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Results for “Fish and Shellfish Immunology”

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26 records · Page 2Linked to original sources
USGS2010

Early viral replication and induced or constitutive immunity in rainbow trout families with differential resistance to Infectious hematopoietic necrosis virus (IHNV)

The main objective of this study was to assess correlates of innate resistance in rainbow trout full-sibling families that differ in susceptibility to Infectious hematopoietic necrosis virus (IHNV). As part of a commercial breeding program, full-sibling families were challenged with IHNV by waterborne exposure at the 1 g size to determine susceptibility to IHNV. Progeny from select families (N = 7 families) that varied in susceptibility (ranging from 32 to 90% cumulative percent mortality (CPM)) were challenged again at the 10 g size by intra-peritoneal injection and overall mortality, early viral replication and immune responses were evaluated. Mortality challenges included 20–40 fish per family while viral replication and immune response studies included 6 fish per family at each time point (24, 48 and 72 h post-infection (hpi)). CPM at the 1 g size was significantly correlated with CPM at the 10 g size, indicating that inherent resistance was a stable trait irrespective of size. In the larger fish, viral load was measured by quantitative reverse-transcriptase PCR in the anterior kidney and was a significant predictor of family disease outcome at 48 hpi. Type I interferon (IFN) transcript levels were significantly correlated with an individual's viral load at 48 and 72 hpi, while type II IFN gene expression was significantly correlated with an individual's viral load at 24 and 48 hpi. Mean family type I but not type II IFN gene expression was weakly associated with susceptibility at 72 hpi. There was no association between mean family susceptibility and the constitutive expression of a range of innate immune genes (e.g. type I and II IFN pathway genes, cytokine and viral recognition receptor genes). The majority of survivors from the challenge had detectable serum neutralizing antibody titers but no trend was observed among families. This result suggests that even the most resistant families experienced sufficient levels of viral replication to trigger specific immunity. In summary, disease outcome for each family was determined very early in the infection process and resistance was associated with lower early viral replication.

Fish and Shellfish Immunology↗
USGS2010

Identification, characterization and genetic mapping of TLR1 loci in rainbow trout (Oncorhynchus mykiss)

Induction of innate immune pathways is critical for early anti-microbial defense but there is limited understanding of how teleosts recognize microbial molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 1 and 2 form a heterodimer involved in recognizing peptidoglycans and lipoproteins of microbial origin. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR1 gene ortholog and its mRNA expression. Two TLR1 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA sequencing and genetic linkage analyses. Full length cDNA clone and direct sequencing of four BACs revealed an intact omTLR1 open reading frame (ORF) located on chromosome 14 and a second locus on chromosome 25 that contains a TLR1 pseudogene. The duplicated trout loci exhibit conserved synteny with other fish genomes that extends beyond the TLR1 gene sequences. The omTLR1 gene includes a single large coding exon similar to all other described TLR1 genes, but unlike other teleosts it also has a 5' UTR exon and intron preceding the large coding exon. The omTLR1 ORF is predicted to encode an 808 amino-acid protein with 69% similarity to the Fugu TLR1 and a conserved pattern of predicted leucine-rich repeats (LRR). Phylogenetic analysis grouped omTLR1 with other fish TLR1 genes on a separate branch from the avian TLR1 and mammalian TLR1, 6 and 10. omTLR1 expression levels in rainbow trout anterior kidney leukocytes were not affected by the human TLR2/6 and TLR2/1 agonists diacylated lipoprotein (Pam2CSK4) and triacylated lipoprotein (Pam3CSK4). However, due to the lack of TLR6 and 10 genes in teleost genomes and up-regulation of TLR1 mRNA in response to LPS and bacterial infection in other fish species we hypothesize an important role for omTLR1 in anti-microbial immunity. Therefore, the identification of a TLR2 ortholog in rainbow trout and the development of assays to measure ligand binding and downstream signaling are critical for future elucidation of omTLR1 functions.

Fish and Shellfish Immunology↗
USGS2010

Transcriptome analysis of rainbow trout infected with high and low virulence strains of Infectious hematopoietic necrosis virus

