Article

Efficacy of a polyvalent injectable vaccine against Flavobacterium psychrophilum administered to rainbow trout (Oncorhynchus mykiss L.)

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  • Veterinary Medicines Directorate
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Abstract

Flavobacterium psychrophilum is one of the most important pathogens affecting cultured rainbow trout (Oncorhynchus mykiss). Recent information from UK salmonid farms showed country‐wide distribution of genetically and serologically divergent clones, which has hampered the development of a vaccine for rainbow trout fry syndrome. The current study assessed the efficacy of an injectable polyvalent vaccine containing formalin‐inactivated F. psychrophilum in rainbow trout. The vaccine was formulated with an oil adjuvant (Montanide ISA 760VG) or formalin‐killed cells alone. Duplicate groups of trout (60 ± 13 g) were given phosphate‐buffered saline or vaccine formulated with Montanide by intra‐peritoneal (i.p.) injection and challenged by intra‐muscular (i.m.) injection with a homologous and a heterologous isolate of F. psychrophilum at 525 degree days post‐vaccination (dd pv). Significant protection was achieved in vaccinated fish (p = 0.0001, RPS 76% homologous, 88% heterologous). Efficacy of the adjuvanted vaccine was also demonstrated by heterologous challenge at 1155 dd pv resulting in 100% protection, whereas survival in the un‐adjuvanted group was not significantly different from control fish. Levels of specific antibody at 1155 dd pv, as measured by ELISA, were significantly higher in the fish vaccinated with adjuvant when compared with unvaccinated fish.

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... Many of these methods have proven efficacious in examples of terrestrial disease [35][36][37] but as of yet, few have been successful in providing consistently effective protection against BCWD/RTFS. In recent studies, there have been vaccine candidates/formulations that have shown promise against BCWD/RTFS [38][39][40] as well as some selective breeding regimes preventing outbreaks of this troublesome disease [41]. For all of these studies, very little is known regarding the cause of this enhanced protection. ...
... This is likely due to the components of the bacterial membrane, thus F. psychrophilum cells alone may not be as immunogenic as those of Vibrio anguillarum. Indeed, this has been observed in recent attempts to develop effective vaccines for BCWD/RTFS where injection with formalin-killed polyvalent F. psychrophilum alone was not protective but an adjuvanted version of the same formulation had much higher efficacy [38]. Additionally, a study by Ref. [40] reported promising results using a live-attenuated immersion vaccine for BCWD/RTFS [39,40]. ...
Article
The life cycle of Flavobacterium psychrophilum (Fp), the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), appears to involve interactions with spleen and head kidney macrophages. To develop an in vitro model for studying this, F. psychrophilum was incubated with a rainbow trout splenic monocyte/macrophage-like cell line (RTS11) and fundamental macrophage functions evaluated. The animal cell basal medium, L15, supplemented with bovine serum (FBS) supports RTS11 maintenance, and surprisingly, L15 with 2% FBS (L15/FBS) also supported F. psychrophilum growth. L15/FBS in which the bacteria had been grown is referred to as F. psychrophilum conditioned medium (FpCM). Adding FpCM to RTS11 cultures caused a small, yet significant, percentage of cells to die, many cells to become more diffuse, and phagocytosis to be temporarily reduced. FpCM also significantly stimulated transcript expression for pro-inflammatory cytokines (IL-1β, TNFα and IL-6) and the anti-inflammatory cytokine (IL-10) after one day of exposure but this upregulation rapidly declined over time. Adding live F. psychrophilum to RTS11 cultures also altered the cellular morphology and stimulated cytokine expression more profoundly than FpCM. Additionally, the phagocytic activity of RTS11 was also significantly impaired by live F. psychrophilum, but not to the same extent as when exposed to FpCM. Adding heat-killed bacteria to RTS11 cultures elicited few changes. These bacteria/RTS11 co-cultures should be useful for gaining a deeper understanding of the pathogenesis of F. psychrophilum and may aid in the development of effective measures to prevent infection and spread of this troublesome disease.
... Vaccination is applied broadly in aquaculture (4), and there remains a need to develop new and improved vaccines for many species and diseases, along with less labor-intensive modes of administration (3). An important part of the development pipeline is vaccine efficacy testing, which can be assessed directly via the extent of protection (e.g. using disease challenge tests), or indirectly, by studying changes in immune parameters, for example by confirming an antigen-specific antibody response (8,9) or recording changes in the expression of immune molecules (e.g. (10,11)). ...
Article
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Vaccination plays a critical role in the protection of humans and other animals from infectious diseases. However, the same vaccine often confers different protection levels among individuals due to variation in genetics and/or immunological histories. While this represents a well-recognized issue in humans, it has received little attention in fish. Here we address this knowledge gap in a proteomic study of rainbow trout (Oncorhynchus mykiss, Walbaum), using non-lethal repeated blood sampling to establish the plasma protein response of individual fish following immunization. Six trout were immunized with adjuvanted hen egg-white lysozyme (HEL) and peripheral blood sampled at ten time points from day 0 to day 84 post-injection. We confirm that an antigen-specific antibody response to HEL was raised, showing differences in timing and magnitude among individuals. Using label-free liquid chromatography-mass spectrometry, we quantified the abundance of 278 plasma proteins across the timecourse. As part of the analysis, we show that this approach can distinguish many (but not all) duplicated plasma proteins encoded by paralogous genes retained from the salmonid-specific whole genome duplication event. Global variation in the plasma proteome was predominantly explained by individual differences among fish. However, sampling day explained a major component of variation in abundance for a statistically defined subset of 41 proteins, representing 15% of those detected. These proteins clustered into five groups showing distinct temporal responses to HEL immunization at the population level, and include classical immune (e.g. complement system members) and acute phase molecules (e.g. apolipoproteins, haptoglobins), several enzymes and other proteins supporting the immune response, in addition to evolutionarily conserved molecules that are as yet uncharacterized. Overall, this study improves our understanding of the fish plasma proteome, provides valuable marker proteins for different phases of the immune response, and has implications for vaccine development and the design of immune challenge experiments.
