Toshiaki Itami

Enhancement of disease resistance of kuruma shrimp, Penaeus japonicus, after oral administration of peptidoglycan derived from Bifidobacterium thermophilum

The potency of oral administration of peptidoglycan (PG) derived from Bifidobacterium thermophilum was examined. PG was administered to kuruma shrimps through the diet at 0.2 mg/kg body weight/day for 7 consecutive days, alternated with 7 days without PG throughout a 95-day test period. The shrimps were sampled on day 65 and 95 and challenged by Vibrio penaeicida, which is highly virulent to shrimps, through a water-borne route. The survival rate of PG-fed group was significantly higher than the control (P<0.01). PG-fed shrimps were challenged by white spot syndrome baculovirus. The survival rate was significantly higher than the control (P<0.01). To clarify the mechanism of defense that was enhanced by oral administration of PG, the phagocytic activity of shrimp granulocytes in vitro expressed by the phagocytic index (PI) was examined. Granulocytes were obtained from shrimps fed with PG and isolated by Percoll continuous density gradient. Fluorescently-labeled latex beads were used for the phagocytosis assay. The PI of PG-fed shrimps was higher than that of the control (P<0.05). These results indicate that the oral administration of PG to kuruma shrimp enhances the phagocytic activity of the granulocytes and increases the disease resistance of shrimps.

Loop-mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens.

Abstract Fish and shellfish diseases are a constant threat to the sustainability and economic viability of aquaculture. Early diagnosis plays a vital role in management of fish and shellfish diseases. Traditionally, various biochemical and serological tests have been used for fish disease diagnosis. However, the time and expertise required for such diagnoses makes it difficult for aquaculturists to easily adopt them under production conditions. Polymerase chain reaction and probe‐based nucleic acid detection have become increasingly popular in fish and shellfish diagnostics. Recently, a novel technique called loop‐mediated isothermal amplification (LAMP) has been developed, which is highly sensitive and rapid. LAMP has been used for the detection of bacterial, viral, fungal and parasitic diseases in both animal and plants. In aquaculture, LAMP‐based detection of pathogens like Edwardsiella tarda, E. ictaluri, Nocardia seriolae, Tetracapsuloides bryosalmonae, white spot syndrome virus and infectious haematopoietic necrosis virus have been reported. In this review, the application of LAMP for the detection of aquaculture‐associated pathogens is discussed.

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.

Detection of white-spot syndrome in cultured penaeid shrimp in Asia: Microscopic observation and polymerase chain reaction

Abstract Serious disease outbreak caused by a new virus has been occurring among cultured penaeid shrimps in Asian countries since 1993. Typical signs include white spots or patches on the inner surface of the shell and carapace and/or reddish coloration of the body. Histopathological changes observed among diseased shrimps collected from various countries exhibited widespread cellular degeneration and severe nuclear hypertrophy in cells of most tissues derived from ectodermal and mesodermal origin. Similar rod-shaped to elliptical virus particles of various sizes of 70–120 nm×240–340 nm surrounded by typical trilaminar envelope were found in the hypertrophied nuclei of affected cells. This virus was tentatively classified as a member of genus non-occluded baculovirus (NOB) of the subfamily Nudibaculovirinae of Baculovirus. A portion of the sequences of specific DNA fragment of an isolate from Thailand was used as a primer and compared to the other isolates collected from various countries. Results indicate that closely related strains of putative baculovirus were the causative agent of the white-spot syndrome of cultured penaeid shrimps occurring in six Asian countries.

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.

Primary culture of lymphoid organ cells and haemocytes of kuruma shrimp, Penaeus japonicus

A primary cell culture system was developed for the cells of lymphoid organ tissue of kuruma shrimp, Penaeus japonicus. Minced tissues of lymphoid organs were seeded and incubated at 30 degrees C in medium 199 supplemented with 20% foetal bovine serum, a salt mixture and a lactalbumin hydrolysate (0.1 g/l). Fibroblast-like cells and epithelioid-like cells survived for 54 days. Cells did not survive after trypsin, collagenase or hyaluronidase treatment used for cell dissociation. Mitogens (Con A, PHA-P, Pokeweed) and insulin did not enhance cell proliferation. When penaeid rod-shaped DNA virus (PRDV) was inoculated into the lymphoid organ cell culture, a cytopathic effect was observed within 8 days. On the other hand, large granular haemocytes that were fractionated using a Percoll continuous density gradient were not infected with PRDV in vitro within 10 days, which was the longest period of haemocyte maintenance.

Real-time quantitative loop-mediated isothermal amplification as a simple method for detecting white spot syndrome virus

Aims:  White spot syndrome virus (WSSV) continues to be the most pathogenic virus among the crustacean aquaculture causing mass mortality. In the present study, we established a one‐step, single tube, real‐time accelerated loop‐mediated isothermal amplification (real‐time LAMP) for quantitative detection of WSSV.

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.

OVARIAN PRIMARY TISSUE CULTURE OF THE KURUMA SHRIMP MARSUPENAEUS JAPONICUS

SummaryCell growth in ovarian primary culture of the kuruma shrimp, Marsupenaeus japonicus, was examined under various culture conditions. The best growth of ovarian cells was obtained in a culture system consisting of double strength of Leibowitz-15 medium supplemented with 10% fetal bovine serum, glucose (1 g/L), proline (0.1 g/L), TC-Yeastolate (1 g/L), and lactalbumin hydrolysate (1 g/L). The cells survived in this medium at 25° C for 45 d. The epithelial-like cells predominated in 10-d-old cultures, covering >80% of the surface area on the bottom of flask. Cells in mitosis were often observed. Cell proliferation was monitored by incorporation of 5-bromo-2′-deoxyuridine (BrdU), an analog of thymidine. 5-Bromo-2′-deoxyuridine-associated cells accounted for 11.5 and 35.0% of cell populations at 2 and 24 h, respectively, after BrdU treatment. Our results provide an improved culture technique for ovarian tissue of the kuruma shrimp.

Survival of Larval Giant Tiger Prawns Penaeus monodon after Addition of Killed Vibrio Cells to a Microencapsulated Diet

Abstract Feeding trials were conducted with microencapsulated test diets supplemented with formalinkilled vibrio cells to increase the number of survivors and to improve the quality of larvae in an early developmental stage of the giant tiger prawn Penaeus monodon. Vibrio cells were added at a level of 0.05, 0.5, or 5%, and the mixture was encapsulated with gelatin by a spray-dry method. Larvae at the zoeal stage were fed the test diet for four consecutive days. The survival of larvae as a percentage of initial larvae was higher in the groups fed bacterial cells than in the control group (P < 0.01 or P < 0.05).