[Show abstract][Hide abstract] ABSTRACT: Recent advances in genotyping-by-sequencing have enabled genome-wide association studies (GWAS) in non-model species including those in aquaculture programs. As with other aquaculture species, rainbow trout and steelhead (Oncorhynchus mykiss) are susceptible to disease and outbreaks can lead to significant losses. Fish culturists have therefore been pursuing strategies to prevent losses to common pathogens such as Flavobacterium psychrophilum (the etiological agent for bacterial cold water disease (CWD)) and infectious hematopoietic necrosis virus (IHNV) by adjusting feed formulations, vaccine development, and selective breeding. However, discovery of genetic markers linked to disease resistance offers the potential to utilize marker assisted selection to increase resistance and reduce outbreaks. For this study we sampled juvenile fish from 40 families from two year classes that either survived or died following controlled exposure to either CWD or IHNV. Restriction-site associated DNA (RAD) sequencing produced 4,661 polymorphic SNP loci after strict filtering. Genotypes from individual survivors and mortalities were then used to test for association between disease resistance and genotype at each locus using the program TASSEL. After accounting for kinship and stratification of the samples, tests revealed 12 SNP markers that were highly associated with resistance to CWD and 19 markers associated with resistance to IHNV. These markers are candidates for further investigation and are expected to be useful for marker assisted selection in future broodstock selection for various aquaculture programs.
[Show abstract][Hide abstract] ABSTRACT: The mucosal surfaces of all vertebrates have been exposed to similar evolutionary pressures for millions of years. In terrestrial vertebrates such as birds and mammals, the nasopharynx-associated lymphoid tissue (NALT) represents a first line of immune defence. Here we propose that NALT is an ancient arm of the mucosal immune system not restricted to terrestrial vertebrates. We find that NALT is present in rainbow trout and that it resembles other teleost mucosa-associated lymphoid tissues. Trout NALT consists of diffuse lymphoid cells and lacks tonsils and adenoids. The predominant B-cell subset found in trout NALT are IgT(+) B cells, similar to skin and gut. The trout olfactory organ is colonized by abundant symbiotic bacteria, which are coated by trout secretory immunoglobulin. Trout NALT is capable of mounting strong anti-viral immune responses following nasal delivery of a live attenuated viral vaccine. Our results open up a new tool for the control of aquatic infectious diseases via nasal vaccination.
[Show abstract][Hide abstract] ABSTRACT: Rosemary Rosmarinus officinalis oil has shown potential for use as a phytobiotic fish feed supplement with antioxidant properties that can inhibit the growth of Flavobacterium psychrophilum, the pathogen that causes bacterial cold-water disease (BCWD). To determine the in vivo effectiveness of rosemary oil in preventing or minimizing BCWD, Rainbow Trout Oncorhynchus mykiss were fed commercial feed top-coated with one of two concentrations of rosemary oil (1% or 3% of the feed) or a control diet for 14 d. Fish from each feed treatment were then challenged with one of two doses of F. psychrophilum via subcutaneous injection, and mortality was monitored for 28 d. In both F. psychrophilum challenges, fish treated with feed coated with rosemary oil at the 1% and 3% levels experienced significantly higher mortality than fish treated with only soybean oil–coated feed. While the use of rosemary oil as a top-coat on feed increased mortality among Rainbow Trout subjected to a disease challenge in the current study, the mechanism for this result has not been established.Received January 22, 2014; accepted April 18, 2014
North American Journal of Aquaculture 10/2014; 76(4). · 0.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The genome sequence of Flavobacterium psychrophilum strain CSF259-93, isolated from rainbow trout (Oncorhynchus mykiss), consists of a single circular genome of 2,900,735 bp and 2,701 predicted open reading frames (ORFs). Strain CSF259-93 has been used to select a line of rainbow trout with increased genetic resistance against bacterial cold water disease.