... From a BCWD prevention perspective in relation to vaccine development, characterizing the antigenic profiles of the predominating F. psychrophilum strains behind the disease outbreaks is crucial (16). Indeed, recent studies in the United Kingdom have demonstrated the promise that whole-killed F. psychrophilum vaccine preparations (i.e., bacterins) hold when guided by serotyping data (41). In this context, data from this study will be useful as future research takes aim at developing vaccine preparations, which likely will need to be region-and/or fish species-specific in some cases. ...
Article
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Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, causes great economic losses in salmonid aquaculture worldwide. Recent molecular studies have uncovered important epidemiological and ecological aspects of this pathogen; however, such data are lacking for F. psychrophilum populations affecting aquaculture in China. Herein, F. psychrophilum phenotype, genotype, and virulence were characterized for isolates recovered from epizootics in multiple salmonid aquaculture facilities across China. Thirty-one F. psychrophilum isolates, originating from four provinces and three host fish species, were predominantly homogeneous biochemically but represented 5 sequence types (STs) according to multilocus sequence typing (MLST) that belonged to clonal complex CC-ST10 or 3 newly recognized singleton STs. PCR-based serotyping classified 19 and 12 F. psychrophilum isolates into molecular serotypes 1 and 0, respectively, showing an obvious relationship with host species. Antimicrobial susceptibility analysis via broth microdilution revealed reduced susceptibility to enrofloxacin, flumequine, and oxolinic acid, moderate susceptibility to gentamicin, erythromycin, and florfenicol, and variable susceptibility to ampicillin and oxytetracycline. In vivo challenge experiments confirmed the ability of two representative Chinese F. psychrophilum isolates to induce typical signs of BCWD and mortality in 1-year-old rainbow trout (Oncorhynchus mykiss). Findings collectively demonstrate (i) that BCWD outbreaks in China studied thus far are caused by F. psychrophilum lineages that are common on other continents (e.g., CC-ST10) and others that have not been reported elsewhere (e.g., ST355, ST356, ST357), (ii) that F. psychrophilum molecular serotypes distinguish isolates from different host fish species, even within STs, and (iii) reduced F. psychrophilum antimicrobial susceptibility against compounds used for BCWD control in China. IMPORTANCE Flavobacterium psychrophilum causes substantial economic losses in salmonid aquaculture worldwide. Although this bacterium is also believed to be a disease source in China, published reports of its presence do not yet exist. Herein, F. psychrophilum was linked to multiple disease outbreaks in several salmonid aquaculture facilities within four Chinese provinces, and polyphasic characterization revealed that most isolates were genetically distinct from strains recovered on other continents. Analyses further revealed the predominating molecular serotypes, antimicrobial susceptibility profiles, and pathogenic potential of two representative recovered isolates. Collectively, the results presented here provide important data on the epidemiology and disease ecology of F. psychrophilum in China and pave the way for targeted prevention and control methods to be pursued in the future.
... This mirrors the difficulties and variable success observed when attempting to develop a fish vaccine for R. salmoninarum, an intracellular bacterial pathogen (Burnley et al., 2010). Despite all that is unknown regarding F. psychrophilum, there have been some promising vaccine candidates developed recently for BCWD (Fredriksen et al., 2013;Ma et al., 2019;Hoare et al., 2019) but these studies must be repeated and tested during natural outbreak/exposure conditions. For many bacterial pathogens, developing an effective vaccine or improving current formulations has required an understanding of the bacterial pathogenesis. ...
... Vaccination is applied broadly in aquaculture (4), and there remains a need to develop new and improved vaccines for many species and diseases, along with less labor-intensive modes of administration (3). An important part of the development pipeline is vaccine efficacy testing, which can be assessed directly via the extent of protection (e.g. using disease challenge tests), or indirectly, by studying changes in immune parameters, for example by confirming an antigen-specific antibody response (8,9) or recording changes in the expression of immune molecules (e.g. (10,11)). ...
Chapter
Globally bacterial diseases cause significant economic losses to aquaculture production. Vaccination against bacterial pathogens is one of the most effective measures for preventing bacterial diseases, especially in finfish aquaculture. Both live and inactivated vaccines are predominantly available for use in aquaculture against bacterial diseases in many countries. Most of these vaccines are known to provide high efficacy in eliciting long-term immunity. Besides, several new approaches such as sub-unit vaccines and DNA vaccines have also been successfully demonstrated to provide protection against bacterial pathogens of fish. A summary of bacterial vaccines either in use in aquaculture or under research is compiled in the chapter.
