Explorative study on scale cortisol accumulation in wild caught common dab (Limanda limanda).

BACKGROUND: Flatfish live in a diverse marine ecosystem that is changing due to natural variations and anthropogenic influences. These changes can evoke a stress response mainly resulting in production of the glucocorticoid cortisol, which mediates effects on various levels of biological organization. The finding that cortisol accumulates in fish scales, offering a retrospective view on cortisol production, provides opportunities to use this matrix for chronic stress assessment. The present study is the first to gather information on scale cortisol concentration in wild-caught common dab (Limanda limanda), based on a two-pronged approach using (1) field measurements and (2) a laboratory in vivo-study where wild-caught dab were fed by cortisol-spiked feed during 30 or 90 days to demonstrate the possible accumulation of cortisol in the scales and to evaluate its impact on fish health. RESULTS: Based on the field measurements, the average scale cortisol concentration in wild-caught fish was 0.0034 ± 0.0046 µg kg-1 scale (n = 67). This indicates that wild common dab is indeed able to incorporate cortisol in the scales. Based on the experimental data, the cortisol-fed fish showed an increased plasma cortisol concentration (80.16 ± 82.58 µg L-1) compared to the control group (4.54 ± 9.57 µg L-1) after 30 days of cortisol feeding. The increase in plasma cortisol concentration was positively correlated with an increased cortisol concentration in the scale after 30 days of cortisol-spiked feeding. This correlation was, however, no longer observed after 90 days of cortisol-spiked feeding. Interestingly, cortisol concentration of the scales on the pigmented side was significantly higher compared to the non-pigmented side. Some health parameters such as epidermal thickness, body condition and Ichthyobodo sp. parasitic infection showed a correlation with scale cortisol concentration after 30 days. CONCLUSIONS: We have demonstrated that common dab is able to accumulate cortisol in its scales. This seems to occur proportionally to circulating concentrations of plasma cortisol in fish fed with cortisol supplemented feed after 30 days.

INFLUENCE OF PATHOGENS, FISH-RELATED CHARACTERISTICS, AND ENVIRONMENTAL FACTORS ON THE DEVELOPMENT OF SKIN ULCERATIONS IN WILD COMMON DAB (LIMANDA LIMANDA) FROM THE NORTH SEA.

Environmental changes or stressors can result in the development of diseases. Through regular fish disease surveys in the Belgian part of the North Sea, attention was drawn to a sudden increase of skin ulceration prevalence between 2011 and 2014 in common dab (Limanda limanda). Information on prevalence, ulceration, bacteriology, fish-related (e.g., length, age, and sex) and (spatial and temporal) environmental factors, and fishing intensity were gathered. This detailed investigation was framed within a long-term monitoring program, executed every spring-autumn from 2000 to present. Ulcerations were observed in 1.3% of fish (n=3,999). Spatial and temporal differences were evident, and highest prevalence was found in summer. Vibrio was the dominant cultivated bacterial genus present in the lesions. Skin ulcerations appeared to be correlated with length and body condition of the fish, as well as with temperature and pH of the seawater and fishing vessel density. Our research suggested the involvement of multiple factors in the development of skin ulcerations in common dab and endorsed the effects of changing environment and human influence on the marine ecosystem through activities such as fishing.

Comparative genomics of Flavobacterium columnare unveils novel insights in virulence and antimicrobial resistance mechanisms.