[Show abstract][Hide abstract] ABSTRACT: To determine the potential for protection against Flavobacterium psychrophilum infection, a study was conducted where a probiotic, Enterobacter sp. strain C6-6, was delivered to rainbow trout, Oncorhynchus mykiss, via injection. Two separate studies were conducted. In a preliminary study groups of rainbow trout (mean weight, 2 g) were either left unhandled or intramuscularly (IM) injected with a standardized concentration of either a 48 or 72 h culture of C6-6 and subsequently challenged with two different doses of F. psychrophilum 7, 28 and 56 days post-injection (PI). The relative survival ranged from 66 to 87%, 42 to 53% and 0 to 18% at 7, 28 and 56 days PI, respectively. In the second study groups of rainbow trout (mean weight, 1.3 g) received either an intraperitoneal injection (IP) of phosphate buffered saline (PBS; negative control), supernatant from a C6-6 culture, formalin killed C6-6, or live C6-6 and were subsequently challenged with F. psychrophilum 7 or 28 days PI. Log rank survival analysis showed a significant (P < 0.05) reduction in mortality for fish receiving these treatments at both 7 and 28 days PI. Injection of live C6-6 resulted in the lowest mortality at either time point and cumulative mortality at 7 days PI was significantly reduced (P < 0.05) in fish receiving formalin killed (62.7 ± 7.4%) or live Enterobacter C6-6 (48 ± 6.9%) when compared to the negative controls (92 ± 2.3%). Additionally, at 28 days PI, fish receiving the formalin killed or live C6-6 had significantly increased antibody titers against F. psychrophilum. This was not expected and suggests that protection observed at 28 days could be in part due to a cross protective adaptive immune response. Antibody titers were not detected at 7 days PI but significant protection was observed and indicates that innate immunity was responsible for this. Taken together, results from this study indicate that protection against F. psychrophilum after either IM or IP injection of this naturally occurring bacterium, either alive or dead, is at least in part dependent on the enhanced immune function(s) of the treated fish. This may shed light on protective mechanisms associated with the use of the Enterobacter sp. and may be useful as a potential alternate strategy for reducing the impacts from F. psychrophilum infection through non-specific immune-enhancement during times of increased fish stress or as a possible adjuvant.
[Show abstract][Hide abstract] ABSTRACT: C5a, the most potent anaphylatoxin generated during complement activation, has important pro-inflammatory actions and has also been shown to enhance antigen-specific antibody response in mammals, thereby acting as a molecular adjuvant. In rainbow trout, C5a has been shown to have a chemoattractant ability and its receptor has also been found on potential APCs. In this study, we tested the possible role of trout C5a as a molecular adjuvant. We demonstrated the presence of native C5a in trout serum using the antibody generated by recombinant trout C5a, and then we generated recombinant infectious hematopoietic necrosis virus glycoprotein (G), and a G-C5a fusion protein to test the adjuvant activity of trout C5a. Recombinant G-C5a displayed a potent chemoattractant activity in contrast to G alone, indicating that the C5a portion of the fusion protein was functional. Thereafter, G-C5a, partially emulsified in a small quantity of IFA, was injected into one group of trout, while the other group of trout was inoculated with the same dose of recombinant G. At four to sixteen weeks post-injection, the serum IgM antibody levels of the fish injected with recombinant G-C5a were obviously higher than those injected with G protein alone. Thus, these results suggest, for the first time, that C5a acts as molecular adjuvant in teleost fish by enhancing antibody response to a soluble antigen.
