Alexandra Adams

Chinese herbs (Astragalus radix and Ganoderma lucidum) enhance immune response of carp, Cyprinus carpio, and protection against Aeromonas hydrophila

The effect of Chinese herbs (Astragalus radix and Ganoderma lucidum) on immune response of carp was investigated. Fish were fed diets containing Astragalus (0.5%), Ganoderma (0.5%) and combination of two herbs (Astragalus 0.5% and Ganoderma 0.5%) for 5 weeks. Other groups of fish were vaccinated (i.p.) against Aeromonas hydrophila/Aeromonas salmonicida (Shering Plough, Essex, U.K.) at the beginning of the experiment and fed the same diets as described above. Control fish (negative control) and fish vaccinated only (positive control) were fed basal diets without supplements of herbs. The respiratory burst activity, phagocytosis, lysozyme activity and circulatory antibody titres in plasma were monitored. Following 5 weeks after feeding, fish were infected with A. hydrophila and mortalities were recorded. The results of this study showed that feeding non-vaccinated and vaccinated carp with combination of Astragalus and Ganoderma stimulated respiratory burst activity, phagocytosis of phagocytic cells in blood and lysozyme and circulatory antibody titres in plasma in vaccinated carp. Fish challenged with A. hydrophila had variable survival. The best survival (60%) was in vaccinated group fed with both herbs, while almost 90% of control fish (negative control) and 60% of fish vaccinated only (positive control) died.

Response of penaeid shrimp to exposure to Vibrio species

Greater than 99% of Vibrio alginolyticus were cleared from the haemolymph of Penaeus monodon within 4 h of exposure to the heat-killed bacteria. Bactericidins and other humoral factors (possibly lectins) appear to be induced within 1 day post-treatment. The appearance of ‘lectins’ was short-lived and only detected in bacteria-treated shrimps. Bactericidins, however, peaked at day 2 and persisted in the haemolymph until day 5. They showed partial specificity. In addition, bactericidins were briefly induced at day 2 in control shrimps which had been injected with saline. Haemagglutinins active against sheep red blood cells were detected at a variety of titres in all groups of shrimps. These results are discussed with reference to the responses of other crustacea and of insects to exposure to bacteria.

Survival and replication of Piscirickettsia salmonis in rainbow trout head kidney macrophages

Piscirickettsia salmonis is pathogenic for a variety of cultured marine fish species worldwide. The organism has been observed within host macrophages in natural disease outbreaks among coho salmon and European sea bass. In vitro studies, incorporating transmission electron microscopy (TEM) and ferritin loading of lysosomes, have confirmed that P. salmonis is capable of surviving and replicating in rainbow trout macrophages. Certain features of this intracellular survival underline its difference to other intracellular pathogens and suggest that a novel combination of defence mechanisms may be involved. Escape into the macrophage cytoplasm is not used as a means to avoid phago-lysosomal fusion and the organism remains at least partly enclosed within a vacuole membrane. While the piscirickettsial vacuole is often incomplete, survival and replication appear to require occupation of a complete, tightly-apposed, vacuolar membrane which does not fuse with lysosomes. Unlike some mammalian rickettsiae, actin-based motility (ABM) is not used as a means of intercellular spread. It is postulated that the presence of numerous small vesicles within vacuoles, and at gaps in the vacuolar membrane, may result from the blebbing of the piscirickettsial outer membrane seen early in the infection.

In situ hybridisation identifies the gill as a portal of entry for PKX (Phylum Myxozoa), the causative agent of proliferative kidney disease in salmonids.

Abstract PKX (Phylum Myxozoa) is an important pathogen affecting salmonid culture in Western Europe and North America. All of the available oligonucleotide probes developed for the PCR amplification of PKX DNA were examined for their ability to detect PKX in fixed tissue sections using in situ hybridisation. Out of the 12 probes examined, only four stained PKX in tissue sections. The specificity of these probes to PKX was examined by testing them individually against a range of myxosporean infections. One of the probes (1032) cross-reacted with Sphaerospora truttae infecting brown trout kidney and stained this parasite in tissue sections, while probe 6R stained stickleback DNA. The results from these studies allowed for an optimised, relatively rapid, in situ hybridisation protocol to be developed for PKX detection. Using this protocol, a preliminary study was conducted on the life history of the parasite in the rainbow trout Oncorhynchus mykiss. This demonstrated the presence of PKX in the gill arch 3 days after initial exposure in an enzootic river. It is suggested that a portal of entry for PKX is the gill. From here, it migrates to the kidney where the disease progresses as previously described.