This study reports the comparative analyses of four Flavobacterium columnare isolates that have different virulence and antimicrobial resistance patterns. The main research goal was to reveal new insights into possible virulence genes by comparing the genomes of bacterial isolates that could induce tissue damage and mortality versus the genome of a non-virulent isolate. The results indicated that only the genomes of the virulent isolates possessed unique genes encoding amongst others a methyl-accepting chemotaxis protein possibly involved in the initial colonization of tissue, and several VgrG proteins engaged in interbacterial competition. Furthermore, comparisons of genes unique for the genomes of the highly virulent (HV) carp and trout isolates versus the, respectively, low and non-virulent carp and trout isolates were performed. An important part of the identified unique virulence genes of the HV-trout isolate was located in one particular gene region identified as a genomic island. This region contained araC and nodT genes, both linked to pathogenic and multidrug-resistance, and a luxR-gene, functional in bacterial cell-to-cell communication. Furthermore, the genome of the HV-trout isolate possessed unique sugar-transferases possibly important in bacterial adhesion. The second research goal was to obtain insights into the genetic basis of acquired antimicrobial resistance. Several point-mutations were discovered in gyrase-genes of an isolate showing phenotypic resistance towards first and second-generation quinolones, which were absent in isolates susceptible to quinolones. Tetracycline-resistance gene tetA was found in an isolate displaying acquired phenotypic resistance towards oxytetracycline. Although not localized on a prophage, several flanking genes were indicative of the gene's mobile character.

First report of an idiopathic partial tail myonecrosis in European brown shrimp Crangon crangon caught in the North Sea.

The European brown shrimp Crangon crangon is an abundant and commercially important species in the North Sea. Currently, there is interest in landing live shrimp to provide fresh animals for a growing market in live brown shrimp. During 4 survival studies between 2014 and 2016, shrimp were collected from commercial trawlers and maintained alive in off-shore facilities. From Day 1 onwards, a minority of shrimp (~2.24%) developed a white discoloration of the abdominal muscles and a depigmentation of the distal part of the abdomen, along with paralysis of the affected tissues and appendages. As the symptoms progressed, a circumferential blackish delineation appeared, creating a distinct boundary between healthy and necrotic tissue. Affected shrimp survived up to 3 wk, although in several animals the distal part of the tail was completely lost. Histological and electron microscopical examination confirmed the myonecrosis. A secondary bacterial invasion of the necrotic muscle was observed in some animals. RT-PCR for infectious myonecrosis virus was negative. The condition appears not to be contagious, based on the feeding of healthy shrimp with necrotic tissue of affected shrimp. Based on these observations, a mechanical cause inflicted during the catching process is proposed.

Pinpointing the role of Aeromonas salmonicida in the development of skin ulcerations in common dab (Limanda limanda).

Aeromonas salmonicida was isolated from ulcerations in common dab (Limanda limanda). An experiment was performed to pinpoint its role in ulceration development, considering the importance of the skin barrier and the pigmented and non-pigmented sides. The skin of dab was treated in three zones, one where scales and epidermis were removed, one where mucus was discarded and one non-treated zone. Fish were tagged to allow individual identification and challenged with A. salmonicida. Mortality and severity of the developing lesions were recorded for 21 days post-inoculation. Starting 12 days post-inoculation, mortality occurred gradually in challenged fish; however, no direct cause could be established. Both control fish and challenged fish developed ulcerations containing A. salmonicida. Sequencing of vapA gene revealed that isolates retrieved from both groups were distinct, suggesting the presence of A. salmonicida prior to the trial. Most ulcerations developed in zones where skin was removed, suggesting that abrasion might be a predisposing factor in ulceration development. Ulcerations were also observed at the insertion site of the tag, where exposed muscle tissue might have favoured the development of ulcerations. In conclusion, A. salmonicida seems to be involved in the development of skin ulcerations in dab, although the exact pathogenesis needs to be elucidated.

Scrutinizing the triad of Vibrio tapetis, the skin barrier and pigmentation as determining factors in the development of skin ulcerations in wild common dab (Limanda limanda).