Fish & Shellfish Immunology 08/2014; · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Flavobacterium psychrophilum gliding motility N (GldN) protein was investigated to determine its ability to elicit antibody responses and provide protective immunity in rainbow trout Oncorhynchus mykiss (Walbaum). GldN was PCR amplified and cloned into pET102/D-TOPO and expressed in Escherichia coli. Bacteria expressing recombinant GldN (rGldN) were formalin inactivated and injected intraperitoneally (i.p.) into rainbow trout with Freund's Complete Adjuvant (FCA) in 4 separate studies that used two different immunization protocols followed by challenge evaluations. Fish injected with E. coli only in FCA served as the control. Antibody responses to F. psychrophilum whole cell lysates measured by ELISA were low in all 4 studies. Protection against F. psychrophilum challenge was observed in the first study but not in the 3 following studies. The discrepancies in results obtained in the later studies are unclear but may relate to formalin treatment of the antigen preparations. Overall it appeared that rGldN delivered i.p. as a crude formalin killed preparation is not a consistent vaccine candidate and more work is required. Additionally, this study illustrates the importance of conducting multiple in-vivo evaluations on potential vaccine(s) before any conclusions are drawn.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Cyprinid herpesvirus 3 (CyHV-3), commonly known as koi herpesvirus (KHV), is a member of the Alloherpesviridae, and is a recently discovered emerging herpesvirus that is highly pathogenic for koi and common carp. Our previous study demonstrated that CyHV-3 becomes latent in peripheral white blood cells (WBC). In this study, CyHV-3 latency was further investigated in IgM(+) WBC. The presence of the CyHV-3 genome in IgM(+) WBC was about 20-fold greater than in IgM(-) WBC. To determine if CyHV-3 expressed genes during latency, transcription from all 8 ORFs in the terminal repeat was investigated in IgM(+) WBC from koi with latent CyHV-3 infection. Only a spliced ORF6 transcript was found to be abundantly expressed in IgM(+) WBC from CyHV-3 latently infected koi. The spliced ORF6 transcript was also detected in vitro during productive infection as early as 1 day post-infection. The ORF6 transcript from in vitro infection begins at -127 bp upstream of the ATG and ends +188bp downstream of the stop codon, +20 bp downstream of the polyadenylation signal. The hypothetical protein of ORF6 contains a consensus sequence with homology to a conserved domain of EBNA-3B and ICP4 from Epstein Barr Virus and Herpes simplex virus 1, respectively, both members of the Herpesviridae. This is the first report of latent CyHV-3 in B cells and identification of gene transcription during latency for a member of the Alloherpesviridae.
[Show abstract][Hide abstract] ABSTRACT: Flavobacterium columnare is the etiologic agent of columnaris disease, a pervasive disease of fresh water finfish. During the past 4 years, losses that ranged from 5 to 50% in rainbow trout (Oncorhynchus mykiss) fry being reared at a constant 14.5 °C (mean weight, 0.2 g; ~ 400°days post-fertilization), have been occurring in Hagerman Valley, Idaho, USA. A total of 70 different F. columnare isolates were obtained from diseased fish and the water they were reared in. All of the isolates were confirmed to be genomovar I by 16S rRNA restriction fragment length polymorphism. Sequencing of the 16S rRNA, 16S–23S rDNA spacer region and the gyrase B subunit genes from these 70 strains revealed no sequence differences among these isolates. Whole-cell protein profiling by SDS-PAGE also indicated low variation between isolates. Virulence was assessed for a representative isolate and demonstrated a high degree of pathogenicity against rainbow trout fry at 15 °C. These results suggest the emergence of a highly successful F. columnare strain that can affect very early life stages of fish being reared at a constant 14.5 °C at a commercial rainbow trout farm in Idaho.
Aquaculture 01/2014; s 418–419:126–131. · 1.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Commensal microorganisms live in association with the mucosal surfaces of all vertebrates. The skin of teleost fish is known to harbor commensals. In this study we report for the first time the presence of an intracellular Gram positive bacteria, Staphylococcus warneri that resides in the skin epidermis of rainbow trout (Oncorhynchus mykiss). S. warneri was isolated from healthy hatchery trout skin epithelial cells. In situ hybridization confirmed the intracellular nature of the bacterium. Skin explants exposed in vitro to S. warneri or the extracellular pathogen Vibrio anguillarum show that S. warneri is able to induce an anti-inflammatory cytokine status via TGF-β1b compared to the pro-inflammatory responses (IL-1β, IL-6 and TNF-∝) elicited by V. anguillarum. In vivo experiments showed that S. warneri is not pathogenic to rainbow trout when injected intraperitoneally at high concentrations. However, S. warneri is able to stimulate V. anguillarum growth and biofilm formation on rainbow trout scales. Our results demonstrate that rainbow trout skin commensals such as S. warneri have the potential to become indirect pathobionts by enhancing growth and biofilm formation of pathogens such as V. anguillarum. These results show that fish farming practices (i.e. handling and other manipulations) can alter the skin microbiota and compromise the skin health of rainbow trout.
