Innate immunity of fish (overview)

The innate immune system is the only defence weapon of invertebrates and a fundamental defence mechanism of fish. The innate system also plays an instructive role in the acquired immune response and homeostasis and is therefore equally important in higher vertebrates. The innate system's recognition of non-self and danger signals is served by a limited number of germ-line encoded pattern recognition receptors/proteins, which recognise pathogen associated molecular patterns like bacterial and fungal glycoproteins and lipopolysaccharides and intracellular components released through injury or infection. The innate immune system is divided into physical barriers, cellular and humoral components. Humoral parameters include growth inhibitors, various lytic enzymes and components of the complement pathways, agglutinins and precipitins (opsonins, primarily lectins), natural antibodies, cytokines, chemokines and antibacterial peptides. Several external and internal factors can influence the activity of innate immune parameters. Temperature changes, handling and crowding stress can have suppressive effects on innate parameters, whereas several food additives and immunostimulants can enhance different innate factors. There is limited data available about the ontogenic development of the innate immunological system in fish. Active phagocytes, complement components and enzyme activity, like lysozyme and cathepsins, are present early in the development, before or soon after hatching.     

Immunostimulants,adjuvants, and vaccine carriers in fish: Applications to aquaculture

Use of immunostimulants, adjuvants, and vaccine carriers in fish culture offers a wide range of attractive methods for inducing and building up protection against diseases. Immunostimulants and adjuvants can be administered before, with, or after vaccines to amplify the specific immune response generating elevations of circulating antibody titers and numbers of plaque-forming cells. Special applications of immunostimulants include assisting shower or other regimens to increase topical uptake of vaccines. In addition, immunostimulants may be used alone, inducing elevated activities in the nonspecific defense mechanisms such as increased oxidative activity of neutrophils, augmented engulfment activity of phagocytic cells, or potentiating cytotoxic cells. In cases where disease outbreaks are cyclical and can be predicted, losses may be reduced by elevating the nonspecific defense mechanisms, and the immunostimulants may be used in anticipation of events to prevent losses from diseases. Complete Freund's adjuvant was one of the first immunostimulants used in animals to elevate the specific immune response, and it has also been successfully used in conjunction with injection of fish bacterins. Other adjuvants, immunostimulants, and biological response modifiers that have been used in fisheries research include levamisole, salt baths, and bacterial lipopolysaccharides. Vaccines have been adsorbed to inert particles, such as bentonite on latex beads, to carry the immunogens to maximize in vivo uptake for bath immunization and to facilitate in vitro phagocytosis. Each substance presents special problems in timing and method of administration (injection, immersion, oral—by feed—or flush treatments), dosage adjustments for size and fish species, storage stability, and cost. An additional consideration is that the nonspecific defense mechanisms and immune responses in fish are highly variable among individuals and statistical validation requires appropriate sample numbers and carefully controlled experiments.This article reviews the literature and present concepts of use of immunostimulants, adjuvants, and vaccine carriers in fish. Cautions for use are noted, as some of these potent substances can suppress or alter biological pathways if used inappropriately. Recent research, defining pathways of the action of immunostimulants, adjuvants, and vaccine carriers, helps explain how these substances activate the protective mechanisms in fish. In addition, immunostimulants used alone hold tremendous potential for use in fish farms, hatcheries, and aquaculture facilities to reduce losses from infectious diseases. Research on the immunostimulant, levamisole, and the light oil adjuvants for use in food fish is in progress. Applications for use of these immunostimulants are proposed.     