Chapter
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Technical Report
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Availability of effective treatments for control of infectious diseases is a critical requirement of the Scottish and wider UK rainbow trout industry. The purpose of this project was to: identify the key diseases that affect freshwater aquaculture operations in Scotland, particularly the trout sector, and determine their relative impact; to identify the main methods used to control these diseases; identify the potential consequences if any of the main control methods were to be withdrawn; and finally, to identify any new potential treatments that could be used instead, if any of the main treatments were to be withdrawn. Producers, vets and health professionals surveyed confirmed that production was constrained by a limited group of common diseases that affected rainbow trout producers in England and Scotland. These included rainbow trout fry syndrome (RTFS) caused by the bacterium Flavobacterium psychrophilum, white spot disease caused by the endoparasite Ichthyophonus multifiliis, enteric redmouth disease (ERM) caused by the bacterium Yersinia ruckeri, proliferative kidney disease caused by the myxozoan parasite Tetracapsuoidesa bryosalmonae, red mark syndrome (RMS) and bacterial gill disease (BGD). The main treatments available to control these conditions were limited, with florfenicol reportedly used by all producers to control RTFS, formalin used extensively to control white spot and a range of parasites and chloramine T to treat bacterial gill disease. ERM was mainly controlled by vaccination, particularly via dip vaccination of fry with the Relera dual antigen vaccine. Other licensed antibiotics (oxytetracycline, amoxicillin and oxolinic acid) were used to treat sporadic outbreaks of ERM, in fish where vaccine protection had waned, and furunculosis. The major reliance of the industry on florfenicol and formalin was concerning. Firstly there were limited identified alternatives to control RTFS in the event of RTFS-causing strains of F. psychrophilum developing resistance to florfenicol. There is also pressure at an EU level to withdraw formalin from sale as a biocide. Possible alternatives to the use of formalin products purchased for biocidal applications were reviewed in the event of their withdrawal from sale. For control of white spot it may be possible to use a licensed product marketed in Spain for the control of parasites of turbot under the veterinary cascade. The bronopol containing medicine Pyceze is one identified alternative that may be used. Where systems can be engineered to allow its use, Salt (sodium chloride), either via low concentration continuous dosing for several days, or short duration high concentration flushes is also a potential treatment. Practical issues with regards either maintaining low concentrations of salt, or dealing with high concentration effluents, may limit the use of this treatment strategy though. Project staff also consulted with Danish producers who are trialing the use of peracetic acid. For control of some ectoparasites, particularly flukes (e.g. trichodina), praziquantel, either as a water-based or in feed treatment, may also be an option to explore. Review of the literature suggested that caprylic acid, green tea extract and epigallocatechin gallate (EGCG), Piscidin 2, quinine, Triclabendazole and potassium ferrate may all have some promise as alternative treatments. Selection of any alternative treatments should be guided by whether they are likely to be readily useable. In this regards, products that already have approval for use in food animal production, either as biocides, feed additives or as medicines should be preferred in the first instance. Recommendations  Undertake further controlled studies (laboratory and field based) on the effectiveness of peracetic acid for the control of white spot and other production diseases.  Obtain further information on the margin of safety of peracetic acid at different temperatures via target animal safety studies, at both a farm and laboratory scale.  Continue to support efforts to develop alternatives vaccines for the control of RTFS.  Determine the effectiveness of alternative antibiotics to florfenicol to control RTFS infections caused by Flavobacterium psychrophilum.  Explore practicalities of importing formalin-containing medicinal products licensed in other Ms for control of fish diseases for controlling white spot and other diseases.  Investigate use of mechanical control measures to reduce the impact of white spot in rainbow trout production systems.  In vivo trials are needed to follow up some of the potential alternative chemical treatments identified (e.g. caprylic acid, green tea extract and epigallocatechin gallate (EGCG), Piscidin 2, quinine, Triclabendazole and potassium ferrate). Conclusions The survey clearly demonstrates that the rainbow trout industry is heavily reliant on a very limited range of treatment options to control major production diseases. Discussions with fish medicine producers and veterinarians also suggest that the freshwater stage of the Atlantic salmon industry is similarly reliant on a small range of similar treatments to those used in the trout industry. In particular, there is also heavy reliance on formalin to control white spot disease and Costia in some hatcheries, and similar reports that florfenicol is the only effective treatment for the control of Flavobacterium psychrophilum. They also report that formalin is used quite extensively to control saprolegniasis in vaccinated salmon smolts prior to seawater transfer. These findings are collectively concerning as either the withdrawal of formalin from sale, or the development of resistance to florfenicol in Flavobacterium psychrophilum, could affect the viability of both industries.
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A key hallmark of the vertebrate adaptive immune system is the generation of antigen-specific antibodies from B cells. Fish are the most primitive gnathostomes (jawed vertebrates) possessing an adaptive immune system. Vaccination of rainbow trout against enteric redmouth disease (ERM) by immersion in Yersinia ruckeri bacterin confers a high degree of protection to the fish. The immune mechanisms responsible for protection may comprise both cellular and humoral elements but the role of specific immunoglobulins in this system has been questioned and not previously described. The present study demonstrates significant increase in plasma antibody titers following immersion vaccination and significantly reduced mortality during Y. ruckeri challenge. Rainbow trout were immersion-vaccinated, using either a commercial ERM vaccine (AquaVac™ ERM vet) or an experimental Y. ruckeri bacterin. Half of the trout vaccinated with AquaVac™ ERM vet received an oral booster (AquaVac™ ERM Oral vet). Sub-groups of the fish from each group were subsequently exposed to 1x109 CFU Y. ruckeri/ml either eight or twenty-six weeks post vaccination (wpv). All vaccinated groups showed 0% mortality when challenged, which was highly significant compared to the non-vaccinated controls (40 and 28% mortality eight and twenty-six weeks post vaccination (wpv), respectively) (P<0.0001). Plasma samples from all groups of vaccinated fish were taken 0, 4, 8, 12, 16 and 26 wpv. and Y. ruckeri specific IgM antibody levels were measured with ELISA. A significant increase in titers was recorded in vaccinated fish, which also showed a reduced bacteremia during challenge. In vitro plasma studies showed a significantly increased bactericidal effect of fresh plasma from vaccinated fish indicating that plasma proteins may play a role in protection of vaccinated rainbow trout.
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Teleost fish are the most primitive bony vertebrates that contain immunoglobulins. In contrast to mammals and birds, these species are devoid of immunoglobulin A (IgA) or a functional equivalent. This observation suggests that specialization of immunoglobulin isotypes into mucosal and systemic responses took place during tetrapod evolution. Challenging that paradigm, here we show that IgT, an immunoglobulin isotype of unknown function, acts like a mucosal antibody. We detected responses of rainbow trout IgT to an intestinal parasite only in the gut, whereas IgM responses were confined to the serum. IgT coated most intestinal bacteria. As IgT and IgA are phylogenetically distant immunoglobulins, their specialization into mucosal responses probably occurred independently by a process of convergent evolution.
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Experiments were done in order to achieve a reproducible method that can be used to infect rainbow trout Oncorhynchus mykiss with Flavobacterium psychrophilum, the causal agent of coldwater disease and rainbow trout fry syndrome. The main method investigated was intraperitoneal injection, and this method was tested using isolates with different elastin-degrading profiles and representing different serotypes. Injecting trout, average weight 1 g, with 10(4) CFU (colony-forming units) per fish caused cumulative mortalities around 60 to 70%. The virulent strains belonged to certain serotypes and degraded elastin. The intraperitoneal injection challenge method could be used on larger fish, but the infection dose was 10(7) CFU per fish before mortalities occurred. Bath infection and bath infection in combination with formalin treatment (stress) seemed to be reproducible methods that could be used as alternatives to the intraperitoneal method, although the mortalities among infected trout were lower. The results of investigated methods were influenced by parameters such as the challenge isolate, number of fish in the tank affecting the infection pressure, origin of fish and weight of fish.