[Show abstract][Hide abstract] ABSTRACT: Yersinia ruckeri is a well-established bacterial pathogen for many salmonid species, against which a formalin-killed bacterin vaccine has been effective in reducing disease outbreaks. Previous studies have reported conflicting results about the protective value of the systemic humoral response to Yersinia ruckeri vaccination. Here we directly demonstrate that plasma contains the long-term protective component elicited by both immersion and intraperitoneal injection vaccination of rainbow trout. A total of 0.5μL of plasma from vaccinated fish provided almost complete protection against experimental challenge. Conversely, the cells obtained from peripheral blood conferred little or no protection in naïve recipients. The protective component of immune sera was IgM based on size exclusion chromatography and recognition by monoclonal antibody Warr 1-14. Immune plasma generated against a Y. ruckeri biotype 1 strain protected equally against challenges with Y. ruckeri biotype 1 and 2 strains. These results illustrate the importance of the humoral IgM response against Y. ruckeri and the use of doubled haploid rainbow trout (Oncorhynchus mykiss) and transfer of plasma/serum and cells into F1 outcross progeny as a model system for dissection of the mechanism(s) of vaccine-induced protection.
Developmental and comparative immunology 12/2013; · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Skin homeostasis is critical to preserve animal integrity. Although the skin of most vertebrates is known to contain a skin-associated lymphoid tissue (SALT), very little is known about skin B-cell responses as well as their evolutionary origins. Teleost fish represent the most ancient bony vertebrates containing a SALT. Due to its lack of keratinization, teleost skin possesses living epithelial cells in direct contact with the water medium. Interestingly, teleost SALT structurally resembles that of the gut-associated lymphoid tissue, and it possesses a diverse microbiota. Thus, we hypothesized that, because teleost SALT and gut-associated lymphoid tissue have probably been subjected to similar evolutionary selective forces, their B-cell responses would be analogous. Confirming this hypothesis, we show that IgT, a teleost immunoglobulin specialized in gut immunity, plays the prevailing role in skin mucosal immunity. We found that IgT(+) B cells represent the major B-cell subset in the skin epidermis and that IgT is mainly present in polymeric form in the skin mucus. Critically, we found that the majority of the skin microbiota are coated with IgT. Moreover, IgT responses against a skin parasite were mainly limited to the skin whereas IgM responses were almost exclusively detected in the serum. Strikingly, we found that the teleost skin mucosa showed key features of mammalian mucosal surfaces exhibiting a mucosa-associated lymphoid tissue. Thus, from an evolutionary viewpoint, our findings suggest that, regardless of their phylogenetic origin and tissue localization, the chief immunoglobulins of all mucosa-associated lymphoid tissue operate under the guidance of primordially conserved principles.