Recently, Vibrio tapetis was isolated for the first time from skin ulcerations in wild-caught common dab (Limanda limanda). To further examine its role in the development of these skin lesions, an in vivo experiment was performed. The significance of the skin barrier and in addition the difference between pigmented and non-pigmented side were investigated. Hence, the skin of common dab was treated in three different ways on both the pigmented and non-pigmented side. On a first "treatment zone", the scales and overlying epidermal tissue were removed whereas in a second zone only the mucus was discarded. The third zone served as a non-treated zone. Thereafter, fish were challenged with V. tapetis. The control group was sham treated. Mortality, clinical signs, severity and size of the developing lesions were recorded. All animals were sacrificed and sampled 21 days post-inoculation. Significantly more fish of the group challenged with V. tapetis died compared to the control group with the highest incidence occurring 4 days post-inoculation. Fish challenged with V. tapetis developed more severe skin ulcerations. In zones where scales and epidermal tissue were removed, the ulcerations were more severe compared to zones where only mucus was eliminated. Ulcerations occurred more frequently, were more severe and larger on the pigmented side. Our data represents prove of V. tapetis as causative agent of ulcerative skin lesions although prior damage of the skin seems to be a major contributing factor. Furthermore, the pigmented side seemed predisposed to the development of skin ulcerations.

Evidence that the stress hormone cortisol regulates biofilm formation differently among Flavobacterium columnare isolates.

The impact of cortisol on Flavobacterium columnare biofilm formation was explored. Firstly, the dynamics of biofilm formation by one highly (HV) and one low virulent (LV) F. columnare isolate with and without the stress hormone cortisol under microfluidic flow conditions was characterized. This to confirm that F. columnare cells could form biofilm under cortisol supplementation, and to compare the temporal and structural differences between different treatment groups. One trial revealed that in both isolates cell aggregates resembling biofilms occurred within 7-h post-inoculation. Consequently, cell clusters were sloughed away, followed by a rebuilding of bacterial cell aggregates, suggestive for a high spreading capacity. While the HV isolate revealed cell aggregates formed upstream at all time-points, for the LV isolate this was only seen upon cortisol supplementation. Secondly, the transcriptional effect of genes (gldK, gldL, gldM, gldN, sprA, sprE, sprT, and porV) belonging to the Type IX secretion system involved in gliding motility was investigated in planktonic and biofilm cells of a HV and LV isolate to which no, a low (LD) or high (HD) dose of cortisol was added. Significantly lower expression of gliding genes gldK, gldL, gldM and gldN, and of protein secretion regulator porV was seen in the LV isolate planktonic cells supplemented with a HD-cortisol. The LV isolate biofilm cells treated with the HD-cortisol showed a significant upregulation of sprT, encoding mobile surface adhesion important in bacterial colonization. This is the first evidence for the co-regulatory effect of cortisol on biofilm formation and F. columnare gliding gene expression.

Experimental infection model for vibriosis in Dover sole (Solea solea) larvae as an aid in studying its pathogenesis and alternative treatments.

Severe economic losses due to diseases in marine larviculture may be linked to vibriosis. To better understand the pathogenesis of vibriosis and evaluate new ways to prevent and combat this important disease, there is a great need for reliable and reproducible experimental infection models. The present study aimed at developing a challenge model for vibriosis in Dover sole larvae and testing its applicability to study the effect of the probiotic treatment. For that purpose, larvae were challenged at 10 days post hatching with Vibrio anguillarum WT, V. anguillarum HI610 or V. harveyi WT. Following administration of V. anguillarum WT via immersion at 1 × 107 colony forming units/mL, a larval mortality of 50% was observed at 17 days post-inoculation. In a next step, the probiotic potential of 371 isolates retrieved from Dover sole was assessed by screening for their inhibitory effects against Vibrio spp. and absence of haemolytic activity. One remaining isolate (V. proteolyticus) and V. lentus, known for its protective characteristics in seabass larvae, were further tested in vivo by means of the pinpointed experimental infection model. Neither isolate provided via the water or feed proved to be protective for the Dover sole larvae against challenge with V. anguillarum WT. This developed challenge model constitutes a firm basis to expedite basic and applied research regarding the pathogenesis and treatment of vibriosis as well as for studying the impact of (a)biotic components on larval health.