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Rainbow trout fry syndrome (RTFS) is a septicaemic infection of young rainbow trout Oncorhynchus mykiss occurring at low temperatures and responsible for severe economic losses in European fish farming. The causative agent, Flavobacterium psychrophilum, is a gliding bacterium, and difficulties in culturing it have long been an impediment to investigations on pathogenesis and immunity. Successful attempts at experimentally inducing the disease have been reported, but no experimental model resulting in well-controlled and quantitatively reproducible effects has been described. Recent improvements in F. psychrophilum cultivation made it possible to produce bacterial suspensions with nearly constant viability and to complete challenge injections in rainbow trout fingerlings, using accurately adjusted infective doses. Parenteral injection resulted in significant mortality, which was higher when administered intramuscularly (IM) than intraperitoneally (IP). Lethal doses 50 % lower than 10(3) colony forming units were consistently obtained in trout weighing 3 to 5 g, and the regular shape of the cumulative mortality curves appeared to lend itself to quantitative analyses. Bath experiments produced milder effects, although mortality ranging between 45 and 60 % was obtained in 6 g trout when skin lesions or stressors were induced along with bacterial exposure. Temperature, salinity and the process of preserving isolates (at least over short periods of time) did not seem to be associated with the severity of infection. Nevertheless, infection trials performed at 2 different locations differing both in water quality and in the system of fish maintenance resulted in different mortalities. These findings notwithstanding, the proposed IM model appears easy to apply under standardized experimental conditions and should contribute to effective advances in the study of the disease.
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Flavobacteria are a poorly understood and speciated group of commensal bacteria and opportunistic pathogens. The psychrotroph Flavobacterium psychrophilum is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic diseases that heavily impact salmonids. Consequently, two verified but geographically diverse isolates were characterized phenotypically and biochemically. A facile typing system was devised which readily discriminated between closely related species and was verified against a pool of recent prospective isolates. F. psychrophilum was found to be enveloped in a loosely attached, strongly antigenic outer layer comprised of a predominant, highly immunogenic, low-molecular-mass carbohydrate antigen as well as several protein antigens. Surface-exposed antigens were visualized by a combination of immunoflourescence microscopy, immunogold transmission, and thin-section electron microscopy and were discriminated by Western blotting using rabbit antisera, by selective extraction with EDTA-polymyxin B agarose beads, and by extrinsic labeling of amines with sulfo-N-hydoxysuccinimide-biotin and glycosyl groups with biotin hydrazide. The predominant approximately 16 kDa antigen was identified as low-molecular-mass lipopolysaccharide (LPS), whereas high-molecular-mass LPS containing O antigen was not as prevalent on whole cells but was abundant in culture supernatants. Rainbow trout convalescent antisera recognized both molecular mass classes of LPS as well as a predominant approximately 20-kDa protein. This study represents the first description at the molecular level of the surface characteristics and potential vaccine targets of confirmed F. psychrophilum strains.
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Flavobacterium psychrophilum is the causative agent of coldwater disease, which is responsible for serious losses in fish aquaculture in several parts of the world. No commercial vaccines are currently available for the prevention of coldwater disease. The present study sought to assess the efficacy of a F. psychrophilum vaccine based on the antigenic outer membrane fraction (OMF). This fraction induced significantly higher protection against coldwater disease in both rainbow trout (Oncorhynchus mykiss) and ayu (Plecoglossus altivelis) compared to inactivated whole cell F. psychrophilum bacterin. Coincident with higher protection, sera of fish immunised with the OMF vaccine had higher agglutination titres than those of fish immunised with inactivated whole cell F. psychrophilum.
Chapter
The purpose of this chapter is to provide readers with a general overview of the fish immune system, including commonalities and particularities in comparison with higher vertebrate systems. Throughout this chapter, we will review the current knowledge of the immune functionality of fish organs, tissues, structures, cells, molecules, and mechanisms, mainly focusing on current knowledge of the cultivable teleost species on which most of the immunological research has focused in recent years. A comparison with mammalian systems will also be addressed, especially in cases where teleost immunity seems to challenge mammalian paradigms.
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On the basis of the 16S rRNA sequence data analysis among the closely related species, the specific primers for Cytophaga psychrophila were constructed. The specificity in amplifying the 16S rRNA of C. psychrophila was confirmed by using some selected strains of the related species. In addition, it was revealed that these specific primers distinguished C. psychrophila from other principal fish pathogens. The present PCR technique is expected to be a powerful tool for the diagnosis of cold-water disease.
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Prophylactic measures against Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis, have been an active field of research for decades, with studies mainly focused on Atlantic salmon (Salmo salar). In the present study we have examined the protective and adverse effects of mineral oil-adjuvanted injection vaccines on rainbow trout (Oncorhynchus mykiss). A commercial vaccine and an experimental auto vaccine, as well as their respective adjuvant formulations alone were used to evaluate their individual effects, both prior to and during an experimental waterborne infection challenge. Macro- and microscopic examination revealed signs of vaccine-induced adverse effects from 10 weeks to 14 months post vaccination. Both vaccines induced statistically significant protection during the experimental challenge (P=0.018 for both vaccines), as well as significantly elevated levels of specific circulating antibodies prior to and during the experimental challenge when compared to an unvaccinated control group. During the early, critical time points of the infection, both vaccines appeared to protect against pathological changes to the liver and spleen, which provides a probable explanation for the reduced mortality seen in the vaccinated groups. A significant correlation was found between the level of A. salmonicida-specific antibodies measured prior to challenge and the endpoint survival of each group after the experimental infection, and furthermore, the levels of these antibodies remained elevated for at least 14 months post vaccination.
