Terutoyo Yoshida

Identification of cDNA encoding Toll receptor, MjToll gene from kuruma shrimp, Marsupenaeus japonicus.

Toll receptors are cell-surface receptors acting as pattern recognition receptors (PRRs) that are involved in the signaling pathway for innate immunity activation and are genetically conserved from insects to mammals. Tolls from penaeid shrimp are found in white leg shrimp Litopenaeus vannamei (lToll) and black tiger shrimp Penaeus monodon (PmToll). However, the molecular ligand-recognition patterns and identification of these penaeid Toll classes remain unknown. Here, we report cDNA cloning of a new type of Toll receptor gene (MjToll) from kuruma shrimp, Marsupenaeus japonicus, and the modulation of expression by immunostimulation. The full length cDNA of MjToll gene has 3095 nucleotides coding for a putative protein of 1009 amino acids. The MjToll gene is constitutively expressed in the gill, gut, lymphoid organ, heart, hematopoietic organ, hemocyte, ventral abdominal nerve cord, eyestalk neural ganglia and brain tissues. The MjToll gene expression was significantly increased (76-fold) as compared to a control in lymphoid organ stimulated with peptidoglycan at 12h, in vitro. lToll gene showed high similarity to PmToll gene with 96.9% identity; however, MjToll gene exhibited a percentage identity of 59% with that of penaeid Toll homologues. Therefore, this suggests that the identified MjToll gene belongs to the other class of Toll receptors in shrimp.

Efficacy of taurine supplementation for preventing green liver syndrome and improving growth performance in yearling red sea bream Pagrus major fed low-fishmeal diet

This study was performed to evaluate the efficacy of taurine supplementation for preventing green liver syndrome and improving growth performance in red sea bream Pagrus major fed a low-fishmeal (FM) diet. Yearling red sea bream were fed for 34 weeks on low-FM diets either supplemented with taurine, or without taurine, and the tissue taurine and bile pigment concentrations were measured. Compared to the fish fed the FM diet, fish fed the low-FM diet without taurine supplementation resulted in inferior feed performances and higher incidence of green liver related to the morphological transformation of the erythrocytes. In these fish, the hepatopancreatic taurine concentration was significantly lower and hepatopancreatic biliverdin concentration was high compared to the fish fed the FM diet. These parameters were markedly improved by taurine supplementation of the low-FM diet and were similar in levels to the fish fed the FM diet. These results indicate that green liver appearance and inferior feed performances of red sea bream fed the low-FM diet without taurine supplementation were caused by dietary taurine deficiency, and indicate the requirement of taurine supplementation to low-FM diets for red sea bream.

Protective efficacy ofAeromonas hydrophila vaccines in Nile tilapia

Protection and serum antibody production against Aeromonas hydrophila was examined in nile tilapia, Tilapia nilotica (L.). Intraperitoneally injected formal in-killed and Freunds complete adjuvant vaccines were compared using different doses (2.9 X 10(7) and 2.9 X 10(9) cfu/ml). Upon challenge, the protective ability and antibody titers resulting were significantly different between vaccinated and unvaccinated groups. A relative level of protection of 100% was obtained within two-weeks, and a maximum level of 53-61% protection was found one-week post-vaccination.

Immunomodulatory effects of the fermented products of chicken egg, EF203, on rainbow trout, Oncorhynchus mykiss

Abstract Immunomodulatory effects of immunoactive peptides from the fermented products of chicken egg (EF203) administered orally to rainbow trout, Oncorhynchus mykiss , were investigated. The chemiluminescent responses of kidney phagocytes after treatment with EF203 were significantly increased. Fish administered EF203 showed high phagocytic activities as compared to controls, and immunomodulatory effects were found to be dose dependent. Fish treated with EF203 displayed an increased resistance to both natural and experimental beta-haemolytic streptococcal infection.