Predation on Multiple Trophic Levels Shapes the Evolution of Pathogen Virulence

The pathogen virulence is traditionally thought to co-evolve as a result of reciprocal selection with its host organism. In natural communities, pathogens and hosts are typically embedded within a web of interactions with other species, which could affect indirectly the pathogen virulence and host immunity through trade-offs. Here we show that selection by predation can affect both pathogen virulence and host immune defence. Exposing opportunistic bacterial pathogen Serratia marcescens to predation by protozoan Tetrahymena thermophila decreased its virulence when measured as host moth Parasemia plantaginis survival. This was probably because the bacterial anti-predatory traits were traded off with bacterial virulence factors, such as motility or resource use efficiency. However, the host survival depended also on its allocation to warning signal that is used against avian predation. When infected with most virulent ancestral bacterial strain, host larvae with a small warning signal survived better than those with an effective large signal. This suggests that larval immune defence could be traded off with effective defence against bird predators. However, the signal size had no effect on larval survival when less virulent control or evolved strains were used for infection suggesting that anti-predatory defence against avian predators, might be less constrained when the invading pathogen is rather low in virulence. Our results demonstrate that predation can be important indirect driver of the evolution of both pathogen virulence and host immunity in communities with multiple species interactions. Thus, the pathogen virulence should be viewed as a result of both past evolutionary history, and current ecological interactions.     

Preparation and Characterization of Alginate Microparticles Containing a Model Protein for Oral Administration in Gnotobiotic European Sea Bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>) Larvae

Aquaculture is the fastest growing animal production sector. However, the production of marine fish is still hampered by the high mortality rate in the first few weeks after hatching. Mortality in larvae is often caused by microbial infections. Today, the incorporation of immunostimulants into microparticles provides us new tools to enhance disease resistance in marine larviculture. In this study, we prepared alginate microparticles loaded with the model antigen fluorescein isothiocyanate conjugated-bovine serum albumin. Optimum concentrations of alginate and CaCl₂, the correct alginate viscosity and the appropriate preparatory conditions led to the creation of desirable microparticles with the correct size for oral feeding in gnotobiotic European sea bass larvae. The prepared alginate microparticles were stable in sea water and were successfully ingested by gnotobiotic sea bass larvae at day after hatching 7 without causing any negative effects. Results suggest the suitability of this drug delivery system for targeting the innate immune system of fish larvae in order to enhance disease resistance and thus reduce mortality in larviculture.     

Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.     

Immunological Control of Fish Diseases

All metazoans possess innate immune defence system whereas parameters of the adaptive immune system make their first appearance in the gnathostomata, the jawed vertebrates. Fish are therefore the first animal phyla to possess both an innate and adaptive immune system making them very interesting as regards developmental studies of the immune system. The massive increase in aquaculture in recent decades has also put greater emphasis on studies of the fish immune system and defence against diseases commonly associated with intensive fish rearing. Some of the main components of the innate and adaptive immune system of fish are described. The innate parameters are at the forefront of immune defence in fish and are a crucial factor in disease resistance. The adaptive response of fish is commonly delayed but is essential for lasting immunity and a key factor in successful vaccination. Some of the inherent and external factors that can manipulate the immune system of fish are discussed, the main fish diseases are listed and the pathogenicity and host defence discussed. The main prophylactic measures are covered, including vaccination, probiotics and immunostimulation. A key element in the immunological control of fish diseases is the great variation in disease susceptibility and immune defence of different fish species, a reflection of the extended time the present day teleosts have been separated in evolution. Future research will probably make use of molecular and proteomic tools both to study important elements in immune defence and prophylactic measures and to assist with breeding programmes for disease resistance.     

Positive selection pressure within teleost toll-like receptors tlr21 and tlr22 subfamilies and their response to temperature stress and microbial components in zebrafish

Toll-like receptors (TLRs) play a crucial role in host defence, since they trigger immune response following recognition of pathogen-associated molecular patterns (PAMPs) in potential infectious agents. TLRs have been found in numerous organisms, including mammals, birds and teleosts. Some TLR members are commonly retained across all species, whilst others were lost, gained or diverged independently during evolution. Our knowledge about the evolution and specific functions of tlr21, tlr22 and tlr23 in teleosts are still scarce. Phylogenetic analysis of 18 tlr13, tlr21, tlr22 and tlr23 genes from 9 different fish species divided them in two groups. All tlr21 genes were under the first clade, while the second comprised tlr22, tlr23 and tlr13 from Atlantic salmon. Evidence of positive selection was detected at three sites within the leucine-rich repeat regions of Tlr22, which may influence PAMP recognition. Immunostimulation experiments revealed that expression of zebrafish tlr22 is modulated by several unrelated PAMPs. Up to a 3-fold increase in tlr21 and tlr22 expression was detected in larvae exposed to immunostimulants such as lipopolysaccharide, peptidoglycan or poly I:C. We found that zebrafish tlrs are expressed mainly in immune-related organs, such as spleen and kidney as well as in testis and temperature stress did not have an effect on the expression of tlr21 and tlr22 in the early stages of development in zebrafish larvae. Our data indicates that these teleost tlrs may play a role in innate host defence. In particular, tlr22 is evolving under positive selection, which indicates functional diversification and adaptation of the response to different PAMPs.     