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Pattern recognition receptors (PRRs) recognize the conserved molecular structure of pathogens and trigger the signaling pathways that activate immune cells in response to pathogen infection. Toll-like receptors (TLRs) are the first and best characterized innate immune receptors. To date, at least 20 TLR types (TLR1, 2, 3, 4, 5M, 5S, 7, 8, 9, 13, 14, 18, 19, 20, 21, 22, 23, 24, 25, and 26) have been found in more than a dozen of fish species. However, of the TLRs identified in fish, direct evidence of ligand specificity has only been shown for TLR2, TLR3, TLR5M, TLR5S, TLR9, TLR21, and TLR22. Some studies have suggested that TLR2, TLR5M, TLR5S, TLR9, and TLR21 could specifically recognize PAMPs from bacteria. In addition, other TLRs including TLR1, TLR4, TLR14, TLR18, and TLR25 may also be sensors of bacteria. TLR signaling pathways in fish exhibit some particular features different from that in mammals. In this review, the ligand specificity and signal pathways of TLRs that recognize bacteria in fish are summarized. References for further studies on the specificity for recognizing bacteria using TLRs and the following reactions triggered are discussed. In-depth studies should be continuously performed to identify the ligand specificity of all TLRs in fish, particularly non-mammalian TLRs, and their signaling pathways. The discovery of TLRs and their functions will contribute to the understanding of disease resistance mechanisms in fish and provide new insights for drug intervention to manipulate immune responses.
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Live attenuated vaccine is one of the efficient vaccine candidates in aquaculture, which can be easily delivered to fish via bath-vaccination. An outstanding advantage of bath-vaccination is that vaccine delivery is through the same route as that utilized by many fish pathogens, generating specific mucosal immune responses. In this work, we investigated the mucosal immune responses induced by a live attenuated Vibrio anguillarum vaccine in zebrafish via bath-vaccination. Bacteria proliferated rapidly in 3 hours after vaccination and maintained at a high level until 6 hours in the intestine. Besides, bacteria persisted in the intestine for a longer time whereas decreased rapidly in the skin and gills. Moreover, a significant up-regulation of TLR5 triggering a MyD88-dependent signaling pathway was observed in the intestine, which implied that flagella were the crucial antigenic component of the live attenuated vaccine. And macrophages and neutrophils showed active responses participating in antigen recognition and sampling after vaccination. Furthermore, an inflammation was observed with plenty of lymphocytes in the intestine at 24 h post vaccination but eliminated within 7 days. In conclusion, the live attenuated V. anguillarum vaccine induced notable mucosal immune responses in the intestine which could be used as a mucosal vaccine vector in the future.
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The immune response and morphological changes in the gills of rainbow trout fry after immersion in hydrogen peroxide (H2 O2 ), Flavobacterium psychrophilum or combined exposure were examined. The gills were sampled 4, 48, 125 and 192 h after exposure, and the regulation of expression of the following genes was investigated using qPCR: IgT, IgM, CD8, CD4, MHC I, MHC II, IL-4/13A, TcR-β, IL-10, IL-1β, IL-17, SAA and FoxP3. Bacteria were not observed in haematoxylin-and-eosin-stained gill tissue, but the presence of F. psychrophilum 16S rRNA was detected using qPCR. The 16S rRNA levels were correlated with gene expression. Although pretreatment with H2 O2 before immersion in F. psychrophilum did not significantly alter the amount of bacteria found in the gill, the immune response was influenced: exposure to F. psychrophilum resulted in a negative correlation with expression of IL-17c1, MHC I and MHC II, while pretreatment with H2 O2 resulted in a positive correlation with IL-4/13A and IgM. Exposure to either H2 O2 or F. psychrophilum influenced the regulation of gene expression and damaged tissue. Exposure to both combined altered the immune response to infection and postponed healing of gill tissue.
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The efficacy of immersion vaccination Yersinia ruckeri bacterin containing Montanide(TM) IMS 1312 VG was evaluated in 100-120 g rainbow trout against yersiniosis. Healthy fish were vaccinated by immersion vaccination with inactivated whole cells (1 × 10(8) cells/ml) of a virulent strain of Y. ruckeri biotype I with and without Montanide (1:1; Montanide/antigen) for 2 min at 12-14°C. Control group was immersed in sterile PBS. Leukocyte counts, serum lysozyme assay, alternative hemolytic complement (ACH50) assay, antibody titration and relative percent survival (RPS) were measured on 2-10 weeks post-immunization. No significant difference was seen in leucocyte population of trout immunized either with Y. ruckeri antigen or Y. ruckeri antigen containing Montanide (P>0.05), while leucocyte and heterophil populations in control group were significantly lower and higher, respectively, than both immunized groups (P<0.05). In addition, there was no significant difference in lymphocyte population of trout immunized either with Y. ruckeri antigen or Y. ruckeri antigen containing Montanide (P>0.05), while lymphocyte population in control group was significantly lower than both immunized groups (P<0.05). Lysozyme activity in immunized fish with Y. ruckeri containing Montanide was higher than the immunized fish with Y. ruckeri antigen alone during 8 weeks post-immunization ((P<0.0.5).Also, level of lysozyme in control fish was generally lower than both immunized groups (P<0.05). The level of ACH50 between both immunized groups was insignificant (P>0.05) but these were significantly higher than control group through the experiment (P<0.05). The lowest anti-Y .ruckeri antibody titers in both immersion vaccination groups were significantly higher through 2-8 weeks post-vaccination compared to the control group (P<0.05). In the group immersion vaccinated with Y. ruckeri bacterin plus Montanide the titers 2-8 weeks post-vaccination were significantly higher the titer in the immersion vaccinated with Y. ruckeri bacterin (P<0.05).Fish vaccinated with antigen without Montanide resulted in RPS of 80-82% on 2-10 weeks post-vaccination, while those for antigen containing montanide gave RPSs of 93.8-100% 2-10 weeks post-immunization (P<0.05).