Development of a reliable experimental set-up for Dover sole larvae Solea solea L. and exploring the possibility of implementing this housing system in a gnotobiotic model.

Due to the increasing importance of the aquaculture sector, diversification in the number of cultured species imposes itself. Dover sole Solea solea L. is put forward as an important new aquaculture candidate due to its high market value and high flesh quality. However, as for many other fish species, sole production is hampered by amongst others high susceptibility to diseases and larval mortality, rendering the need for more research in this area. In this respect, in first instance, a housing system for Dover sole larvae was pinpointed by keeping the animals individually in 24-well plates for 26days with good survival rates and initiating metamorphosis. This ensures a standardised and reliable experimental set-up in which the possible death of one larva has no effect on the other larvae, rendering experiments adopting such a system more reproducible. In addition to proving valuable in many other applications, this multi well system constitutes a firm basis to enable the gnotobiotic rearing of larvae, which hitherto is non-existing for Dover sole. In this respect, secondly, a large number of disinfection protocols were tested, making use of widely employed disinfectants as hydrogen peroxide, glutaraldehyde and/or ozone whether or not combined with a mixture of antimicrobial agents for 24h. Although none of the tested protocols was sufficient to reproducibly generate a gnotobiotic model, the combination of glutaraldehyde and hydrogen peroxide resulted in hatchable, bacteria-free eggs in some cases.

Ultrastructural morphology of the envelope of Dover sole Solea solea eggs from fertilization until hatching with emphasis on sample preparation.

This study is the first to describe the ultrastructural morphology of the envelope of Solea solea eggs from fertilisation until hatching. Defining the ultrastructural morphology of fish eggs is important for species identification and may assist in predicting the effect of external influences on these early life stages. In first instance, various fixation and embedding protocols were assessed to explore the morphology of the egg envelope, whereby the encountered difficulties were highlighted. The successful protocol for SEM proved to be combined fixation with 4% glutaraldehyde in 0.1M cacodylate buffer for minimum 4h with post-fixation of 2h with 1% OsO4 in 0.1M cacodylate buffer. For TEM, puncturing the egg envelope during the first steps of the fixation protocol was necessary to allow the embedding medium to penetrate through the egg envelope. Based on both scanning and transmission electron microscopical examination, three distinct layers were discerned in the egg envelope. During the development of the fish embryo, a change in the outer structure of the egg was observed. Scanning electron microscopical examination of one day post-fertilisation eggs (DPF) revealed a homogeneous outer layer, displaying a large number of pores uniformly distributed on the surface of the egg envelope. Starting from 2 DPF parts of the outermost layer or two outer layers peeled off. The second deeper layer showed larger pores, with less defined edges. In the third innermost layer irregular indentations were noted. On transmission electron microscopy the first outermost layer of 1 DPF eggs clearly folded into the pores. The second layer was more electron dense, had a uniform appearance and did not cover the surface of the pores. The third innermost layer was much thicker and possessed indentations. A total number of 12 undulating zones were discriminated based on different degrees of electron density. Prior to hatching, the compact structure of the innermost layer was distorted by dispersed holes and tears.

Cellular and transcriptomic response to treatment with the probiotic candidate Vibrio lentus in gnotobiotic sea bass (Dicentrarchus labrax) larvae.