Detection of yellow head virus in shrimp by loop-mediated isothermal amplification (LAMP)

Abstract Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the structural glycoprotein gene of yellow head virus (YHV). The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. The whole procedure is very simple and rapid, and reaction time and temperatures were optimized for 60min at 65°C, respectively. Detection of gene amplification could be accomplished by agarose gel electrophoresis. The standardized RT-LAMP procedure was used to detect YHV in the heart and gill from infected shrimp. Thus, the RT-LAMP assay is extremely rapid, cost-effective, sensitive and specific and has potential usefulness for rapid diagnosis for YHV detection in shrimp.

Differences between Lactococcus garvieae isolated from the genus Seriola in Japan and those isolated from other animals (trout, terrestrial animals from Europe) with regard to pathogenicity, phage susceptibility and genetic characterization

Aims:  To clarify the epidemiological relationship between Lactococcus garvieae isolates from the Seriola in Japan and isolates from other animals.

Detection of bacterial antigens using immuno‐PCR

E. KAKIZAKI, T. YOSHIDA, H. KAWAKAMI, M. OSETO, T. SAKAI AND M. SAKAI. 1996. A new and very sensitive antigen detection technique, immuno‐polymerase chain reaction (immuno‐PCR), was developed. This method is basically similar to the enzyme‐linked immunosorbent assay which detects an antigen‐antibody reaction, but instead of an enzyme being conjugated to an antibody, a DNA fragment is used and this DNA can be amplified by PCR. We applied this method to the detection of the fish pathogen, Pasteurella piscicida, in naturally infected yellowtail. Using immuno‐PCR, 3.4 cfu ml−1 of bacteria could be detected. In comparison, ELISA detected only 3.4 × 104 cfu ml−1. Immuno‐PCR is a powerful method for detection of pathogens in host tissues.

Characterization of Lactococcus garvieae isolated from radish and broccoli sprouts that exhibited a KG(+) phenotype, lack of virulence and absence of a capsule

Aims:  To identify Lactococcus garvieae isolates from radish and broccoli sprouts and compare them with virulent and less virulent mutant strains obtained from yellowtails with regard to KG phenotype, presence of a capsule and virulence towards yellowtails and mice.

Genetic and phenotypic comparison of Nocardia seriolae isolated from fish in Japan

The phenotypic and genetic characterizations of 58 isolates of the fish pathogen Nocardia seriolae, from amberjack, Seriolae dumerili, yellowtail, Seriola quinqueradiata, Japanese flounder, Paralichthys olivaceus, and chub mackerel, Scomber japonicus, in Japan from 1970-2005, were examined to investigate the epidemiological relationship between isolates. The phenotypic and genetic characterizations were determined by alpha-glucosidase activity and biased sinusoidal field gel electrophoresis (BSFGE) analysis, respectively. There was no alpha-glucosidase activity in strains isolated from 2000-05 (n = 50) with a few exceptions (n = 3), while all strains isolated from 1970-90 (n = 8) were positive. In BSFGE analysis, digestions with restriction enzymes Xba I and Ase I produced 15 and 16 restriction patterns, respectively. All restriction patterns obtained from 50 strains isolated during 2000-05 were unrelated to those obtained from eight strains isolated during 1970-90, with the exception of two strains isolated during recent outbreaks. Based on the phenotypic and genetic characterizations, recent outbreaks of nocardiosis in Japan are suggested to be epidemiologically unrelated to earlier outbreaks in Japan. Although a low genetic relationship was observed in the restriction pattern between recent and earlier isolates, identity was confirmed between these groups of isolates because five representative strains showed 99.9% homology with N. seriolae ATCC43993(T) in the 16S rRNA sequence.

Partial sequencing of sodA gene and its application to identification of Streptococcus dysgalactiae subsp. dysgalactiae isolated from farmed fish

Aims:  To investigate the difference between Lancefield group C Streptococcus dysgalactiae (GCSD) strains isolated from diseased fish and animals by sequencing and phylogenetic analysis of the sodA gene.