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Flavobacterium psychrophilum is one of the most threatening bacterial infections for freshwater fish species in the world and is of considerable economic significance to aquaculture producers. Up to now, antimicrobial therapies represent the only recourse to control the disease in farmed fish. In this study, a total of 40 F. psychrophilum isolates obtained from Chilean salmonid farms were tested to determine their antimicrobial susceptibility using minimum inhibitory concentration (MIC) determinations and disk diffusion tests, for oxytetracycline (OTC), florfenicol (FLO) and oxolinic acid (OXA) and also to examine the plasmid profile to assist in the characterization of the strains isolated from different salmonid species. In addition, the presence of mutations in the quinolone resistance‐determining region (QRDR) was evaluated. Normalized resistance interpretation (NRI) of MIC data suggested cut-off values for non-susceptible at R > 0.203, and 2.35 μg mL− 1 for OTC, and FLO, respectively. For OXA a slightly larger coefficient of variation in the calculations required a conservative estimate at R ≥ 16 μg mL− 1. Of the 40 Chilean isolates studied, 90%, 92.5%, and 85% F. psychrophilum were resistant to the three antimicrobials. In the disk diffusion tests, all isolates produced zones of inhibition to OTC30 and FLO30. However, there was a low or no correlation between MIC results and inhibition zone diameter values and valid NRI calculations were not possible to perform. For OXA2 disks, no zones were observed (< 7 mm). No correlation was found between quinolone resistance and PCR-RFLP analysis; whereas 5 isolates had mutations in the QRDR of gyrA. Thirty-nine of 40 isolates possessed a single plasmid (15 isolates with 3.5 kb) or combinations of two plasmids (17 and 7 isolates of sizes 1.5 and 3.5 kb and 2.7 and 3.5 kb, respectively), but a relationship between plasmid and resistance could not be established. In summary, we demonstrate in this work by MIC assays that high levels of resistance to OTC, FLO and OXA exist among Chilean isolates, which could be associated with the high amounts of antimicrobials applied at the Chilean farms to control outbreaks caused by F. psychrophilum. Disk contents for disk diffusion tests were not suitable for this bacterium. Surveillance in the use of drugs is recommended in cultured freshwater fish to prevent resistance problem.
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The vaccinal efficacy of a heat-inactivated strain of Flexibacter psychrophilus, the bacterium responsible in France for the visceral form of coldwater disease, was tested after intraperitoneal (IP) or bath vaccination. The level of protection was determined by the survival of fish after IP injection challenge with the live bacterium. After IP vaccination of 2.2 g fish, a relative percent survival value of 80 was obtained. Bath vaccination provided a lower but still significant protection in fish vaccinated at 50 d post-hatch or later. No protection was observed in fish vaccinated at 30 or 40 d post-hatch.
Article
This study aimed to assess systemic and mucosal immune responses of Atlantic salmon (Salmo salar) exposed to two protein-hapten antigens - dinitrophenol (DNP) and fluorescein isothiocyanate (FITC) each conjugated with keyhole limpet haemocyanin (KLH) - administered using different delivery strategies. Fish were exposed to the antigens through different routes, and were given a booster 4 weeks post initial exposure. Both systemic and mucosal antibody responses were measured for a period of 12 weeks using an enzyme-linked immunosorbent assay (ELISA). Only fish exposed to both antigens via intraperitoneal (IP) injection showed increased systemic antibody response starting 6 weeks post immunization. No treatment was able to produce a mucosal antibody response; however there was an increase in antibody levels in the tissue supernatant from skin explants obtained 12 weeks post immunization from fish injected with FITC. Western blots probed with serum and culture supernatant from skin explants showed a specific response against the antigens. In conclusion, IP injection of hapten-antigen in Atlantic salmon was the best delivery route for inducing an antibody response against these antigens in this species. Even though IP injection did not induce an increase in antibody levels in the skin mucus, there was an increased systemic antibody response and an apparent increase of antibody production in mucosal tissues as demonstrated by the increased level of specific antibody levels in supernatants from the tissue explants.
Article
The aim of the present study was to assess the prevalence of the flavobacteria within farmed trout and to quantify their bacterial burden. A total of 61 fish were sampled from seven farms, and were distributed in two groups: (1) visibly diseased fish suffering from the rainbow trout fry syndrome or the bacterial cold water disease caused by the bacteria Flavobacterium psychrophilum and (2) normally appearing fish. F. psychrophilum cells were titered by qPCR, targeting a specific area of the 16S rRNA gene in skin, muscle, gills, liver, spleen and kidney from all fish. The pathogen was detected in these organs whatever the health status, with titers ranging from 10(4) to 6×10(7)bacteria/g of tissue in normally appearing fish, thus showing they were bacterial carriers. Two organs allowed differentiation between diseased and normally appearing fish: spleen and kidney, with titers ranging from 10(6) to 10(7)bacteria/g of tissue in normally appearing fish vs 10(11) to 10(12)bacteria/g of tissue in diseased fish. No relationship was found between immunoglobulin M-like titer in plasma and health status. Gene expression analysis in fish organs revealed two genes that were markers of the bacterial infection: mt-a and il-1β genes encoding the metallothionein A and the interleukin1-β, respectively. These genes were both over-expressed in gills, liver, spleen and kidney of diseased fish. Four genes encoding immunity markers were down-regulated in spleen (a key organ implicated in immunity) of diseased fish: tgf-β, cd8-α, mhc2-β and igt, suggesting a weakened immune system in diseased fish.
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The factors influencing the uptake of a 14C-labelled Vibrio anguillarum vaccine in direct immersion experiments with rainbow trout, Salmo gairdneri were studied. Immersion times of longer than 10 s did not increase vaccine uptake. A pre-immersion dip in a hyperosmotic solution had no effect on uptake, even at lower vaccine bath concentrations. It appeared that the head end of the fish was implicated in uptake. Vaccine uptake decreased at lower temperatures, whereas the use of an adjuvanted vaccine and a soluble vaccine preparation led to increased uptake. Larger fish took up more vaccine.
Article
Serum and mucosal antibody responses of juvenile rainbow trout, Oncorhynchus mykiss, were characterized by enzyme-linked immunosorbent assay (ELISA) following immunization with various preparations of formalin-killed Flavobacterium psychrophilum cells. The protective nature of these preparations was then determined by immunizing rainbow trout fry and challenging with the bacterium. Juvenile rainbow trout immunized intraperitoneally (i.p.) with formalin-killed F. psychrophilum emulsified with Freund's complete adjuvant (FCA), and i.p. with formalin-killed F. psychrophilum either with or without culture supernatant generated significant serum antibody responses by 6 and 9 weeks, respectively. Significant mucosal antibody responses were detected by 9 weeks only in fish immunized i.p. with killed F. psychrophilum/FCA. Following immunization and bacterial challenge of rainbow trout fry, protective immunity was conferred in F. psychrophilum/FCA and saline/FCA groups with relative per cent survival values of up to 83 and 51, respectively. Significant protection was not observed in treatment groups immunized by immersion or i.p. without adjuvant at the challenge doses tested. Results suggest that stimulation of non-specific immune factors enhances the ability of fish to mount a protective immune response, but specific antibody appears necessary to provide near complete protection. In this study, an ELISA was developed to monitor anti-F. psychrophilum antibody production in trout. The relationship of such responses to protective immunity suggests that future vaccination strategies against coldwater disease may require stimulation of both the innate and adaptive arms of the immune response.
