Kiyokuni Muroga

Vaccination trials with Penaeus japonicus to induce resistance to white spot syndrome virus

Crustaceans do not possess an adaptive immune response with immunoglobulins; however, recently, “quasi-immune response” has been reported by which kuruma shrimp (Penaeus japonicus) surviving from natural or experimental white spot syndrome virus (WSSV) infections possess a resistance against challenge with WSSV. In this study, efficacy of vaccines made of inactivated WSSV with or without immunostimulants (β-1,3-glucan or killed Vibrio penaeicida) and of recombinant proteins of WSSV (rVP26, rVP28) were tested by intramuscular vaccination followed by intramuscular challenge of kuruma shrimp with WSSV. The shrimp vaccinated with formalin-inactivated WSSV showed a resistance to the virus on 10th day post-vaccination (dpv) but not on 30th dpv. Heat-inactivated WSSV did not induce a resistance in the shrimp even on 10th dpv. Additional injections with glucan or V. penaeicida enhanced the efficacy of formalin-inactivated WSSV vaccine; however, the relative percent survival (RPS) values did not exceed 60% even when shrimp were vaccinated three times. On the other hand, two injections with rVP26 or rVP28 induced a higher resistance, with RPS values 60% and 95%, respectively, in the shrimp on 30th dpv. These results indicate the possibility of vaccination of kuruma shrimp with recombinant proteins against WSSV.

The isolation of intestinal microflora of farmed red seabream (Pagrus major) and black seabream (Acanthopagrus schlegeli) at larval and juvenile stages

The aerobic bacterial flora in the intestine of farmed red seabream and black seabream was investigated at their larval and juvenile stages in relation to the microflora of ambient water and diets. The total bacterial number in the intestine was 7.4×104 and 3.4×104 colony-forming units (CFU)/fish for red seabream and black seabream, respectively. Among the intestinal flora, Vibrio (ca. 45%) and Pseudomonas (30%) were the most dominant bacteria. The total bacterial counts were 3.1×104 CFU/ml for rearing water, 2.1×107 CFU/g for live diets (rotifer and brine shrimp) and 1.2×104 CFU/g for artificial diets. Among the water bacteria, Pseudomonas (22%) and Moraxella (29%) constituted dominant groups, and in the live diets Pseudomonas (48%) was predominant. The incidence of Vibrio in waters and live diets was 7% and 11%, respectively.

Viral and bacterial diseases of marine fish and shellfish in Japanese hatcheries

In Japan, seed production techniques have been exploited for about 80 species of marine fish and shellfish. However, mass mortalities due to infectious and non-infectious diseases have often occurred in larvae and juveniles reared in marine hatcheries. Among these problems the viral and bacterial diseases with their control measures are reviewed in this paper. Since around the middle of the 1980s, viral diseases such as viral epidermal hyperplasia (herpesvirus infection) in the Japanese flounder, viral ascites (birnavirus infection) in yellowtail, viral nervous necrosis (VNN) (nodavirus infection) in Japanese parrotfish and striped jack, and baculoviral mid-gut gland necrosis (BMN) (baculovirus infection) and penaeid acute viremia (PAV) (unclassified virus infection) in kuruma prawn have been reported. It was demonstrated that the selection of virus-free spawners based on the detection of the pathogen by polymerase chain reaction (PCR) serves as an effective control measure against vertical transmission of the pathogen in VNN and PAV. Vibriosis, pasteurellosis, gliding bacterial infection and other bacterial diseases have occurred in various marine fishes at their juvenile stages. On the other hand, larval fish most frequently develop intestinal infections represented by bacterial enteritis with Vibrio ichthyoenteri in the Japanese flounder. In the last part of the article, control measures for viral and bacterial diseases were discussed in each step of seed production process.