There are three main genetic lineages or genogroups of Infectious hematopoietic necrosis virus (IHNV) in N. America. Strains representing the M genogroup are more virulent in rainbow trout relative to the U genogroup. In this study, we used microarray analysis to evaluate potential mechanisms responsible for host-specific virulence in rainbow trout that were given intraperitoneal injections of buffer or a representative M or U type virus strain. Reverse transcriptase quantitative PCR (RT-qPCR) was used to assess viral load and gene expression of select immune genes. Viral load was significantly higher in trout infected with the M virus starting at 24 h post-infection (p.i.) and continuing until 72 h p.i. Microarray analysis of the 48 h time point revealed 153 up-regulated and 248 down-regulated features in response to M virus infection but only 62 up-regulated and 49 down-regulated features following U virus infection. Translation and transcription features were among the most frequent down-regulated features in response to M virus infection and may be associated with the host cell shutoff phenomenon. A greater host cell shutoff response by the M virus may facilitate subversion of the host cell transcriptional machinery and enhance viral replication, suggesting the M virus may be better optimized to manipulate the rainbow trout transcriptional and translational machinery. Anti-viral associated features were the most commonly up-regulated features. A common set of features were up-regulated in both the M and U infection groups, but were induced to a higher magnitude in the M infection group. Gene expression of the anti-viral genes Mx-1 and Vig-1 was correlated but not entirely dependent on viral load in the anterior kidney. Slower replication of the U virus may allow the host more time to induce protective anti-viral immune mechanisms.

Fish and Shellfish Immunology↗
USGS2011

Specificity of DNA vaccines against the U and M genogroups of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss)

Infectious hematopoietic necrosis virus (IHNV) is a fish rhabdovirus that causes significant mortality in salmonid species. In North America IHNV has three major genogroups designated U, M, and L. Host-specificity of the M and U genogroups of IHNV has been established both in the field and in experimental challenges, with M isolates being more prevalent and more virulent in rainbow trout (Oncorhynchus mykiss), and U isolates being more prevalent and highly virulent in sockeye salmon (Oncorhynchus nerka). In this study, efficacy of DNA vaccines containing either M (pM) or U (pU) virus glycoprotein genes was investigated during intra- and cross-genogroup challenges in rainbow trout. In virus challenges at 7 days post-vaccination (early antiviral response), both pM and pU were highly protective against either M or U IHNV. In challenges at 28 days post-vaccination (specific antiviral response), both pM and pU were protective against M IHNV but the homologous pM vaccine was significantly more protective than pU in one of two experiments. At this stage both pM and pU induced comparably high protection against U IHNV challenge. Correlates of protection were also investigated by assessing the expression of the interferon-stimulated gene Mx-1 and the production of neutralizing antibodies (NAbs) following pM or pU DNA vaccination. Mx-1 gene expression, measured at 4 and 7 days post-vaccination as an indicator of the host innate immune response, was found to be significantly higher after pM than pU vaccination in some cases. Neutralizing antibody was produced in response to the two vaccines, but antibody titers did not show consistent correlation with protection. The results show that the rainbow trout innate and adaptive immune responses have some ability to distinguish between the U and M genogroup IHNV, but overall the pM and pU vaccines were protective against both homologous and cross-genogroup challenges.

Fish and Shellfish Immunology↗
USGS2012

Production and characterization of monoclonal antibodies to IgM of Pacific herring ( Clupea pallasii )

Pacific herring ( Clupea pallasii ) have a central role in the North Pacific ecosystem as a forage fish species and are natural reservoirs of several important finfish pathogens, including Viral hemorrhagic septicemia virus (VHSV). Here, we report the identification of the gene encoding the immunoglobulin mu (IgM) heavy chain, as well as the development and characterization of monoclonal antibodies (MAbs) that specifically react with Pacific herring IgM. Pacific herring immunoglobulin was purified and consisted of heavy and light chains of approximately 80 and 25 kDa. Three hybridoma clones were initially identified by ELISA as reactive with purified immunoglobulin but only one clone was able to detect an 80 kDa protein in Pacific and Atlantic herring (Clupea harengus) whole plasma by denaturing western blot. However, all three MAbs were able to precipitate an 80 kDa protein from Pacific herring and LCMS sequencing of peptide fragments derived from this protein matched the predicted amino acid sequence of the cloned, heavy chain gene. In addition, two of the MAbs stained cells within the putative lymphocyte gates for the spleen, anterior kidney and posterior kidney but were not reactive for myeloid/granulocyte gates, which is consistent with these MAbs reacting with surface IgM + B-cells. To our knowledge, this is the first report of IgM-related gene sequences and anti-IgM monoclonal antibodies from any member of the family Clupeidae. The antibodies produced in this study are critical for achieving our long-term goal of conducting serological surveillance to assess pathogen exposure in natural populations of Pacific herring.