Tolerance of whitefish embryos to Pseudomonas fluorescens linked to genetic and maternal effects,and reduced by previous exposure

Juvenile or adult fish can alter their behaviour and rely on an innate and adaptive immune system to avoid/counteract pathogens, while fish embryos have to depend on egg characteristics and may be partly protected by their developing immune system that is building up from a certain age on. We developed an infection protocol that allows testing the reaction of individual whitefish embryos (Coregonus palaea) to repeated exposures to Pseudomonas fluorescens, an opportunistic bacterial fish pathogen. We used a full-factorial in vitro breeding design to separately test the effects of paternal and maternal contributions to the embryos' susceptibility to different kinds of pathogen exposure. We found that a first non-lethal exposure had immunosuppressive effects: pre-exposed embryos were more susceptible to future challenges with the same pathogen. At intermediate and high levels of pathogen intensity, maternal effects turned out to be crucial for the embryos' tolerance to infection. Paternal (i.e. genetic) effects played a significant role at the strongest level of infection, i.e. the embryos' own genetics already explained some of the variation in embryo susceptibility. Our findings suggest that whitefish embryos are largely protected by maternally transmitted substances, but build up some own innate immunocompetence several days before hatching.     

Intracellular innate resistance to bacterial pathogens

Mammalian innate immunity stimulates antigen-specific immune responses and acts to control infection prior to the onset of adaptive immunity. Some bacterial pathogens replicate within the host cell and are therefore sheltered from some protective aspects of innate immunity such as complement. Here we focus on mechanisms of innate intracellular resistance encountered by bacterial pathogens and how some bacteria can evade destruction by the innate immune system. Major strategies of intracellular antibacterial defence include pathogen compartmentalization and iron limitation. Compartmentalization of pathogens within the host endocytic pathway is critical for generating high local concentrations of antimicrobial molecules, such as reactive oxygen species, and regulating concentrations of divalent cations that are essential for microbial growth. Cytosolic sensing, autophagy, sequestration of essential nutrients and membrane attack by antimicrobial peptides are also discussed.     

Bacterial Interactions in Early Life Stages of Marine Cold Water Fish

Abstract The intensive rearing of various fish species in aquaculture has revealed intimate relationships between fish and bacteria that eventually may affect establishment of a ``normal' mucosal microflora or result in disease epizootics. Interactions between bacteria and mucosal surfaces play important roles both at the egg and larval stages of marine fish. Bacterial adhesion and colonization of the egg surface occur within hours after fertilization. The diverse flora which eventually develops on the egg appears to reflect the bacterial composition and load of the ambient water, but species-specific adhesion at the egg surface may also play a role in development of the egg epiflora. Proteolytic enzymes produced by members of the adherent epiflora may cause serious damage to the developing egg and may also affect further adhesion of the epiflora. Ingestion of bacteria at the yolk sac stage results in establishment of a primary intestinal microflora which seems to persist beyond first feeding. Establishment of a gut microflora is likely to undergo several stages, resulting in an ``adult' microflora weeks to months after first feeding. Ingested bacteria may serve as an exogenous supply of nutrients or essential factors at an early life stage. Early exposure to high bacterial densities is probably important for immune tolerance, and thus for the establishment of a protective intestinal microflora. Successful rearing of early life stages of several marine fish species depends on knowledge of the complex interactions among the cultured organisms and the bacterial communities which develop at the mucosal surfaces and in the ambient water and rearing systems. The routine use of antibiotics during rearing of fish larvae is not advisable, since it may increase the risk of promoting antibiotic resistance and adversely affect the indigenous microflora of the larvae. The use of probiotics has proven advantageous in domestic animal production, and the search for effective probiotics may have a great potential in aquaculture of marine organisms. Bacteria with antagonistic effects against fish pathogens have been successfully administered to several fish species, resulting in decreased mortality or increased growth rate. Received: 14 December 1998; Accepted: 7 April 1999     