Proceedings of the National Academy of Sciences 07/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Approximately 8 weeks after a chlorine insult associated with the city water supply, shortnose sturgeon, Acipenser brevirostrum (L.), from one group presented with small (3-4 mm) irregular foci of cutaneous pallor that involved the dorsocranial integument with progressive ulceration of the nascent lesions. Various bacterial organisms were isolated from the cutaneous lesions, but not from the internal viscera. Histologically, the nuclei of the intralesional and perilesional epidermal cells often exhibited margination of the chromatin that resulted in a homogenous, pale, amphophilic, tinctorial quality of the nucleoplasm consistent with a herpesvirus infection. In addition, rare lamellar epithelial cells were prominently enlarged due to an abundant, dense, basophilic cytoplasm characteristic of an iridovirus infection. Inoculation of cutaneous lesion and kidney, spleen, liver sample pools from affected shortnose sturgeon onto white sturgeon spleen (WSS-2) cell line induced cytopathic effect characterized by syncytia formation. Ultrastructural analysis of infected WSS-2 cells revealed viral particles with a characteristic herpesvirus morphology. Intranuclear hexagonal capsids had a diameter of 95-108 nm, and enveloped particles present in the cytoplasm of infected cells had a diameter of 176-196 nm. This is the first report of a herpesvirus and a possible iridovirus-like infection in shortnose sturgeon.
Journal of Fish Diseases 07/2013; · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the potential for improving the conversion and deposition of the omega-3 fatty acids docosahexaenoic acid (DHA; 22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3) in fish, 44 families of rainbow trout were fed a diet low in these components and then evaluated for their ability to convert and store plant oils that did not contain DHA and EPA in their muscle tissue. The range for EPA and DHA detected in the tissues of different families varied between 1.2 and 2.9%, and 3.8 and 7.1% total fatty acid, respectively. After adjusting for covariates, an averaged heritability of 0.78 ± 0.11 for DHA and 0.61 ± 0.17 for EPA was calculated. Expression analysis of genes related to the elongation and conversion of lipids were also analyzed, and significant differences were found in the expression of some genes between groups of families that were delineated as having relative high, medium, and low capabilities of depositing EPA and DHA in their muscle tissue after being reared on the mainly soy/flax oil containing diet. However, none of these genes showed a positive correlation with the high conversion/deposition group.
Journal of the World Aquaculture Society 06/2013; 44(3). · 0.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Selective fish breeding programs for disease resistance comprise an increasingly important role in aquaculture production and offer an additional management tool for reducing bacterial-caused disease losses. Bacterial cold water disease (BCWD) is one of the most frequent causes of elevated mortality in juvenile salmonids, and we have selectively bred three genetic lines of rainbow trout for varying resistance to BCWD. These lines, designated ARS-Fp-R (resistant), ARS-Fp-C (control) and ARS-Fp-S (susceptible), differ in survival following standardized laboratory challenges with the causative agent of BCWD, Flavobacterium psychrophilum. This study evaluated survival of the genetic lines in laboratory challenges and in a production environment. Evaluations of disease resistance demonstrated a reproducible, 30% or greater, survival difference between ARS-Fp-R and ARS-Fp-S lines at body weights ranging from 0.7 to 13 g. Farm trials were performed to evaluate survival over an 80-day growth period starting after the trout began feeding. After a BCWD epizootic, the ARS-Fp-R line displayed significantly greater risk-adjusted survival (95.7%) than the ARS-Fp-S line (91.2%, P < 0.0001) and the ARS-Fp-C line (92.4%, P < 0.0001). Phenotype stability in farm-trial fish was also evaluated using laboratory challenges. The ARS-Fp-R line consistently displayed a higher, but not always statistically significant, survival percentage compared to the other lines and the data suggest that the magnitude of the survival phenotype difference is sensitive to environmental influence. In summary, the overall greater survival of the ARS-Fp-R line provides evidence of genetic improvement under production conditions.
Aquaculture 04/2013; s 388–391:128–136. · 1.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Farmed and wild salmonids are affected by a variety of skin conditions, some of which have significant economic and welfare implications. In many cases, the causes are not well understood, and one example is cold water strawberry disease of rainbow trout, also called red mark syndrome, which has been recorded in the UK since 2003. To date, there are no internationally agreed methods for describing these conditions, which has caused confusion for farmers and health professionals, who are often unclear as to whether they are dealing with a new or a previously described condition. This has resulted, inevitably, in delays to both accurate diagnosis and effective treatment regimes. Here, we provide a standardized methodology for the description of skin conditions of rainbow trout of uncertain aetiology. We demonstrate how the approach can be used to develop case definitions, using coldwater strawberry disease as an example.