Mode of Transmission of Vibriosis among Ayu Plecoglossus altivelis

Abstract The mode of transmission of Vibrio anguillarum infection in ayu (Plecoglossus altivelis) and the portal of entry for the pathogen were investigated by the use of various challenge methods. The methods employed were immersion, direct contact, cohabitation, patch contact (filter paper soaked in bacterial suspension placed on part of a test fish for 1 min), and intubation. Based on the results of immersion, direct contact, and cohabitation challenges, it was concluded that water-borne infection is the primary mode of transmission of the disease. The patch contact challenge revealed that V. anguillarum penetrated the host through the skin, fins, anus, and gills. Among those, the skin and anus were determined to be the essential portals of entry for the pathogen. Artificial wounds of the skin surface caused by swabbing or scale removal facilitated invasion by the pathogen. Infection also was established by oral and anal intubations.

Vibrio penaeicida sp. nov., a Pathogen of Kuruma Prawns (Penaeus japonicus)

Six similar strains which were isolated as pathogens of cultured kuruma prawns (Penaeus japonicus) in Japan had characteristics of the genus Vibrio. These organisms were distinguished from previously described Vibrio species by their positive results in tests for gelatinase and lipase activities and their negative reactions in tests for arginine dihydrolase and lysine and ornithine decarboxylase activities, growth in the presence of 6% NaCl and at 35°C, acid production from sucrose, utilization of d-glucuronate and propionate, and luminescence. The G+C contents of the DNAs of these organisms were 46.2 to 47.0 mol%. The levels of DNA relatedness among these six strains were 87 to 99% (as determined by the membrane filter method), while the levels of DNA relatedness between prawn pathogen strain KH-1T (T = type strain) and members of 28 previously described Vibrio species were less than 18%. The name Vibrio penaeicida sp. nov. is proposed for the prawn-pathogenic strains. The type strain is strain KH-1 (= IFO 15640 = JCM 9123).

Characteristics of the causative bacterium of vibriosis in the kuruma prawn, Penaeus japonicus

Abstract Seventy-five strains of bacteria, which were isolated from diseased kuruma prawns ( Penaeus japonicus ) in several prefectures of western Japan from 1985 to 1990, were examined for their morphological, biochemical, physiological, serological, genetic, and pathological characteristics. All of these strains were classified as members of one species which seems to be identical to the Vibrio sp. reported as the causative agent of vibriosis in the kuruma prawn. Some selected strains of our isolates were confirmed to be pathogenic to the kuruma prawn by intramuscular injection. The bacterium is a gram-negative, motile, and 0 129 ( Vibrio static compound) sensitive short rod. It does not decarboxylate arginine, lysine or ornithine. The growth ranges (optimum) of temperature, pH and NaCl are 13–31°C (27°C), 6–9 (7–8), and 1–5% (2–4%), respectively. The GC content of DNA was 46.7±0.7 mol%. There was a major common O-antigen among all the tested strains of the pathogen, but it was possible to differentiate the present pathogen from fish pathogenic vibrios by slide agglutination tests. It is proposed that this pathogen is named Vibrio sp. PJ (PJ: Penaeus japonicus ).

Vibrio ichthyoenteri sp. nov., a pathogen of Japanese flounder (Paralichthys olivaceus) larvae

Seven similar strains which were pathogens of Japanese flounder (Paralichthys olivaceus) larvae with opaque intestines had characteristics of the genus Vibrio. These strains were divided into two genomic species (species 1 containing six strains, and species 2 containing one strain) on the basis of the results of DNA-DNA hybridization experiments in which the membrane filter method was used, and these two species could be differentiated from each other by the following characteristics: acid production from D-galactose and utilization of D-glucuronate and β-hydroxybutyrate. Strain F-2, the type strain of species 1, exhibited levels of DNA relatedness with 29 previously described Vibrio species of 5 to 18%. The flounder isolates belonging to species 1 were also differentiated from the previously described Vibrio species phenotypically by the following characteristics: they were nitrate reduction positive; each cell had a single polar flagellum; they did not produce arginine dihydrolase, chitinase, gelatinase, and lipase; they did not utilize D-cellobiose and citrate; and they did not grow at 35°C. The G+C contents of the DNAs of four species 1 strains were 43 to 44 mol%. The name Vibrio ichthyoenteri sp. nov. is proposed for genomic species 1. The type strain of V. ichthyoenteri is strain F-2 (= IFO 15847). Species 2 was also considered a new genomic species, but a species name is not proposed in this paper because only one strain is available and the phenotypic variability of the species is not known.