Article
Rainbow trout (Oncorhynchus mykiss) were either injected twice intraperitoneallywith 0·1 ml of a 1:10 dilution from broth culture of acetone-killed Flavobacterium branchiophilum, immersed twice for 1 h in a 1:10, 1:100 or 1:1000 dilution from a broth culture of acetone-killed F. branchiophilum, or utilized as unexposed controls. After bath challenge with live F. branchiophilum the percent cumulative mortality of each group was as follows; controls, 45·3%, i.p.-injected, 32·1%; immersed in a 1:1000 dilution, 38·0%; immersion in a 1:100, 40·9%; and immersion in a 1:10 dilution, 11·7%. There was no relationship between the group treatment and the levels of F. branchiophilum antigen detected by enzyme immunoassay following challenge. The amount of gill-associated and serum antibody to F. branchiophilum detected in the immersion immunized groups before challenge increased with increasing concentration of antigen in the bath. The i.p.-injected group had the highest serum antibody of any group while gill-associated antibody was comparable to those groups which were bath exposed with the lower concentrations of F. branchiophilum antigen. A similar pattern of antibody response was seen with five groups of rainbow trout treated exactly as the five groups above, in which antibody was monitored over time, but which were not challenged. Gill-associated and serum antibody were detectable following primary antigen exposure regardless of route. The serum and gill-associated antibody responses were present for longer following a second antigen exposure by the same route, and the gill-associated but not the serum antibody was increased over those following primary antigen exposure. The greatest increase in gill-associated antibody response was seen in the group which received the highest concentration of antigen via bath.
Article
In lifetesting, medical follow-up, and other fields the observation of the time of occurrence of the event of interest (called a death) may be prevented for some of the items of the sample by the previous occurrence of some other event (called a loss). Losses may be either accidental or controlled, the latter resulting from a decision to terminate certain observations. In either case it is usually assumed in this paper that the lifetime (age at death) is independent of the potential loss time; in practice this assumption deserves careful scrutiny. Despite the resulting incompleteness of the data, it is desired to estimate the proportion P(t) of items in the population whose lifetimes would exceed t (in the absence of such losses), without making any assumption about the form of the function P(t). The observation for each item of a suitable initial event, marking the beginning of its lifetime, is presupposed. For random samples of size N the product-limit (PL) estimate can be defined as follows: List and label the N observed lifetimes (whether to death or loss) in order of increasing magnitude, so that one has \(0 \leqslant t_1^\prime \leqslant t_2^\prime \leqslant \cdots \leqslant t_N^\prime .\) Then \(\hat P\left( t \right) = \Pi r\left[ {\left( {N - r} \right)/\left( {N - r + 1} \right)} \right]\), where r assumes those values for which \(t_r^\prime \leqslant t\) and for which \(t_r^\prime\) measures the time to death. This estimate is the distribution, unrestricted as to form, which maximizes the likelihood of the observations. Other estimates that are discussed are the actuarial estimates (which are also products, but with the number of factors usually reduced by grouping); and reduced-sample (RS) estimates, which require that losses not be accidental, so that the limits of observation (potential loss times) are known even for those items whose deaths are observed. When no losses occur at ages less than t the estimate of P(t) in all cases reduces to the usual binomial estimate, namely, the observed proportion of survivors.
Article
Freshwater fish are able to mount a protective immune response against the parasite Ichthyophthirius multifiliis (Ich) following a non-lethal exposure. Factors involved in immunity comprise cellular and humoral factors, but antibodies have been suggested to play a prominent role in protection. However, host antibodies have not yet been demonstrated to bind to the parasite in situ. By the use of immunohistochemical techniques, this study demonstrated that IgT and IgM bind to surface structures, including cilia, on the early feeding stage of the parasite in the gills of immune rainbow trout, Oncorhynchus mykiss, shortly (2 h) after invasion. No binding of IgT and no or only a weak binding of IgM was observed on the parasites in the gills of similarly exposed but naïve rainbow trout. This study indicates that antibodies play an important part in the protection of immune fish against Ich although additional humoral and cellular factors may contribute to this reaction.
Article
Flavobacterium psychrophilum, the causative agent of RTFS or rainbow trout fry syndrome, causes high mortality among hatchery reared rainbow trout (Oncorhynchus mykiss) fry in Europe and the USA. Despite several attempts, no efficient vaccines have yet been developed, the main obstacle being that the fry have to be vaccinated very early, i.e. around 0.2-0.5 g, where RTFS usually starts to give problems in the fish farms. Consequently, only oral or bath vaccines are relevant. Immersion of fry in inactivated or attenuated bacteria has resulted in RPS values of less than 50%. However, the results are biased by the fact that the fish have been challenged by intraperitoneal (ip) or subcutaneous (sc) injection against which an immersion/oral vaccine may not protect. Therefore, the present study was undertaken in order to investigate whether the presumably most potent immersion immunization, i.e. bathing in high titres of non-attenuated isolates of F. psychrophilum, was able to induce immunity to a subsequent ip challenge. Immersion in live bacteria for 30 or 50 min caused no mortality and protected a major fraction of the fry against challenges 26 and 47 days later with RPS values of 88.2 and 60.3%, respectively. Increased specific antibody titres suggested that adaptive immune mechanisms were involved in the protection.
