Changes in drug resistance of Vibrio anguillarum in cultured ayu, Plecoglossus altivelis Temminck and Schlegel, in Japan

Abstract. Two-hundred-and-fifty-nine strains of Vibrio anguillarum isolated from diseased cultured ayu, Plecoglossus altivelis in various districts of Japan from 1974 to 1977 were studied for their sensitivity to 12 different chemotherapeutic agents: chloramphenicol (CM), tetracycline (TC), streptomycin (SM), kanamycin (KM), aminobenzyl penicillin (ABP), colistin (CL), nalidixic acid (NA), oxolinic acid (OA), piromidic acid (PA), furazolidone (NF), sulpharaonomethoxine (SA) and trimethoprim (TMP). One-hundred-and-thirty-four of the strains were additionally tested for sensitivity to O/129(2,4-diamino-6,7-diisopropyl pteridine) and ormethoprim (OMP). All strains were susceptible to SM, KM and ABP and also highly sensitive to CL. A correlation of resistance was found among the chemically-related drugs NA, OA and PA, and TMP, OMP and O/129, Nine strains were sensitive to all the drugs tested. The remaining 250 strains were resistant to various combinations of six drugs (CM, TC, NA, NF, SA and TMP). In particular, strains resistant to NA and NF (20.1%), to CM, TC, SA, NA and NF (29.7%) and to all six drugs (28.2%) were frequently detected. NA- or NF-resistant strains have increased since 1974 and TMP-resistant strains have increased since 1976. Transferable R plasmids were detected in 165 out of 250 resistant strains. The most common type of R plasmid determined resistance to CM, TC and SA. These multiple drug-resistant strains with transferable R plasmids were isolated from many different districts.     

Analysis of protective mechanisms in cultured ayu, Plecoglossus altivelis Temminck and Schlegel, administered Vibrio vaccine by the immersion method

Abstract. The protective effect of Vibrio angnillarum immunization administered by the direct immersion technique in ayu, Plecoglossus altivelis Temminck and Schlegel, was investigated. The activities of phagoeytic cells were suppressed at the time of vaccination by previous bath exposures to a cyclophosphamide solution containing a concentration of 500 mg/kg/ml on days 2 and 3 before vaccination. An artificial challenge was carried out 3 weeks after vaccination by bath exposure. The survival rate of the vaccine group pretreated with cyclophosphamide was 96%. In the group given traditional vaccine only, 73% survived; and in the non-vaccine control group, 10% survived. These results suggest that protective immunity of ayu vaccinated by the direct immersion technique may not implicate the early activities of phagoeytic cells.     

Modification of the hyperosmotic infiltration method of vaccination against vibriosis in cultured ayu, Plecoglossus altivelis Temminck and Schlegel

Abstract. A vaccine solution of a formalin-killed culture of Vibrio anguillarum cells was observed to be toxic to young ayu when administered by the hyperosmotic infiltration method. The toxin was present in the culture broth. After the toxin was removed from the broth by centrifugation, the fish were dipped in 5.32% NaCl solution for 2 min and then in a solution containing precipitated cells for 3 min. The immunized fish were protected against vibriosis when challenged one month after immersion. The bacterin was administered to ayu by a further two methods, both using lyophilized whole cells of formalin-killed V. anguillarum. In one method, the fish were placed in a 5.32% NaCl solution for 2 min and then in a solution containing lyophilized cells at 2 g/l of well water for 3 min (two-step immersion). In the other method, the fish were placed in a 5.32% NaCl solution containing lyophilized cells also at 2 g/l for 3 min (one-step immersion). A high level of protection against artificial challenge was achieved with either method. No agglutinating antibodies to V. anguillarum were detected in either the serum or mucus of fish dipped in a vaccine solution, a supernatant, or a precipitated solution, one month after immersion. On the other hand, serum titres were detected in fish vaccinated by injection, although no titres were detected in mucus. LD₅₀ values are presented for the virulence of the V. anguillarum strain. Compared to the original strain, virulence increased after the third passage in ayu, but decreased after the thirteenth passage in medium.     