The present study aimed at evaluating the cellular and transcriptomic responses induced by the probiotic candidate Vibrio lentus with gnotobiotic European sea bass (Dicentrarchus labrax, Linnaeus 1785) larvae. For this, a histomorphological analysis was performed using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and the anti-proliferating cell nuclear antigen (PCNA) assay. In addition, a global transcriptomic approach was adopted to study the whole body mRNA changes upon administration of V. lentus by microarrays with the custom Agilent sea bass oligonucleotide-microarray v2.0 (4 × 44 K). Following V. lentus administration, the apoptotic and cell proliferative indexes did not show significant differences between treatments for hindgut nor for midgut. However, V. lentus treatment did significantly modify the gene expression related not only to cell proliferation and cell death, but also to cell adhesion, reactive oxygen species metabolism, iron transport, and immune response. Our data represent the first global analysis of the effects of the probiotic candidate V. lentus on the gene expression profile in gnotobiotic European sea bass, and as such, provides a first delineation of the mechanisms by which this agent interacts with its host and exerts its beneficial effects.

Cortisol directly impacts Flavobacterium columnare in vitro growth characteristics.

Teleost fish faced with stressful stimuli launch an endocrine stress response through activation of the hypothalamic-pituitary-interrenal axis to release glucocorticoids, in particular cortisol, into the blood. For the majority of bacterial fish pathogens, stress is considered a key factor in disease outbreaks. Based upon studies in mammals, there is considerable evidence to suggest that, besides impairing the immune system, cortisol can have a direct effect on bacterial cells. Hitherto, this intriguing field of microbial endocrinology has remained largely unexplored in aquatic diseases. The present study investigated in vitro the impact of cortisol on phenotypic traits of the fresh water fish pathogen Flavobacterium columnare. Colonies obtained from the highly virulent (HV) isolates resulted in significantly larger and more spreading colonies compared to those from the low virulent (LV) isolates. High cortisol doses added displayed a direct effect on the bacterial cells and induced a significant decrease in colony size. An additional intriguing finding was the inverse relationship between cortisol concentrations added to the broth and the spreading character of colonies retrieved, with higher cortisol doses resulting in less rhizoid to rough and even smooth colony formation (the latter only in the LV trout isolate), suggesting a dose-response effect. The loss of the rhizoid appearance of the F. columnare colonies upon administration of cortisol, and hence the loss of motility, might indicate a phenotypic change to the biofilm state. These findings form the basis for further research on the impact of glucocorticoids on other virulence factors and biofilm formation of F. columnare.

Scales tell a story on the stress history of fish.

Fish faced with stressful stimuli launch an endocrine stress response through activation of the hypothalamic-pituitary-interrenal (HPI-) axis to release cortisol into the blood. Scientifically validated biomarkers to capture systemic cortisol exposure over longer periods of time are of utmost importance to assess chronic stress in governmental, wildlife, aquaculture and scientific settings. Here we demonstrate that cortisol in scales of common carp (Cyprinus carpio L.) is the long-sought biomarker for chronic stress. Undisturbed (CTR) and daily stressed (STRESS) carp were compared. Dexamethasone (DEX) or cortisol (CORT) fed fish served as negative and positive controls, respectively. Scale cortisol was quantified with a validated ultra-performance liquid chromatography tandem mass spectrometry method. An increase in scale cortisol content was found in STRESS and CORT but not in CTR and DEX fish. Scale cortisol content reflects its accumulation in a stressor and time dependent manner and validates the scale cortisol content as biomarker for chronic stress. Plasma analyses confirmed that (i) CTR, DEX and CORT treatments were effective, (ii) plasma cortisol of STRESS fish showed no signs of chronic HPI-axis activation, and (iii) plasma cortisol is a poor predictor for chronic stress. The expression of HPI key genes crf, pomc, and star were up-regulated in STRESS fish in the absence of a plasma cortisol response, as was the target gene of cortisol encoding subunit α1 of the Na+/K+-ATPase in gills. When lost, scales of fish regenerate fast. Regenerated scales corroborate our findings, offering (i) unsurpassed time resolution for cortisol incorporation and as such for stressful events, and (ii) the possibility to investigate stress in a well defined and controlled environment and time frame creating novel opportunities for bone physiological research. We conclude that the cortisol content in ontogenetic and regenerated scales is an innovative biomarker for chronic stress offering ample applications in science and industry.