Journal of Fish Diseases 03/2013; · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Three cohorts of farmed yellowtail kingfish (Seriola lalandi) from South Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To characterise the bacteria, 38 gill samples were processed for histopathology, electron microscopy and 16S ribosomal RNA amplification, sequencing and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolisation and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the order Chlamydiales. A novel 1393 bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a three year period that corresponded to the 16S rRNA sequence amplified directly from laser dissected cysts. This sequence was only 87% similar to the reported 'Candidatus Piscichlamydia salmonis' (AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35 Chlamydia and Chlamydia-like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the order Chlamydiales. Based on these observations, we propose this bacterium of yellowtail kingfish be known as 'Candidatus Parilichlamydia carangidicola' and the new family to be known as 'Candidatus Parilichlamydiaceae'.
Applied and Environmental Microbiology 12/2012; · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease and has significant economic impacts on aquaculture production worldwide. Molecular analyses have demonstrated that there is genetic diversity among F. columnare isolates. A review of the published literature that used restriction fragment length polymorphism analysis of the 16S rRNA gene revealed that all isolates typed from salmonids were Genomovar I. Our objective was to develop a laboratory challenge model for F. columnare in rainbow trout Oncorhynchus mykiss (Walbaum) and use the model to determine the virulence of Genomovar I and II isolates. Six F. columnare isolates were obtained from rainbow trout experiencing losses due to columnaris disease and were determined to be Genomovar I. Three of these were chosen for a preliminary assessment of virulence, and isolate 051-10-S5 was chosen for additional experiments to determine the reproducibility of the waterborne challenge model. In 2 independent experiments, cumulative percent mortalities (CPM) were 49 ± 10% and 50 ± 19%. Challenge of rainbow trout with Genomovar I and II isolates demonstrated a difference in the CPM, with the Genomovar II isolates inducing significantly higher CPM. This reproducible waterborne challenge model for columnaris disease in rainbow trout will be useful to investigate host-pathogen interactions, vaccine development, and other potential control strategies. This research also provides a basis for further defining the molecular diversity and virulence associated with F. columnare genomovars in rainbow trout and other salmonid species.
Diseases of Aquatic Organisms 11/2012; 101(2):115-22. · 1.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Small amoeboid cells, believed to be the infectious stage of Ichthyophonus sp., were observed in the bolus (stomach contents) and tunica propria (stomach wall) of Pacific staghorn sculpins and rainbow trout shortly after they ingested Ichthyophonus sp.-infected tissues. By 24-48 hr post-exposure (PE) the parasite morphed from the classically reported multi-nucleate thick walled schizonts to 2 distinct cell types; a larger multinucleate amoeboid cell surrounded by a narrow translucent zone and a smaller spherical cell surrounded by a "halo" and resembling a small schizont. Both cell types also appeared in the tunica propria, indicating that they had recently penetrated the columnar epithelium of the stomach. No Ichthyophonus sp. pseudo-hyphae ("germination tubes") were observed in the bolus or penetrating the stomach wall. Simultaneously, Ichthyophonus sp. was isolated in vitro from aortic blood, which was consistently positive from 6 -144 hr PE, then only intermittently for the next 4 wks. Small PAS-positive cells observed in blood cultures grew into colonies consisting of non-septate tubules (pseudo-hyphae) terminating in multinucleated knob-like apices similar to those seen in organ explant cultures. Organ explants were culture-positive every day; however, typical Ichthyophonus sp. schizonts were not observed histologically until 20-25 days PE. From 20 to 60 days PE, schizont diameter increased from < 25 µm to ≥ 82 µm. Based on the data presented herein, we are confident that we have resolved the life cycle of Ichthyophonus sp. within the piscivorous host.
Journal of Parasitology 08/2012; · 1.32 Impact Factor