A visceral mycosis in ayu fry, Plecoglossus altivelis Temminck & Schlegel, caused by a species of Phoma

Abstract. Epizootics and histopathology of a new visceral mycosis due to fungi impefecti which occurred in farmed ayu, Plecoglossus altivelis Temminck & Schlegel, in Tokushima Prefecture, Japan are described. Histopathological examination suggested that fungal infection occurred primarily in the airbladder. A fungus with characteristics of the genus Phoma isolated from diseased fish is described and its taxonomic position discussed.     

Histopathological studies of bacterial haemorrhagic ascites of ayu, Plecoglossus altivelis (Temminck & Schlegel)

A histopathological study was carried out on ayu, Plecoglossus altivelis, with bacterial haemorrhagic ascites. The fish were obtained from culture ponds in Wakayama Prefecture in 2003. The causative agent was identified as Pseudomonas plecoglossicida by a slide agglutination test using anti-P. plecoglossicida FPC941 serum. Histopathological studies revealed lesions in spleen, kidney, liver, intestine, heart and gills. Lesions in the spleen and haematopoietic tissue were prominent and invaded by P. plecoglossicida. Necrotic lesions accompanied by haemorrhage, fibrin deposition and oedema occurred in the splenic pulp and sheathed tissue, and in the kidney. The liver also had necrotic lesions and abscess formations. However, the intestine, heart and gills were only slightly invaded by P. plecoglossicida. No lesions or bacteria were observed in the brain.     

A novel genotyping technique for distinguishing between Flavobacterium psychrophilum isolates virulent and avirulent to ayu, Plecoglossus altivelis altivelis (Temminck & Schlegel)

We developed a simple genotyping method for Flavobacterium psychrophilum for analysing two single nucleotide polymorphisms (SNPs) in the gyrA gene and to distinguish between isolates that are virulent and avirulent to ayu, Plecoglossus altivelis altivelis (Temminck & Schlegel). The genotyping method is an on/off switch assay and is based on the polymerase chain reaction technique with phosphorothioated primers. We classified 232 isolates from four families of fish (i.e. Plecoglossidae, Osmeridae, Cyprinidae and Salmonidae) into four genotypes (G‐C, A‐T, A‐C and G‐T). The G‐C type isolates exhibited strong pathogenicity to ayu, whereas the A‐T and G‐T types did not show any pathogenicity to this species. The A‐C type exhibited no or weak pathogenicity to ayu. These results indicate that genotyping F. psychrophilum isolates with two SNPs from gyrA can clearly distinguish between isolates potentially harmful to ayu (G‐C type) and those that are potentially not harmful or less harmful (A‐C, A‐T and G‐T type). The on/off switch assay provides a quick, simple, and very powerful DNA genotyping technique for F. psychrophilum isolates.     

Changes in oocyte and blood plasma osmotic components of ayu, Plecoglossus altivelis Temminck & Schlegel during oocyte maturation

Oocyte maturation in teleosts is characterized by a complex series of cytoplasmic and nuclear changes. The purpose of this study is to present sequential changes in the oocyte constituents and regulatory mechanisms involved in the hydration and osmotic regulation of oocytes during final maturation of ayu, Plecoglossus altivelis Temminck & Schlegel. Oocyte volumes and water contents in ayu (P. altivelis) markedly increased from 0.372 mm³ and 60.5% at the mature stage to 0.712 mm³ and 79.4% at the time of oviposition. In the same period, net increases in total free amino acids and electrolytes were noted, and involvements of these constituents in the water uptake by oocytes are discussed. Moderate oocyte hydration in ayu is accompanied by steady increases in Na⁺, but not K⁺, and these observations are in contrast to results for all other marine teleosts reported elsewhere. In the present study, a steady increase in oocyte Na⁺ during maturation until ovulation pre‐dominantly results from diffusional leakage due to concentration gradients, while at the time of oviposition, both active transport and diffusional pathways play important roles in osmotic regulation in ayu oocytes.     