Purification and antibacterial characterization of a novel isoform of the Manila clam lectin (MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum

In many bivalve molluscs, lectins are present in the hemolymph and are thought to be important for internal host defense mechanisms. For this study, we purified a novel isoform of the Manila clam lectin (designated MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum, using affinity chromatography and gel filtration. Native PAGE results showed that the MCL-4 consisted of 70 kDa protein. MCL-4 was found to be composed of 58-kDa and 43-kDa bands when examined using SDS-PAGE under reducing and non-reducing conditions. The native MCL-4 was revealed as a 147 kDa molecular mass protein by gel filtration. The purified MCL-4 agglutinates calcium-dependently in the erythrocytes of sheep and rabbit, but not in cells of the three species of marine bacteria tested. However, the phagocytic ability of the R. philippinarum hemocytes for the MCL-4-opsonized Vibrio tubiashii cells was significantly greater than that for the BSS-treated bacterial cells. Addition of purified MCL-4 markedly suppressed Alteromonas haloplanktis growth. These results suggest that MCL-4, because of its opsonizing and bacteriostatic properties, might contribute to the host defense mechanisms against invading microorganisms in R. philippinarum.

Pasteurellosis in Cultured Black Seabream (Mylio macrocephalus)

1976年8月,岡山県水産試験場において種苗生産に関する一連の実験のために,沖出し後海面小割中で飼育されていたクロダイ(Mylio macrocephalus)幼魚に細菌性疾病と思われる流行病が発生し,約2週間の間に飼育されていた9,000尾のうち約8,000尾が死亡した。 それらの病魚には遊泳及び摂餌活動の低下ならびに体色の黒化が認められたが,そのほかの目立った病微は認められなかった。検査したほとんどの病魚の内臓諸器官から一種の非運動性細菌がほぼ純粋に分離された。 分離菌の形態学的・生理学的及び生化学的性状を調べた結果,本菌はPasteurella piscicidaに同定された。また分離菌をクロダイに接種したところ,比較的短時間で死亡せしめることも確認された。 なお,これらのクロダイが飼育されていた水域ではここ数年ハマチ養殖は行なわれておらず,また少なくとも沖出し後はこれらの魚にペレットのみを餌として与えており,今回の流行の感染源を明らかにすることはできなかった。 P. piscicidaは従来我が国ではハマチの類結節症の原因菌としてよく知られてきたが,今後はクロダイにおいても本菌感染症の発生に対する注意が必要と考えられた。

Failure of the Polymerase Chain Reaction (PCR) Method to Detect Striped Jack Nervous Necrosis Virus (SJNNV) in Striped Jack Pseudocaranx dentex Selected as Spawners

Abstract The causative agent of viral nervous necrosis (VNN) in striped jack Pseudocaranx dentex is transmitted from female and male spawners to larvae, and elimination of carrier spawners, determined by the detection of striped jack nervous necrosis virus (SJNNV) via polymerase chain reaction (PCR), has been used to prevent transmission of the disease in larval production facilities. However, some outbreaks of VNN occurred in larvae obtained from SJNNV-negative spawners. We compared the occurrence of infection between groups of larvae obtained from SJNNV-negative spawners and those from SJNNV-positive spawners. Viral nervous necrosis occurred in all seven groups of larvae obtained from the virus-positive spawners between the 3rd and 7th day of rearing. The virus was detected only occasionally after 2 weeks in four of six groups obtained from the virus-negative spawners. These results confirmed vertical transmission of the virus and revealed that a very small amount of SJNNV in spawners escaped PCR detect...