Article
Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome has become a widespread fish pathogen in freshwater aquaculture worldwide. In this study, a low molecular mass fraction (P25-33), with an approximate weight of 25-33 kDa, was identified among F. psychrophilum strains in an immunoblotting analysis with anti-F. psychrophilum sera. The immunogenic efficacy of the isolated and extracted P25-33 was investigated in two intraperitoneal immunization trials with rainbow trout, Oncorhynchus mykiss (Walbaum). The first trial included immunizations using P25-33 with Freund's complete adjuvant (FCA) and the second trial included immunizations using P25-33, formalin-inactivated whole and sonicated F. psychrophilum cell preparations without FCA. In both trials, antibody titres against F. psychrophilum were analysed with an enzyme-linked immunosorbent assay and the efficacy of the immunizations was determined by a challenge with F. psychrophilum. The P25-33 was shown to give rise to a protective immune response in rainbow trout after immunization with FCA, but not without FCA when a low concentration of P25-33 was used. Instead formalin-inactivated whole and sonicated cells of F. psychrophilum were able to protect the immunized fish more effectively when immunized without FCA. The results suggest that whole or sonicated F. psychrophilum cells could be better candidates for a cost-effective water-based injection vaccine than the immunogenic fraction.
Article
Previous studies have demonstrated that passage of pathogenic bacteria on increasing concentrations of the antibiotic rifampicin leads to the attenuation of virulence and these resistant strains may serve as live attenuated vaccines. Two rifampicin resistant strains of Flavobacterium psychrophilum, 259-93A.16 and 259-93B.17, were generated by passage on TYES plates containing increasing concentrations of rifampicin. Electrophoretic analysis of whole-cell lysates prepared from the parent and resistant strains identified five differentially expressed proteins between the 259-93B.17 strain and parent strain, while there were no differences identified between the 259-93A.16 and parent strain. The LPS banding patterns were identical between all three strains. Bacterial challenges of rainbow trout (Oncorhynchus mykiss Walbaum) with the resistant strains demonstrated that the 259-93B.17 strain was highly attenuated and the 259-93A.16 strain was modestly attenuated at the challenge doses tested. Immunization of rainbow trout with the live attenuated 259-93B.17 strain by intraperitoneal injection resulted in significant protection against challenge with the virulent parent F. psychrophilum strain at 8 and 15 weeks post-immunization and fish exhibited elevated specific antibody titers. Importantly, immersion delivery of the 259-93B.17 strain stimulated protective immune responses in fish at 10 weeks post-immunization. The results demonstrate that the rifampicin resistant 259-93B.17 strain may serve as an effective live attenuated vaccine for the prevention of F. psychrophilum infections.
Article
Sodium penicillin was conjugated to sheep erythrocytes and optimal quantities, added to a 5% SRBC suspension, were determined for haemagglutination (12-5 mg/ml) and for haemolysis (50 mg/ml) using carp antibodies and carp complement. The epitope density on the BSA molecule was gradually increased, when increasing amounts of sodium-penicillin were added to a constant quantity of BSA, until a maximum of about thirty penicilloyl groups were bound. Low conjugates, having less than seven haptenic groups per one BSA molecule, were found to stimulate carp for both anti-hapten and anti-carrier antibodies. The higher conjugates having seven and more haptenic groups were found to stimulate carp for anti-panicilloyl antibodies but not for anti-BSA antibodies. A booster dose with native BSA, given to the Pen30 BSA preimmunized carp, gave rise directly to a secondary-like response. In the rabbits, however, both heavy and low conjugates were found to stimulate antibody production for the hapten as well as for the carrier. It was suggested that the modified BSA in the heavy conjugates loses its ability to stimulate B cells, probably due to a decrease in local concentration of antigenic determinants in the BSA molecule, but its ability to stimulate helper cells is not affected for this reason.
Article
Part I of this report appeared in the previous issue (Br. J. Cancer (1976) 34,585), and discussed the design of randomized clinical trials. Part II now describes efficient methods of analysis of randomized clinical trials in which we wish to compare the duration of survival (or the time until some other untoward event first occurs) among different groups of patients. It is intended to enable physicians without statistical training either to analyse such data themselves using life tables, the logrank test and retrospective stratification, or, when such analyses are presented, to appreciate them more critically, but the discussion may also be of interest to statisticians who have not yet specialized in clinical trial analyses.
Article
The susceptibility of fish to disease is partly dependent on their environment, in particular on water temperature. It is generally accepted that lower temperatures adversely affect specific immune responses mediated by T helper cells. The probable mechanisms involved in such suppression in teleost fish are reviewed. Furthermore, the effects of temperature on nonspecific defences, such as phagocytosis and cytotoxicity, are described and total immune competence in teleosts at low environmental temperatures is discussed.
Article
The specific humoral response of teleost fish to extracellular bacteria was examined using a rainbow trout-Vibrio anguillarum model. Treatment groups were immunized by oral, immersion, and injection routes. All 3 delivery methods conferred full protection in controlled laboratory challenges (p < 0.01). Prior to boosting, serum antibody titers did not correlate with protection in the orally and immersion-vaccinated groups, but, contrary to previous studies, titers measured 10 and 17 d after boosting correlated positively with protection in all 3 vaccinated groups. The route of administration strongly affected the magnitude of the antibody response as measured by enzyme-linked immunosorbent assay (ELISA) and Western blots; however, the antigenic epitopes recognized were not substantially altered by delivery method as evidenced in immunoblot patterns. Given that the primary and booster vaccination protocols were identical, the data suggest that all 3 vaccinated groups may have had a specific humoral response following initial immunization but that specific serum antibody levels before boosting were too low to be detected by ELISA in fish vaccinated by oral and immersion routes. An anamnestic response was evident in all 3 groups. The data support the possibility that teleosts, like higher vertebrates, have a protective immune response to extracellular bacteria that is predominantly humoral. Route of delivery may primarily affect the efficiency with which the immunogenic constituents of the vaccine are presented to the relevant recognition and effector components of the immune system.
Article
This study was carried out in order to try to establish an efficacious and reliable experimental infection model for Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome, using contact, oral and anal challenges. Ten F psychrophilum strains of different origin were included. The influence of water temperature, scarification, water quality, stress and growth conditions of the pathogen on the experimental infection was assessed. For each challenge protocol, all strains failed to reproduce disease signs or mortality in rainbow trout (Oncorhynchus mykiss L.) fry. Histological and bacteriological examination of the skin, gills and internal organs of the fish 3 weeks following inoculation were found to be negative. Different hypotheses to explain the inability of the challenge models to reproduce the disease experimentally are discussed.