香鱼(Plecoglossus altivelis Temminck et Schlegel)的生物学特征及其养殖技术探讨

香鱼是一种优良的养殖品种,本文作较较详细介绍了香鱼的生物学特性及其养殖技术,其内容包括：香鱼生物学特性、香鱼养殖、香鱼食法及鱼病防治等。     

Genetic differences in some haematological traits of communally reared clonal ayu, Plecoglossus altivelis Temminck & Schlegel, under stressed and non-stressed conditions

Four clonal lines of ayu, Plecoglossus altivelis Temminck & Schlegel, were produced through gynogenesis, mixed before hatching and reared communally. After 10 months, a randomly taken sample was subjected to a standardized shallow-water stressor. Haematocrit, haemoglobin, red blood cells count (RBC) and mean corpuscular volume (MCV) were obtained from stressed and non-stressed fish. DNA fingerprinting was used to confirm the clonal nature of the organisms and to identify the clonal line to which each fish belonged. We observed significant differences between clonal lines mostly in the haematocrit and MCV measured under no-stress conditions. Such differences are suggested to represent mainly genetic variance, on account of the common environment provided to all the experimental groups. The stress response ratio was lower than expected, mainly due to some unexpectedly high non-stress values. Heritability values (h²) were medium to high for the no-stress measurements (mean 0.238) and very low or zero for the stressed groups’traits (excepting one high value of 0.484). We conclude that the use of communally reared clonal lines represents a good tool for the characterization of the physiological traits, thus allowing for their utilization as genetic selection criteria.     

Effects of bicarbonate ions and pH on acquisition and maintenance of potential for motility in ayu, Plecoglossus altivelis Temminck et Schlegel (Osmeridae), spermatozoa

The effects of extracellular ions on the acquisition and maintenance of the potential for motility in the spermatozoa of the ayu, Plecoglossus altivelis Temminck et Schlegel (Osmeridae), were examined. Testicular spermatozoa and milt spermatozoa were obtained from fully matured males and diluted with buffered solution (BS, 20 mM HEPES–NaOH, pH 7.5). Testicular spermatozoa showed a significantly low rate of motility (0.8 ± 0.4%), whereas milt spermatozoa showed a high rate (89.4 ± 2.1%). The spermatozoa were incubated with various isotonic media for 2 h, diluted with BS, and changes in the rates of motility were then compared. When incubated for 2 h with artificial seminal plasma (ASP), corresponding in terms of ionic constituents to seminal plasma buffered at pH 8.0, both spermatozoa showed a high rate of motility. Testicular spermatozoa acquired and milt spermatozoa maintained the potential for motility in response to the HCO₃^– ion concentrations (between 0 and 20 mM) in the ASP. The differences in the pH of the ASP had a significant effect on the acquisition and maintenance of the potential for motility, and spermatozoa showed the highest rate of motility with the ASP at pH 8.0 and 8.5. These results suggest that the quality of milt in the ayu can be regulated by controlling the concentration of bicarbonate and the pH of the incubating media.     

日本香鱼(Plecoglossus altivelis Temminck et Schlegel)生产及其病害防治技术研究

香鱼是重要的养殖经济鱼类，本文作者详细概述日本香鱼养殖生产的现状、香鱼全雌化生产技术以及香鱼病害防治技术的研究，内容较翔实，对香鱼养殖生产单位有一定的参考作用。     

Efficacy of Vibrio anguillarum antigen administered by intraperitoneal injection route in Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel)

Japanese flounder, Paralichthys olivaceus (Temminck et Schlegel), were immunized by the intraperitoneal (i. p.) injection route with formalin‐killed whole cells of Vibrio anguillarum that originated from a diseased fish. Fifty days later, a booster vaccination was given by the same route. Control fish were similarly treated with sterile phosphate‐buffered saline. The efficacy of vaccination was evaluated based on protection against two bacterial challenges and immune responses (both specific and non‐specific). The challenges were performed by i. p. injection with V. anguillarum or V. parahaemolyticus. The results indicated that the vaccinated fish showed higher non‐specific immune activity than the unvaccinated fish. The effects of vaccinations on the phagocytic activity of phagocyte, bactericidal and lysozyme activities were notable, especially on bactericidal activity. Determined by ELISA, antiserum of vaccinated fish displayed high antibody titres. The vaccination conferred protection against V. anguillarum challenge (81.25–93.75% relative percentage survival (RPS)). The RPS was 46.15–53.85% against V. parahaemolyticus challenge, indicating some degree of cross‐protective immunity.     