Fish and Shellfish Immunology↗
USGS2023

Characterization of maternal immunity following vaccination of broodstock against IHNV or Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss)

Infectious hematopoietic necrosis (IHN) is a significant viral disease affecting salmonids, whereas Flavobacterium psychrophilum (Fp), the causative agent of bacterial coldwater disease (BCWD), remains one of the most significant bacterial pathogens of salmonids. We explored maternal immunity in the context of IHN and BCWD management in rainbow trout ( Oncorhynchus mykiss ) aquaculture. Two experimental trials were conducted where different groups of female broodstock were immunized prior to spawning with an IHNV DNA vaccine or a live attenuated F. psychrophilum (Fp B.17-ILM) vaccine alone, or in combination. Progeny were challenged with either a low or high dose of IHNV at 13 days post hatch (dph) and 32 dph or challenged with F. psychrophilum at 13 dph. Mortality following a low-dose IHNV challenge at 13 dph was significantly lower in progeny from vaccinated broodstock vs. unvaccinated broodstock, but no significant differences were observed at 32 dph. Mortality due to BCWD was also significantly reduced in 13 dph fry that originated from broodstock immunized with the Fp B.17-ILM vaccine. After vaccination broodstock developed specific or neutralizing antibodies respectively to F. psychrophilum and IHNV; however, antibody titers in eggs and fry were undetectable. In the eggs and fry mRNA transcripts of the complement components C3 and C5 were detected at much higher levels in progeny from vaccinated broodstock and showed a significantly increased and rapid response post-challenge compared with unvaccinated broodstock. After challenges pro-inflammatory cytokine expression was immediately and considerably elevated in the fry from vaccinated broodstock vs. unvaccinated broodstock, whereas adaptive immune genes were elevated to a lesser degree. Results suggest that maternal transfer of innate and adaptive factors at the transcript level occurred because development of lymphomyeloid organs is not complete in such young fry. In addition to documenting maternally derived immunity in teleosts, this study demonstrates that broodstock vaccination can confer some degree of protection to progeny against viral and bacterial pathogens.

Fish and Shellfish Immunology↗
USGS2004

Calcein AM release-based cytotoxic cell assay for fish leucocytes

A non-specific cytotoxic cell assay for fish is presented that is based on the release of the activated fluorochrome calcein AM from lysed carp epithelioma papulosum cyprini (EPC) cells. To establish the suitability of treating EPC cells with calcein AM the uptake and spontaneous release of the calcein AM by the EPC cells was evaluated. Incubation of 5 μM calcein AM in culture medium with 1×10 5 EPC cells well −1 for a minimum of 3 h provided sufficient labelling. Spontaneous release of fluorescence from the labelled EPC cells during 10 h of post labelling incubation ranged from 30 to 39% of the total observed fluorescence. Cytotoxic activity of trout leucocytes was evaluated at three leucocyte to target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8, and 10 h) with calcein AM-labelled EPC cells at 15 °C. In some instances, the monoclonal antibody specific for the NCC surface receptor NCCRP-1 (MAb5C.6) was included in the cultures. The activity of NCC cells was significantly inhibited in the presence of 0.25 μg well −1 of MAb5C.6 relative to no antibody ( P ≤0.013) or an equal amount of an unrelated antibody ( P ≤0.001). Average maximum observed percent cytotoxic cell activity of ∼18% was observed following 8 h of incubation at the 2:1 and 1:1 leucocyte to target cell ratios. Percent cytotoxic cell activity using calcein AM was similar to values reported for rainbow trout leucocytes using the 51 Cr-release assay.

Fish and Shellfish Immunology↗
USGS2014

To understand coral disease, look at coral cells

Diseases threaten corals globally, but 40 years on their causes remain mostly unknown. We hypothesize that inconsistent application of a complete diagnostic approach to coral disease has contributed to this slow progress. We quantified methods used to investigate coral disease in 492 papers published between 1965 and 2013. Field surveys were used in 65% of the papers, followed by biodetection (43%), laboratory trials (20%), microscopic pathology (21%), and field trials (9%). Of the microscopic pathology efforts, 57% involved standard histopathology at the light microscopic level (12% of the total investigations), with the remainder dedicated to electron or fluorescence microscopy. Most (74%) biodetection efforts focused on culture or molecular characterization of bacteria or fungi from corals. Molecular and immunological tools have been used to incriminate infectious agents (mainly bacteria) as the cause of coral diseases without relating the agent to specific changes in cell and tissue pathology. Of 19 papers that declared an infectious agent as a cause of disease in corals, only one (5%) used microscopic pathology, and none fulfilled all of the criteria required to satisfy Koch’s postulates as applied to animal diseases currently. Vertebrate diseases of skin and mucosal surfaces present challenges similar to corals when trying to identify a pathogen from a vast array of environmental microbes, and diagnostic approaches regularly used in these cases might provide a model for investigating coral diseases. We hope this review will encourage specialists of disease in domestic animals, wildlife, fish, shellfish, and humans to contribute to the emerging field of coral disease.

EcoHealth↗