Effects of traditional Chinese medicines on immunity and culturable gut microflora to Oncorhynchus masou.

The present study was conducted to evaluate the effect of dietary traditional Chinese medicines on the growth, immunity, and composition of culturable gut microflora in Oncorhynchus masou. Diets were formulated to contain no medicine (control), antitoxic decoction (A), general antiphlogistic decoction (B), or Herbae Artemisiae Capillariae decoction (C). Fish were manually fed twice daily till apparent satiation for 30 days. Compared with that in the control group, supplementation with the three kinds of Chinese herbal medicine enhanced fish growth significantly (P < 0.05). The activities of liver superoxide dismutase and glutathione peroxidase in the treatment groups were significantly higher compared with those in the control group (P < 0.05). The quantity of intestinal microflora was higher in the treatment groups compared with that in the control group. Moreover, there were some effects of dietary Chinese herbal medicine on the composition of intestinal microflora. Microflora of Pseudomonas sp., Psychrobacter sp., Microbacterium sp., Macrococcus sp., Burkholderia sp., and Arthrobacter sp. were found in the treatment groups, whereas there were none in the control group. There was a significant increase in their amounts in the treatment groups (P < 0.05). The three kinds of traditional Chinese medicines can improve the growth and immunity of Oncorhynchus masou and affect the quantity and composition of intestinal microflora.

Increasing versatility of the DNA vaccines through modification of the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells.

The route of administration of DNA vaccines can play a key role in the magnitude and quality of the immune response triggered after their administration. DNA vaccines containing the gene of the membrane-anchored glycoprotein (gpG) of the fish rhabdoviruses infectious haematopoietic necrosis virus (IHNV) or viral haematopoietic septicaemia virus (VHSV), perhaps the most effective DNA vaccines generated so far, confer maximum protection when injected intramuscularly in contrast to their low efficacy when injected intraperitoneally. In this work, taking as a model the DNA vaccine against VHSV, we focused on developing a more versatile DNA vaccine capable of inducing protective immunity regardless of the administration route used. For that, we designed two alternative constructs to gpG1₋507 (the wild type membrane-anchored gpG of VHSV) encoding either a soluble (gpG1₋462) or a secreted soluble (gpG(LmPle20-462)) form of the VHSV-gpG. In vivo immunisation/challenge assays showed that only gpG(LmPle20-462) (the secreted soluble form) conferred protective immunity against VHSV lethal challenge via both intramuscular and intraperitoneal injection, being this the first description of a fish viral DNA vaccine that confers protection when administered intraperitoneally. Moreover, this new DNA vaccine construct also conferred protection when administered in the presence of an oil adjuvant suggesting that DNA vaccines against rhabdoviruses could be included in the formulation of current multicomponent-intaperitoneally injectable fish vaccines formulated with an oil adjuvant. On the other hand, a strong recruitment of membrane immunoglobulin expressing B cells, mainly membrane IgT, as well as t-bet expressing T cells, at early times post-immunisation, was specifically observed in the fish immunised with the secreted soluble form of the VHSV-gpG protein; this may indicate that the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells could be an important factor in determining the ways in which DNA vaccines prime the immune response.

[Comparative analyses of allergens between landlocked and anadromous species of masu salmon Oncorhynchus masou masou].

Although the difference in allergenicity between landlocked and anadromous salmon is little understood, only anadromous salmon are recommended to be labeled in the current allergen labeling system. This study was designed to examine the allergenic potency of landlocked species (yamame) and anadromous species (sakuramasu) of masu salmon Oncorhynchus masou masou, with special reference to parvalbumin, a known major fish allergen. Analysis of the heated extracts by SDS-PAGE suggested that yamame contains parvalbumin in the muscle at considerably higher levels, as compared with sakuramasu. In accordance with this, the parvalbumin content in the muscle of yamame (1.8-7.8 mg/g), determined by visible-light ELISA, was significantly higher than that of sakuramasu (0.28-0.52 mg/g). Furthermore, fluorescence ELISA experiments showed that the heated extract from yamame reacts with serum from fish-allergic patients more strongly than that from sakuramasu. Three parvalbumin isoforms (PA-I, -II and -III) were individually purified from yamame and sakuramasu by gel filtration and reverse-phase HPLC. Based on the retention times in reverse-phase HPLC and the molecular weights estimated by MALDI/TOF-MS, PA-I, -II and -III from yamame were judged to be identical with PA-I, -II and -III from sakuramasu, respectively. Taken together, our data indicate that landlocked masu salmon (yamame) is more allergenic than anadromous salmon (sakuramasu).

Host immunization shapes interspecific associations in trematode parasites.

1. Individuals of free-living organisms are commonly infected by multiple parasite species. Under such circumstances, positive or negative associations between the species are possible because of direct or indirect interactions, details in parasite transmission ecology and host-mediated factors. One possible mechanism underlying these processes is host immunity, but its role in shaping these associations has rarely been tackled experimentally. 2. In this study, we tested the effect of host immunization on associations between trematode parasites infecting eyes of fish. We first analysed the associations between three species (Diplostomum spathaceum, Diplostomum gasterostei and Tylodelphys clavata) in wild hosts, roach (Rutilus rutilus) and perch (Perca fluviatilis). Second, using rainbow trout (Oncorhynchus mykiss) as a model fish species, we experimentally investigated how sequential immunization of the host (i.e. one parasite species infects and immunizes the host first) could affect the associations between two of the species. 3. The results indicated that most of the associations were positive in wild hosts, which supports between-individual variation in host susceptibility, rather than competitive exclusion between the parasite species. However, positive associations were more common in roach than in perch, possibly reflecting differences in ecological conditions of exposure between the host species. The experimental data showed that positive associations between two of the species were eroded by host immunization against one of the parasite species. 4. We conclude that sequential immunization of hosts has a marked effect on interspecific parasite associations and basically can determine if positive associations are detected or not. This implies that correlative results suggesting non-interactive community structure in general may be obscured by the sequence of previous parasite exposure and corresponding dynamics of host immunization.