Pharmacokinetic study of oxytetracycline in healthy and vibriosis-infected ayu (Plecoglossus altivelis)

The pharmacokinetics and bioavailability of oxytetracycline (OTC) were examined in healthy and vibriosis-infected ayu (Plecoglossus altivelis). Water temperature was maintained at 18.0 ± 0.3 °C in all experiments. Serum concentrations of OTC in healthy fish after intravascular administration (25 mg kg⁻¹ body weight) were best described by a two-compartment model, whereas serum concentration-time curves after oral administration (100 mg kg⁻¹ body weight) in healthy and infected ayu could not be fitted by the nonlinear least squares method using one- and two-compartment models with first-order absorption. The estimated bioavailability after oral administration was 9.3% for healthy fish and 3.8% for infected fish. The elimination half-lives of serum, muscle, liver and kidney were 53.1 h, 106 h, 125 h and 117 h for healthy fish, and 63.2 h, 92.9 h, 107 h and 123 h for infected fish, respectively. A significant difference in bioavailability was revealed between healthy fish and infected fish, whereas elimination was similar in both fish. Serum protein binding in vivo of OTC was 68.0 ± 2.8% for healthy fish and 69.9 ± 4.4% for infected fish.     

Duration of immunity in salmonids vaccinated by direct immersion with Yersinia ruckeri and Vibrio anguillarum bacterins

Abstract. The level of protective immunity was determined for several salmonid species following vaccination by the direct immersion method with commercial Vibrio anguillarum (two serotypes) and Yersinia ruckeri (Hagerman strain) bacterins. The duration of protective immunity varied with the bacterin concentration, size and species of fish, but the duration between the two bacterins was comparable. In fish under 1 g duration of protective immunity was longest when the most concentrated bacterin was used. Generally, immunity lasted in 1-g fish for about 120 days, in 2-g fish for about 180 days, but in 4-g fish and larger immunity lasted for a year or longer. Coho salmon ( Oncorhynchus kisutch ) and sockeye salmon ( O. nerka ) retained immunity for a longer time and pink salmon ( O. gorbuscha ) the shortest time. Chinook salmon ( O. tshawytscha ) and rainbow trout ( Salmo gairdneri ) were intermediate. Field data generally followed the laboratory tests, but the duration appeared somewhat shortened. In one test 20-g rainbow trout were vaccinated by the shower method and no loss of immunity occurred after 311 days.     

Food habits of great cormorant Phalacrocorax carbo hanedae at Lake Biwa, Japan, with special reference to ayu Plecoglossus altivelis altivelis

ABSTRACT:   The recent increase in numbers of individuals of a piscivorous bird, the great cormorant Phalacrocorax carbo hanedae , around Lake Biwa has affected the commercial catch of ayu Plecoglossus altivelis altivelis . The food habits of the bird were studied based on 66 stomachs in 1998 and 2001–2002, and a seasonal variation in food contents was observed. The proportion of ayu in stomachs increased after April, and became most dominant during July and August (0.62 in proportion of weight, 0.85 in frequency of occurrence). In contrast, ayu was not found in the stomachs after late October. The increase of ayu in individuals and density in the surface layer of the pelagic zone of the lake during spring was considered as a trigger for the change of the food habits of the birds. The feeding impact of great cormorant on ayu in Lake Biwa was estimated to be approximately 46 t during April to June, and 310 t in July and August. During the main period for the commercial ayu catch (November to the following June), the estimated mass consumed by the bird is much smaller in comparison with the commercial catch.