The leucine-rich repeat domain in plant innate immunity: a wealth of possibilities

The innate immune system of both plants and animals uses immune receptors to detect pathogens and trigger defence responses. Despite having distinct evolutionary origin, most plant and animal immune receptors have a leucine-rich repeat (LRR) domain. The LRR domain adopts a slender conformation that maximizes surface area and has been shown to be ideal for mediating protein–protein interactions. Although the LRR domain was expected to be a platform for pathogen recognition, the NB-LRR class of plant innate immune receptors uses its LRR domain to carry out many other roles. This review discusses the domain architecture of plant LRRs and the various roles ascribed to this motif.     

The protective effect against Vibrio campbellii in Artemia nauplii by pure beta-glucan and isogenic yeast cells differing in beta-glucan and chitin content operated with a source-dependent time lag

In invertebrates the defence system to fight infectious diseases depends mainly on a non-specific or innate immune response, contrary to the vertebrate immune system. The use of natural immunostimulants that enhance the defence mechanism or the immune response of target organisms may be an excellent preventive tool against pathogens. Several strains of baker's yeast Saccharomyces cerevisiae have been found to be good immune enhancers. Previously, it was shown that small quantities of the mnn9 yeast cells and/or glucan particles could protect Artemia nauplii against the pathogenic bacterium Vibrio campbellii in the gnotobiotic Artemia challenge test. Apparently, the higher amount and/or availability of beta-glucans and/or chitin present in mnn9 yeast strain might play an essential role in such protection. The present study reveals that these compounds could only provide protection against the pathogen when they were supplied to Artemia well in advance of the challenge (8-48 h depending on the source). Also the putative immunostimulant did not have a curative action. Moreover, short-time exposure of Artemia to mnn9 strain (priming) did not provide protection against the pathogen longer than two days. Hence, it is hypothesized that the mere stimulation of known biochemical pathways, e.g. prophenoloxidase is not sufficient to explain the mechanisms involved in the observed immunostimulation obtained by beta-glucans and/or mnn9 yeast in Artemia nauplii.     

The efficacy of two immunostimulants against Flavobacterium columnare infection in juvenile rainbow trout (Oncorhynchus mykiss)

Bacterium Flavobacterium columnare is the causative agent of columnaris disease in many wild and farmed fish species. Immunostimulants are used with success in aquaculture against many pathogens, but the ability to improve innate resistance to columnaris disease has not been studied. Fingerling rainbow trout were treated with two immunostimulants, yeast β-glucan and β-hydroxy-β-methylbutyrate (HMB). Selected innate immune function parameters, the production of reactive oxygen species (ROS) by whole blood and by isolated head kidney leukocytes, plasma lysozyme activity and complement bacteriolytic activity, were determined to assess the immune status of fish. The fish were then bath challenged with virulent F. columnare bacteria, and the mortality of fish was recorded. Given orally both stimulants raised the levels of immune function parameters, but did not improve survival in challenge at any concentration of the stimulants used. Intra peritoneal injection of β-glucan increased parameter values several fold, but no beneficial effect of injected glucan on survival was noted. As a control, antibiotic medication administered prior to and during the challenge infection prevented the mortality. Innate immune mechanisms, even when induced to high levels with immunostimulants, as evidenced here, were not able to increase resistance against F. columnare. This may be connected to the external character of the infection. The results from the treatments with β-glucan and HMB suggest that there is little prospect of preventing columnaris disease by means of immunostimulants in early life stage of rainbow trout. However, the efficacy of other immune stimulants remains open.     