Production and efficacy of an Aeromonas hydrophila recombinant S-layer protein vaccine for fish

A recombinant protein for the S-layer protein of Aeromonas hydrophila was produced and its ability to protect common carp Cyprinus carpio L. against six virulent isolates of A. hydrophila was assessed. A group of 120 carp (30-40 g) were vaccinated intra-peritoneally with 0.1 ml of adjuvanted vaccine (30 microg protein per fish). Another group of 120 carp were injected with 0.1 ml of PBS-adjuvant mixture to serve as controls. Twenty fish from each group were challenged with each one of six virulent isolates of A. hydrophila 35 days post-vaccination. The fish were maintained in 12 separate tanks before terminating the experiment at 16 days post-challenge. The relative percentage survival (RPS) for the six isolates of A. hydrophila ranged from 56 to 87%. The difference in survival rate of fish challenged with four of the isolates was statistically significant in vaccinated fish compared to control fish, when analysed using a Chi-square test. The results of the study suggest that the recombinant S-layer protein of A. hydrophila could be useful as a vaccine antigen to protect fish against different isolates of this pathogenic bacterium.

Transmission of Tetracapsuloides bryosalmonae (Myxozoa : Malacosporea), the causative organism of salmonid proliferative kidney disease, to the freshwater bryozoan Fredericella sultana

Proliferative kidney disease (PKD), caused by the malacosporean parasite Tetracapsuloides bryosalmonae, causes significant losses among salmonids in Western Europe and North America. The role of salmonid fish in the life-cycle of this parasite has been conjectured upon for over a quarter of a century. To examine whether fish can transmit the infection to bryozoans, the known invertebrate host, water containing parasitized brown trout Salmo trutta was pumped into tanks containing colonies of Fredericella sultana collected from the wild. The specific parasite-free status of these colonies being first assessed, by PCR and prolonged laboratory culture. After 6 weeks exposure to the brown trout aquarium effluent, portions of these colonies displayed overt infections with T. bryosalmonae. This was in contrast to control bryozoans, derived from the experimental colonies prior to exposure, which remained T. bryosalmonae negative. In addition, spores obtained from the experimentally infected colonies were exposed to naïve rainbow trout, resulting in clinical PKD, thus completing a cycle of transmission. During the experiments, the infection was noted to inhibit statoblast formation within bryozoans and appeared to be pathogenic, finally killing the bryozoan host. These findings indicate that fish can transmit the parasite to bryozoans and are an integral part of this parasites life-cycle.

Immunostimulation of striped snakehead Channa striata against epizootic ulcerative syndrome

Abstract Five immunostimulants were injected intraperitoneally into striped snakeheads (Channa striata). The inhibitory effects of the serum and macrophages collected from the treated fish on the germination and subsequent growth of Aphanomyces invadans (=piscicida), the causative agent of epizootic ulcerative syndrome (EUS), were then assessed. Salar-bec, a vitamin premix, and Ergosan, an alginate, both stimulated the inhibitory effects of serum on the germination and subsequent growth of A. invadans cysts, and the inhibitory effect of macrophages on growth. Betamak C85, a yeast extract containing β glucans and mannans, and Lysoforte, a lysophospholipid biosurfactant, induced little or no improvement in the parameters measured. Oro glo layer dry, a xanthophyll preparation, was rejected because of high mortalities among injected fish. Salar-bec showed the greatest improvement in the inhibition of both germination and growth by serum, and of growth by macrophages. It was selected for a tank trial in which snakeheads were fed on pellets coated with 2 g kg−1 Salar-bec, then injected with A. invadans. Control fish were fed on uncoated feed. Although the incidence of infection was not affected, hyphae appeared later in treated fish and granulomata developed faster at the infection site, suggesting an enhanced ability to contain the infection. Relative percent survival of treated fish was 59.2% higher than the controls over the 40-day trial. Anti-A. invadans antibody concentration was higher in treated fish, which may also have contributed to the containment of the infection.