Interactions of highly and low virulent Flavobacterium columnare isolates with gill tissue in carp and rainbow trout.

The interactions of Flavobacterium columnare isolates of different virulence with the gills of carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum) were investigated. Both fish species were exposed to different high (HV) or low virulence (LV) isolates and sacrificed at seven predetermined times post-challenge. Histopathological and ultrastructural examination of carp and rainbow trout inoculated with the HV-isolate disclosed bacterial invasion and concomitant destruction of the gill tissue, gradually spreading from the filament tips towards the base, with outer membrane vesicles surrounding most bacterial cells. In carp, 5-10% of the fish inoculated with the LV-isolate became moribund and their gill tissue displayed the same features as described for the HV-isolate, albeit to a lesser degree. The bacterial numbers retrieved from the gill tissue were significantly higher for HV- compared to LV-isolate challenged carp and rainbow trout. TUNEL-stained and caspase-3-immunostained gill sections demonstrated significantly higher apoptotic cell counts in carp and rainbow trout challenged with the HV-isolate compared to control animals. Periodic acid-Schiff/alcian blue staining demonstrated a significantly higher total gill goblet cell count for HV- and LV-isolate challenged compared to control carp. Moreover, bacterial clusters were embedded in a neutral matrix while being encased by acid mucins, resembling biofilm formation. Eosinophilic granular cell counts were significantly higher in the HV-isolate compared to LV-isolate inoculated and control carp. The present data indicate a high colonization capacity, and the destructive and apoptotic-promoting features of the HV-isolate, and point towards important dynamic host mucin-F. columnare interactions warranting further research.

Micromelic dysplasia-like syndrome in a captive colony of common marmosets (Callithrix jacchus).

Over several years, 0% to 5% of adolescent animals in a captive colony of common marmosets (Callithrix jacchus) showed severely bended arms and legs over several years. The animals showed no pain, discomfort, or altered behavior but were unable to stretch their distal limbs to their full extent. To characterize the lesion morphologically, the bones of 4 affected marmosets were compared macroscopically and radiographically with those of 6 unaffected animals. The deformities were characterized by mid- to distal diaphyseal bending and pronounced shortening of long bones. The morphology and density of other bones including the skull and vertebrae were unaffected. Although vitamin D values were low in a fifth affected marmoset during 10 to 16 mo of age, lesions associated with rickets were not observed. To our knowledge, this report is the first to describe a micromelic dysplasia-like syndrome comprising severe, idiopathic bending and shortening of long bones in a colony of marmosets.

Fish as research tools: alternatives to in vivo experiments.

The use of fish in scientific research is increasing worldwide, due to both the rapid expansion of the fish farming industry and growing awareness of questions concerning the humane use of mammalian models in basic research and chemical testing. As fish are lower on the evolutionary scale than mammals, they are considered to be less sentient. Fish models are providing researchers, and those concerned with animal welfare, with opportunities for adhering to the Three Rs principles of refinement, reduction and replacement. However, it should be kept in mind that fish should also be covered by the principles of the Three Rs. Indeed, various studies have shown that fish are capable of nociception, and of experiencing pain in a manner analogous to that in mammals. Thus, emphasis needs to be placed on the development of alternatives that replace, as much as possible, the use of all living vertebrate animals, including fish. This review gives the first comprehensive and critical overview of the existing alternatives for live fish experimental studies. The alternative methods described range from cell and tissue cultures, organ and perfusion models, and embryonic models, to in silico computer and mathematical models. This article aspires to guide scientists in the adoption of the correct alternative methods in their research, and, whenever possible, to reduce the use of live fish.

Columnaris disease in fish: a review with emphasis on bacterium-host interactions.

