pathogens suggest that the use of antimicrobial agents in aquaculture has significantly reduced the therapeutic options for treating fish diseases. The data available to assess the impact of the use of these agents in aquaculture on the therapeutic options for the treatment of human infections are incomplete. At present, no quantitative assessment of this risk can be attempted. Considerations of the data on the impact of the veterinary use of these agents on the therapy of human diseases would suggest that the extent of the risk represented by their use in aquaculture is small. The epidemiology of the human pathogens that have been associated with fish would tend to confirm this assessment. There is little data pertaining to the ecology of R plasmids in the natural environment. The significance of these plasmids in transferring resistance determinants from the aquatic compartment to the human compartment can, at present, only be assessed at a theoretical level. However, such a theoretical analysis suggests that the contribution of R plasmids, selected in the aquatic environment, to the frequency of resistance in human pathogens is probably very small. Fish farmers will have to develop methods of husbandry that limit the rate at which resistant strains emerge. Without these changes in husbandry, fish farming will rapidly enter the preantibiotic era. It is probable that these changes will also have the effect of reducing any impact of antimicrobial agents used in aquaculture on the environment outside the fish farm.     

中药抑制嗜水气单胞菌、副溶血弧菌和溶藻弧菌的分子机制研究进展

嗜水气单胞菌（Aeromonas hydrophila）、副溶血弧菌（Vibrio parahaemolyticus）和溶藻弧菌（Vibrio alginolyticus）是常见的鱼类致病菌,导致鱼类患出血性败血症、溃疡等疾病。大黄、金银花、甘草等中药因具有天然活性、不易产生细菌耐药性而有望替代抗生素抑制这些鱼类致病菌。回顾细菌的致病性和中药的药理活性,从蛋白质组学和基因转录水平阐述了中药抑制嗜水气单胞菌、副溶血弧菌和溶藻弧菌的分子机制。这些研究对人类了解和控制致病菌的感染、提高鱼类免疫力以及推广应用中药作为饲料添加剂具有重要的指导意义。     

Outside-Host Growth of Pathogens Attenuates Epidemiological Outbreaks

Opportunist saprotrophic pathogens differ from obligatory pathogens due to their capability in host-independent growth in environmental reservoirs. Thus, the outside-host environment potentially influences host-pathogen dynamics. Despite the socio-economical importance of these pathogens, theory on their dynamics is practically missing. We analyzed a novel epidemiological model that couples outside-host density-dependent growth to host-pathogen dynamics. Parameterization was based on columnaris disease, a major hazard in fresh water fish farms caused by saprotrophic Flavobacterium columnare. Stability analysis and numerical simulations revealed that the outside-host growth maintains high proportion of infected individuals, and under some conditions can drive host extinct. The model can show stable or cyclic dynamics, and the outside-host growth regulates the frequency and intensity of outbreaks. This result emerges because the density-dependence stabilizes dynamics. Our analysis demonstrates that coupling of outside-host growth and traditional host-pathogen dynamics has profound influence on disease prevalence and dynamics. This also has implications on the control of these diseases.