The potency of adjuvanted injected vaccines in rainbow trout (Salmo gairdneri Richardson) and bath vaccines in Atlantic salmon (Salmo salar L.) against furunculosis

Abstract The efficacy of adjuvanted vaccine (administered IP) was evaluated in rainbow trout, Salmo gairdneri Richardson. In general, simple mineral oil-based adjuvants resulted in protection against furunculosis, vibriosis and enteric red mouth (ERM). The addition of whole cell bacterin to the adjuvant doubled protection to about 40 relative percentage protection (RPP). Increasing the administration volume also improved protection. The efficacy of bath vaccine was also evaluated in Atlantic salmon, Salmo salar. Booster vaccinations using Aeromonas salmonicida whole cells were effective against furunculosis (48.5 RPP), when given 4 weeks after the primary vaccination. A single vaccination using whole cells extracted with the non-ionic detergent Triton X-100 resulted in protection against furunculosis (29.9 RPP), while bacteria treated with EDTA gave little or no protection. Untreated whole cells gave 9.9 RPP. Extracellular products (ECP) from Aeromonas salmonicida were effective in protection when presented in particularised form, by binding ECP to polystyrene beads (35.6 RPP). Soluble ECP vaccine provided no protection at all.

The search for the IFN-γ receptor in fish: functional and expression analysis of putative binding and signalling chains in rainbow trout Oncorhynchus mykiss.

Interferons (IFNs), consisting of three major subfamilies, type I, type II (gamma) and type III (lambda) IFN, activate vertebrate antiviral defences once bound to their receptors. The three IFN subfamilies bind to different receptors, IFNAR1 and IFNAR2 for type I IFNs, IFNgammaR1 and IFNgammaR2 for type II IFN, and IL-28R1 and IL-10R2 for type III IFNs. In fish, although many types I and II IFN genes have been cloned, little is known about their receptors. In this report, two putative IFN-gamma receptor chains were identified and sequenced in rainbow trout (Oncorhynchus mykiss), and found to have many common characteristics with mammalian type II IFN receptor family members. The presented gene synteny analysis, phylogenetic tree analysis and ligand binding analysis all suggest that these molecules are the authentic IFNgammaRs in fish. They are widely expressed in tissues, with IFNgammaR1 typically more highly expressed than IFNgammaR2. Using the trout RTG-2 cell line it was possible to show that the individual chains could be differentially modulated, with rIFN-gamma and rIL-1beta down regulating IFNgammaR1 expression but up regulating IFNgammaR2 expression. Over-expression of the two receptor chains in RTG-2 cells revealed that the level of IFNgammaR2 transcript was crucial for responsiveness to rIFN-gamma, in terms of inducing gammaIP expression. Transfection experiments showed that the two putative receptors specifically bound to rIFN-gamma. These findings are discussed in the context of how the IFNgammaR may bind IFN-gamma in fish and the importance of the individual receptor chains to signal transduction.

Sporogony of Tetracapsuloides bryosalmonae in the brown trout Salmo trutta and the role of the tertiary cell during the vertebrate phase of myxozoan life cycles.

Tetracapsuloides bryosalmonae is the myxozoan that causes the commercially and ecologically important proliferative kidney disease of salmonid fish species. Immunohistochemistry and electron microscopy were used to examine the development of this parasite within the kidney of the brown trout Salmo trutta. The main replicative phase of T. bryosalmonae is a cell doublet composed of a primary cell and a single secondary cell. Engulfment of one secondary cell by another to form a secondary-tertiary doublet (S-T doublet) heralded the onset of sporogony whereupon the parasite migrated to the kidney tubule lumen. Within the tubule, the parasite transformed into a pseudoplasmodium and anchored to the tubule epithelial cells via pseudopodial extensions. Within each pseudoplasmodium developed a single spore, composed of 4 valve cells, 2 polar capsules and 1 sporoplasm. The pseudoplasmodia formed clusters suggesting that large numbers of spores develop within the fish. This examination of T. bryosalmonae suggests that the main replicative phase of freshwater myxozoans within vertebrates is via direct replication of cell doublets rather than through the rupturing of extrasporogonic stages, while tertiary cell formation relates only to sporogony. Taken in conjunction with existing phylogenetic data, 5 distinct sporogonial sequences are identified for the Myxozoa.