Flavobacterium columnare (F. columnare) is the causative agent of columnaris disease. This bacterium affects both cultured and wild freshwater fish including many susceptible commercially important fish species. F. columnare infections may result in skin lesions, fin erosion and gill necrosis, with a high degree of mortality, leading to severe economic losses. Especially in the last decade, various research groups have performed studies aimed at elucidating the pathogenesis of columnaris disease, leading to significant progress in defining the complex interactions between the organism and its host. Despite these efforts, the pathogenesis of columnaris disease hitherto largely remains unclear, compromising the further development of efficient curative and preventive measures to combat this disease. Besides elaborating on the agent and the disease it causes, this review aims to summarize these pathogenesis data emphasizing the areas meriting further investigation.

Light and transmission electron microscopy of Vibrio campbellii infection in gnotobiotic Artemia franciscana and protection offered by a yeast mutant with elevated cell wall glucan.

Luminescent vibrios are amongst the most important pathogens in aquaculture, affecting almost all types of cultured organisms. Vibrio campbellii is one of these most important pathogens. In this study, the effects of feeding mnn9 yeast cell wall mutant and wild type yeast strain were investigated in the digestive tract of brine shrimp nauplii, Artemia franciscana, after experimental infection with V. campbellii (LMG 21363). Gnotobiotic A. franciscana nauplii were fed daily with dead Aeromonas hydrophila LVS3, and with either wild type strain of baker's yeast, Saccharomyces cerevisiae, or mutant strain mnn9, of which the cell wall contains elevated chitin and glucan and lower mannose levels. After three days of feeding, some nauplii were challenged with V. campbellii. Mean survival (%), individual length (mm) and total length (mm) at one day and two days after challenge were significantly higher in the group fed mnn9 than in the group fed wild type yeast (81 ± 1.50 and 63 ± 0.49, 1.56 ± 0.07 and 1.13 ± 0.02, 38.21 ± 3.11 and 21.26 ± 0.81 respectively for one day and 50 ± 2.37 and 20 ± 1.41, 2.33 ± 0.01 and 1.24 ± 0.04, 34.97 ± 5.56 and 7.45 ± 1.63 for two days after challenge). Histological examination revealed that the luminal diameter and enterocyte height of both mid- and hindgut were larger in the mnn9-fed group. Colonization of the gut lumen by V. campbellii could be observed by transmission electron microscopy for the group of nauplii fed with wild type yeast. Furthermore, it was observed that V. campbellii caused damage to the gut epithelium including shortening and disappearance of the microvilli, destruction of the apical cell membrane and cell lysis in the nauplii fed wild type yeast. The gut epithelium remained intact in challenged nauplii fed mnn9 yeast. The morphological findings of the present study further substantiate previous studies reporting a protective effect of this yeast cell wall mutant.

Heat shock proteins protect platyfish (Xiphophorus maculatus) from Yersinia ruckeri induced mortality.

The significant disadvantages accompanied with the use of antibiotics in aquaculture, emphasize the need for developing alternative disease control strategies, like novel vaccine approaches and immunostimulating measures. Several studies have already pointed out the ability of heat shock proteins (HSPs) to modulate innate and adaptive immune responses, what makes them potent candidates for the development of a new disease prevention method. In this study, the use of self and non-self heat shock proteins as a new prophylactic treatment against bacterial diseases in freshwater aquaculture was investigated. Therefore, an infection model was developed with platyfish as a host for Yersinia ruckeri infections. In this infection model, the effect of different treatments with HSPs on the survival of the fish after bacterial infection was tested: non-lethal heat shock, intracoelomal injection with two recombinant bacterial HSPs, GroEL and DnaK, and a combination of a non-lethal heat shock and an injection with bacterial HSPs. The results show that a non-lethal heat shock could not protect fish against a subsequent infection with Y. ruckeri. However, when the fish received an injection with bacterial HSPs, Y. ruckeri induced mortality was reduced. This effect became significant when the administration of bacterial HSPs was combined with a non-lethal heat shock. These data suggest a possible role for heat shock proteins as an immunostimulating treatment in fish against bacterial infections.