[Show abstract][Hide abstract] ABSTRACT: Nasal vaccines are very effective but the olfactory organ provides direct access of antigens to the brain. Infectious hematopoietic necrosis virus (IHNV) is known to cause high mortalities in salmonids. The purpose of this study is to evaluate the safety of a live attenuated IHNV nasal (I.N) vaccine in rainbow trout (Oncorhynchus mykiss). In the olfactory organ, the vaccine was detected 1 and 4 days after primary I.N vaccination but not in the intramuscular (i.m) or control groups. In the brain, IHNV was detected by RT-qPCR 4 and 21 days after i.m primary vaccination. One i.m and one I.N vaccinated trout were positive at days 4 and 28 days post-boost, respectively. Presence of IHNV in the brain of i.m vaccinated fish correlated with moderate increases in IL-1β and TNF-α expression in this tissue. These results demonstrate that IHNV vaccine lasts for 4 days in the local nasal environment and that nasal vaccination appears to be safe to the CNS of rainbow trout.
Full-text · Article · Jan 2016 · Fish & Shellfish Immunology
[Show abstract][Hide abstract] ABSTRACT: Enteric redmouth disease (ERM), caused by Yersinia ruckeri, has been controlled successfully using immersion-applied bacterin vaccines for several decades. While the host response to vaccination and the mechanism of protection of this vaccine have been elucidated, the bacterial components eliciting protection have remained unclear. Here we show that highly purified serotype O1 Y. ruckeri lipopolysaccharide (LPS) is sufficient to induce a protective response to experimental challenge in rainbow trout (Oncorhynchus mykiss). Dose response experiments demonstrated that Y. ruckeri LPS at doses of 1 ng/fish and above resulted in essentially complete protection and doses as low as 0.01 ng/fish (1.38 ng/kg) resulted in significant protection, thus demonstrating the extremely high potency of this immunogen. Analysis of the Y. ruckeri genome identified a cluster of putative O-antigen biosynthetic genes specific to serotype O1 strains. This cluster primarily consisted of genes encoding proteins predicted to function in the biosynthesis of legionamic acid, a nonulosonic acid known to be part of the O-polysaccharide repeat of O1 Y. ruckeri. Mutation of the nab2 gene, a nonulosonic acid biosynthesis gene (nab gene), resulted in production of severely truncated forms of LPS. Vaccination with bacterin vaccines derived from the nab2 mutant and its wild type parent strain demonstrated that LPS is a required component of the whole-cell bacterin vaccine and suggests that LPS is the only cellular component contributing to the protective response elicited by this vaccine. We speculate that the exceptionally high potency of Y. ruckeri LPS accounts for the unusual success of this vaccine when delivered by immersion.
No preview · Article · Jan 2016 · Fish & Shellfish Immunology
[Show abstract][Hide abstract] ABSTRACT: The emergence of an infectious viral disease caused by the Chinese giant salamander iridovirus (GSIV) has led to substantial economic losses. However, no more molecular information is available for the understanding of the mechanisms associated with virus–host interaction. In this study, de novo sequencing was used to obtain abundant high-quality ESTs and investigate differentially-expressed genes in the spleen of Chinese giant salamanders that were either infected or mock infected with GSIV. Comparative expression analysis indicated that 293 genes were down-regulated and 220 genes were up-regulated. Further enrichment analysis showed that the most enriched pathway is “complement and coagulation cascades”, and significantly enriched diseases include “inherited thrombophilia”, “immune system diseases”, “primary immunodeficiency”, “complement regulatory protein defects”, and “disorders of nucleotide excision repair”. Additionally, 30 678 simple sequence repeats (SSRs) from all spleen samples, 26 355 single nucleotide polymorphisms (SNPs) from the spleens of uninfected animals and 36 070 SNPs from the spleens of infected animals were detected. The large amount of variation was specific for the Chinese giant salamanders that were infected with GSIV. The results reported herein provided significant and new EST information that could contribute greatly in investigations into the molecular functions of immune genes in the Chinese giant salamander.
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The online version of this article (doi:10.1186/s13567-015-0279-8) contains supplementary material, which is available to authorized users.
Full-text · Article · Dec 2015 · Veterinary Research
[Show abstract][Hide abstract] ABSTRACT: This study describes the effect of increasing exposure-dose on Ichthyophonus prevalence and infection intensity in experimentally infected rainbow trout, Oncorhynchus mykiss. Specific-pathogen free (SPF) trout were exposed per os to increasing numbers of Ichthyophonus schizonts obtained from naturally infected donor fish, then sampled after 30 and 60 days post-exposure. Both in vitro explant culture and histology revealed that as the number of schizonts per dose increased there was a proportionate increase in the number of infected fish, as well as an increase in the number of infected organs; parasite density in individual infected organs also increased with dose. Explant culture revealed that all fish exposed to the highest dose (≥2,080 schizonts) became infected, while only 67% of those exposed to the intermediate dose (1,040 - 1,153 schizonts) were Ichthyophonus-positive after 60 days; Ichthyophonus was not detected in fish exposed to the 2 lowest doses (≤280 schizonts). Histologic examination of individual infected organs also revealed increasing infection prevalence and parasite density in response to exposure to increasing numbers of Ichthyophonus schizonts.