Modelling of fish interleukin-1 and its receptor.

This paper presents original data regarding: the three-dimensional modelling of rainbow trout (Oncorhynchus mykiss) and sea bass (Dicentrarchus labrax) IL-1beta, the modelling of trout IL-1 receptor type I (IL-1RI) and the modelling of trout IL-1beta bound to its receptor. The 3D models of trout and sea bass IL-1beta molecules were predicted by comparison with those already available of human and mouse, and, in both cases, a structure consisting of a beta-trefoil fold was obtained, a motif well conserved during evolution of IL-1beta-related proteins. Moreover, a model for the rainbow trout IL-1 receptor alone and complexed with IL-1beta was predicted and compared to the murine model. Both ligand-receptor complex models were compared with the known crystal structure of the human IL-1beta/IL-1R complex. A cross-interaction of trout IL-1beta with the mouse receptor was also simulated. Such analysis and the predicted interaction energies calculated from the models have helped to explain the different biological efficacies of mammalian and fish IL-1beta molecules in assays based on mammalian target cells. The obtained data are discussed on the basis of evolutionary and applied perspectives.

Graft-versus-host reaction (GVHR) in clonal amago salmon, Oncorhynchus rhodurus.

The graft-versus-host reaction (GVHR) was demonstrated in a salmonid model system of clonal diploid and triploid amago salmon. Triploid operculum grafts on clonal diploid evoked an acute rejection within 12 days. Grafts exchanged among triploid amago salmon exhibited prolonged survival for 18 days. In contrast, diploid grafts on triploid, and allografts among clonal diploid amago salmon were accepted. A typical GVHR was induced in triploid recipients by intraperitonal injection of head kidney cells from sensitised diploid donors. The clinical signs of graft-versus-host disease (GVHD) were observed in the recipients after 1 week of cell injection as a loss of appetite and appearance of solid faeces, followed by haemorrhage, local swelling of ventral skin and an enlarged spleen. Three of six fish died within 1 month. Water temperature and frequency of sensitisation are critical to induce GVHR. Diploid donors had to be sensitised three times at 20 degrees C to induce the typical GVHR. GVHR was most effectively induced by head kidney cells, followed by peripheral blood leucocytes (PBL) and spleen cells. Ploidy analysis by flow cytometry revealed that the donor head kidney cells greatly increased in the recipient liver, head kidney and spleen, and reached the peak after 9 days of donor cell injection. The results in the present study are quite similar to the findings in ginbuna and ginbuna-gold fish hybrid system, suggesting the presence of T cells in salmonid as well as cyprinid fish.

Cloning and expression analysis of rainbow trout Oncorhynchus mykiss tumour necrosis factor-alpha.

A rainbow trout (Oncorhynchus mykiss) gene for tumor necrosis factor (TNF) has been cloned and sequenced. The cDNA contains an open reading frame of 738 nucleotides that translate into a 246 amino-acid putative peptide, with a 5' untranslated region (UTR) of 140 bp and a 3' UTR of 506 bp. Two potential N-linked glycosylation sites exist in the translation. The genomic sequence measures 2007 bp and contains three introns that intercept four coding exons. Expression studies using RT-PCR have shown that the trout TNF gene is constitutively expressed in the gill and kidney of unstimulated fish. Trout TNF expression could be up-regulated by stimulation of isolated head kidney leucocytes with lipopolysaccharide (LPS). Similarly, stimulation of a trout macrophage cell line (RTS11) with LPS resulted in an increased transcript level, as did incubation with recombinant trout interleukin (IL)-1 beta. The optimal timing for induction of TNF expression in trout macrophages was determined using recombinant trout IL-1 beta, where a clear induction was apparent by 2 h and peaked at 4 h. Evidence that this TNF gene is equivalent to mammalian TNF-alpha is discussed.

Modes of salmonid MHC class I and II evolution differ from the primate paradigm.

Rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) represent two salmonid genera separated for 15--20 million years. cDNA sequences were determined for the classical MHC class I heavy chain gene UBA and the MHC class II beta-chain gene DAB from 15 rainbow and 10 brown trout. Both genes are highly polymorphic in both species and diploid in expression. The MHC class I alleles comprise several highly divergent lineages that are represented in both species and predate genera separation. The class II alleles are less divergent, highly species specific, and probably arose after genera separation. The striking difference in salmonid MHC class I and class II evolution contrasts with the situation in primates, where lineages of class II alleles have been sustained over longer periods of time relative to class I lineages. The difference may arise because salmonid MHC class I and II genes are not linked, whereas in mammals they are closely linked. A prevalent mechanism for evolving new MHC class I alleles in salmonids is recombination in intron II that shuffles alpha 1 and alpha 2 domains into different combinations.