Isolation of RAG-1 and IgM transcripts from the striped trumpeter (Latris lineata), and their expression as markers for development of the adaptive immune response.

A partial sequence of the recombination activating gene-1 (RAG-1) and several full length sequences of the immunoglobulin M (IgM) heavy chain mRNA were obtained from the striped trumpeter (Latris lineata). The RAG-1 fragment consisted of 205 aa and fell within the core region of the open reading frame. The complete IgM heavy chain sequences translated into peptides ranging between 581 and 591 aa. Both genes showed good homology to other vertebrate sequences. The expression of the two genes was assessed throughout the early developmental stages of striped trumpeter larvae (5-100 dph) and used as markers to follow the ontogeny of the adaptive immune response. Using RT-PCR, RAG-1 mRNA expression was detectable at 5 dph and remained so until 80 dph, before becoming undetectable at 100 dph. IgM expression was also detectable at 5 dph, and remained so throughout. These patterns of expression may suggest that the striped trumpeter possess mature B cells with surface IgM at 100 dph. However, complete immunological competence is likely not reached until some time later. The early detection of IgM mRNA at 5 dph led to the investigation of its presence in oocytes. Both RAG-1 and IgM mRNA transcripts were detected in unfertilized oocytes, suggesting that they are maternally transferred. The biological significance of such a phenomenon remains to be investigated.

CpG inclusion in feed reduces sea lice, Lepeophtheirus salmonis, numbers following re-infection.

Lepeophtheirus salmonis infections in Atlantic salmon, Salmo salar, have been characterized by little to no hyperplastic response and a biphasic immune response that results in chronic inflammation with tissue repair as the infection progresses. We hypothesized that CpG administration with prior lice exposure would enhance epithelial inflammatory mechanisms and boost the Atlantic salmon immune response to L. salmonis, leading to greater protection against infection. We administered multiple exposures of L. salmonis to two groups of Atlantic salmon and compared responses against first-time exposed Atlantic salmon. Following re-exposure, CpG fed fish exhibited increased skin expression of interleukin (IL)-1ß and IL-12 ß compared to control previously exposed (CPE) and control first-time exposed (CFE) animals, respectively. This inflammatory enhancement occurred with significantly lower expression of matrix metalloproteinase-9 (MMP 9), both systemically (spleen) and locally (skin). Reduced MMP 9 expression was a hallmark of the re-infected fish (occurred in both tissues at both times). When significant differences were present in the skin or spleen, the two re-exposed groups showed greater similarity than with the first exposure group. Lice numbers on CpG fed fish were significantly lower than CFE fish at 7 days post-re-infection (dpri), and although they were not significantly different at 17 dpri, the trend of lower lice levels remained. CpG fed fish also showed nearly twofold greater protection than CPE when compared to the CFE group (48.5% vs. 27.0% reductions at 7 dpri and 27.2% vs. 13.1% reductions at 17 dpri, respectively). The enhanced protection of CpG oligodeoxynucleotide administration to previous exposure was consistent across all body surfaces and suggests that CpG can not only enhance innate responses to L. salmonis in Atlantic salmon, but also further stimulate adaptive responses.

Cloning and expression analysis of three striped trumpeter (Latris lineata) pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-8, in response to infection by the ectoparasitic, Chondracanthus goldsmidi.

This study reports the cloning and sequencing of three striped trumpeter (Latris lineata Forster) pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-8, as well as their differential expression in response to an infection by the ectoparasite Chondracanthus goldsmidi. The striped trumpeter TNF-alpha transcript consisted of 1093 bp, including a 759 bp ORF which translated into a 253 aa transmembrane peptide. The sequence contained a TACE cut site, that would produce a 167 aa soluble peptide containing the TNF ligand family signature. The IL-1beta sequence consisted of 963 bp, including a 774 bp ORF which translated into a 258 aa protein. The protein lacked both a signal peptide and an ICE cleavage site, but did contain the IL-1 family signature. The sequence for the chemokine IL-8 contained 906 bp, with an ORF of 297 bp, which translated into a 99 aa protein. The protein lacked an ELR motif as is common with many teleost IL-8 sequences. The differential expression of the three cytokine genes in parasitized fish was investigated via quantitative real-time PCR. A significant up-regulation of all three pro-inflammatory cytokines was found in the gills, which were the site of parasite attachment. Examination of head kidney cells revealed a significant up-regulation of TNF-alpha, but not IL-1beta or IL-8. Conversely, the spleen cells showed significant up-regulation of both IL-1beta and IL-8, but not TNF-alpha. These findings allow for more detailed investigations of the striped trumpeter immune response.