No preview · Article · Sep 2015 · Journal of Parasitology
[Show abstract][Hide abstract] ABSTRACT: Columnaris disease (CD), caused by Flavobacterium columnare, is an emerging disease affecting rainbow trout aquaculture. Objectives of this study were to 1) estimate heritability of CD resistance in a rainbow trout line (ARS-Fp-R) previously selected 4 generations for improved bacterial cold water disease (BCWD) resistance; 2) estimate genetic correlations among CD resistance, BCWD resistance, and growth to market BW; and 3) compare CD resistance among the ARS-Fp-R, ARS-Fp-S (selected 1 generation for increased BCWD susceptibility), and ARS-Fp-C (selection control) lines. Heritability of CD resistance was estimated using data from a waterborne challenge of 44 full-sib ARS-Fp-R families produced using a paternal half-sib mating design, and genetic correlations were estimated using these data and 5 generations of BCWD resistance, 9-mo BW (approximately 0.5 kg), and 12-mo BW (approximately 1.0 kg) data from 405 ARS-Fp-R full-sib families. The CD and BCWD challenges were initiated at approximately 52 and 84 d posthatch, or approximately 650 and 1,050 degree days (°C × d), respectively. Survival of ARS-Fp-R families ranged from 0 to 48% following CD challenge and heritability estimates were similar between CD (0.17 ± 0.09) and BCWD (0.18 ± 0.03) resistance, and the genetic correlation between these 2 traits was favorable (0.35 ± 0.25). Genetic correlations were small and antagonistic (–0.15 ± 0.08 to –0.19 ± 0.24) between the 2 resistance traits and 9- and 12-mo BW. Two challenges were conducted in consecutive years to compare CD resistance among ARS-Fp-R, ARS-Fp-C, and ARS-Fp-S families. In the first challenge, ARS-Fp-R families (83% survival) had greater CD resistance than ARS-Fp-C (73.5%; P = 0.02) and ARS-Fp-S (68%; P < 0.001) families, which did not differ (P = 0.16). In the second challenge, using an approximately 2.5-fold greater challenge dose, ARS-Fp-R families exhibited greater CD resistance (56% survival) than ARS-Fp-S (38% survival; P = 0.02) families. The favorable genetic correlation between CD and BCWD resistance is supported by greater CD resistance of the ARS-Fp-R line compared to the ARS-Fp-C and ARS-Fp-S lines and suggests that both traits will be improved simultaneously when selection is practiced on only 1 trait. In summary, these data indicate the feasibility of further selective breeding of the BCWD-resistant ARS-Fp-R line for increased CD resistance to produce a double pathogen–resistant line of rainbow trout.
Full-text · Article · Mar 2015 · Journal of Animal Science
[Show abstract][Hide abstract] ABSTRACT: We present the complete, closed, and finished chromosomal and extrachromosomal genome sequences of Yersinia ruckeri strain CSF007-82, the etiologic agent of enteric red mouth disease in salmonid fish. The chromosome is 3,799,036 bp with
a G+C content of 47.5% and encodes 3,530 predicted coding sequences (CDS), 7 ribosomal operons, and 80 tRNAs.
Full-text · Article · Jan 2015 · Genome Announcements
[Show abstract][Hide abstract] ABSTRACT: Chinese giant salamander hemorrhage is a newly emerged infectious disease in China and has caused huge economic losses. The causative pathogen has been identified as the giant salamander iridovirus (GSIV). In this study, the immunological responses and protection in Chinese giant salamander immunized with β-propiolactone inactivated GSIV are reported. Red and white blood cell counting and classification, phagocytic activity, neutralizing antibody titration, immune-related gene expression and determination of the relative percent survival were evaluated after vaccination. The red and white blood cell counts showed that the numbers of erythrocytes and leukocytes in the peripheral blood of immunized Chinese giant salamanders increased significantly on days 4 and 7 post-injection (P < 0.01). Additionally, the differential leukocyte count of monocytes and neutrophils were significantly different compared to the control group (P < 0.01); the percentage of lymphocytes was 70.45 ± 7.52% at day 21. The phagocytic percentage and phagocytic index was 38.78 ± 4.33% and 3.75 ± 0.52, respectively, at day 4 post-immunization which were both significantly different compared to the control group (P < 0.01). The serum neutralizing antibody titer increased at day 14 post-immunization and reached the highest titer (341 ± 9.52) at day 21. The quantitative PCR analysis revealed that the immunization significantly up-regulated the expression of immune related genes TLR-9 and MyD88 the first two weeks after immunization. The challenge test conducted at day 30 post-injection demonstrated that the immunized group produced a relative survival of 72%. These results indicate that the inactivated GSIV could elicit significant non-specific and specific immunological responses in Chinese giant salamander that resulted in significant protection against GSIV induced disease.
Full-text · Article · Nov 2014 · Veterinary Microbiology
[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.
Full-text · Article · Oct 2014 · G3-Genes Genomes Genetics
[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.
Full-text · Article · Oct 2014 · Nature Communications
[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
No preview · Article · Oct 2014 · North American Journal of Aquaculture
[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.
Full-text · Article · Aug 2014 · Fish & Shellfish Immunology
[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: Unlabelled:
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 whether CyHV-3 expressed genes during latency, transcription from all eight open reading frames (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 postinfection. The ORF6 transcript from in vitro infection begins at -127 bp upstream of the ATG codon and ends +188 bp 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.
This is the first demonstration that a member of the Alloherpesviridae, cyprinid herpesvirus 3 (CyHV-3), establishes a latent infection in the B cells of its host, Cyprinus carpio. In addition, this is the first report of identification of gene transcription during latency for a member of Herpesvirales outside Herpesviridae. This is also the first report that the hypothetical protein of latent transcript of CyHV-3 contains a consensus sequence with homology to a conserved domain of EBNA-3B from Epstein-Barr virus and ICP4 from herpes simplex virus 1, which are genes important for latency. These strongly suggest that latency is evolutionally conserved across vertebrates.
Full-text · Article · Jun 2014 · Journal of Virology