Dysregulated haemolysin promotes bacterial outer membrane vesicles‐induced pyroptotic‐like cell death in zebrafish

Inflammasomes are important innate immune components in mammals. However, the bacterial factors modulating inflammasome activation in fish, and the mechanisms by which they alter fish immune defences, remain to be investigated. In this work, a mutant of the fish pathogen Edwardsiella piscicida (E. piscicida), called 0909I, was shown to overexpress haemolysin, which could induce a robust pyroptotic‐like cell death dependent on caspase‐5‐like activity during infection in fish nonphagocyte cells. E. piscicida haemolysin was found to mainly associate with bacterial outer membrane vesicles (OMVs), which were internalised into the fish cells via a dynamin‐dependent endocytosis and induced pyroptotic‐like cell death. Importantly, bacterial immersion infection of both larvae and adult zebrafish suggested that dysregulated expression of haemolysin alerts the innate immune system and induces intestinal inflammation to restrict bacterial colonisation in vivo. Taken together, these results suggest a critical role of zebrafish innate immunity in monitoring invaded pathogens via detecting the bacterial haemolysin‐associated OMVs and initiating pyroptotic‐like cell death. These new additions to the understanding of haemolysin‐mediated pathogenesis in vivo provide evidence for the existence of noncanonical inflammasome signalling in lower vertebrates.     

Bateman's principle and immunity in a sex-role reversed pipefish

In diverse animal species, from insects to mammals, females display a more efficient immune defence than males. Bateman's principle posits that males maximize their fitness by increasing mating frequency whereas females gain fitness benefits by maximizing their lifespan. As a longer lifespan requires a more efficient immune system, these implications of Bateman's principle may explain widespread immune dimorphism among animals. Because in most extant animals, the provisioning of eggs and a higher parental investment are attributes of the female sex, sex-role reversed species provide a unique opportunity to assess whether or not immune dimorphism depends on life history and not on sex per se. In the broad-nosed pipefish Syngnathus typhle, males brood and nourish the eggs in a ventral pouch and thus invest more into reproduction than females. We found males to have a more active immune response both in field data from four populations and also in an experiment under controlled laboratory conditions. This applied to different measures of immunocompetence using innate as well as adaptive immune system traits. We further determined the specificity of immune response initiation after a fully factorial primary and secondary exposure to a common marine pathogen Vibrio spp. Males not only had a more active but also a more specific immune defence than females. Our results thus indeed suggest that the sex that invests more into the offspring has the stronger immune defence.     

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.     

CpG-ODN increases resistance of olive flounder (Paralichthys olivaceus) against Philasterides dicentrarchi (Ciliophora: Scuticociliatia) infection

Unmethylated cytosine-phosphate-guanine (CpG) dinucleotides flanked by specific bases in bacterial DNA induce a favorable immune response by acting as danger signals to the host. Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) also act like the unmethylated CpG oligonucleotides in bacterial DNA. In the present study, we investigated the effects of synthetic CpG-ODN on the protection of olive flounder (Paralichthys olivaceus) against infection by Philasterides dicentrarchi, a pathogen of scuticociliatosis, through two consecutive experiments (trial I and II). Fish were intraperitoneally (i.p.) injected with CpG-ODN 1668 or GpC-ODN 1720 at different doses (3 microg in trial I and 10 microg in trial II), and after one week the fish were i.p. challenged with P. dicentrarchi. In both trial I and II, fish injected with CpG-ODN 1668 showed significantly higher serum scuticocidal activity than fish injected with PBS alone, while the scuticocidal activity disappeared by heat-inactivation. This result suggests that CpG-ODN might activate an alternative pathway of complement of olive flounder, and complement-mediated killing might be an important innate immune factor in the resistance against P. dicentrarchi infection. Although the cumulative mortality was largely different between trials I and II, the relative survival rate of fish injected with a high dose of CpG-ODN 1668 was considerably higher than that of fish injected with a low dose of this ODN, while the relative survival rate was not different between fish injected with the high dose and low dose of GpC-ODN 1720. The results of the present study suggest that CpG-ODNs may be used as potential immunostimulants to lessen cultured fish loss caused by scuticociliates.