Cellular responses of leucocytes isolated from the gut of rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Leucocytes were isolated from the gut of rainbow trout by collagcnase digestion. They were shown to be capable of producing a chemoattractant(s) for head kidney leucocytes and a macrophagc activating factor, following stimulation with calcium ionophore and T-cell mitogens, respectively. They also included low but significant numbers of phagocytes, as determined by phagocytosis of fluorescent latex microspheres and oxygen free radical production. However, migration of isolated gut cells in response to head kidney chemo-attractants was lacking. The significance of these results in relation to mucosal immunity is discussed.     

Enhancement of resistance to bacterial infection in rainbow trout, Oncorhynchus mykiss (Walbaum), by oral administration of bovine lactoferrin

Abstract Bovine lactoferrin was evaluated for its ability to enhance resistance of rainbow trout, Oncorhynchus mykiss (Walbaum), to infection with Vibrio anguillarum. Oral administration of lactoferrin (100 mg kg⁻¹) to fish daily for 3 days prior to an intraperitoneal challenge with V. anguillarum resulted in increased survival rates, and enhanced resistance against Streptococcus sp., although to a lesser extent. In lactoferrin-treated fish, an increase in phagocytic and chemiluminescent (CL) activities of pronephros cells against V. anguillarum was observed. The phagocytic and CL activities of cells against Streptococcus sp. were also significantly increased. However, no in vitro bactericidal activities of lactoferrin against V. anguillarum or Streptococcus sp. were observed. This suggests that the lactoferrin enhanced the resistance of the rainbow trout against bacterial infection through the activation of phagocytes.     

Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.     

Identification and pathogenicity to rainbow trout, Oncorhynchus mykiss (Walbaum), of some aeromonads

Twelve strains of fish pathogenic aeromonads were identified by 16S rRNA sequencing as Aeromonas bestiarum , A. hydrophila , A. hydrophila subsp. dhakensis , A. salmonicida subsp. salmonicida , A. sobria biovar sobria and A. veronii biovar sobria. Following intramuscular injection, A. hydrophila subsp. dhakensis caused dark liquefying, raised furuncle-like lesions in rainbow trout within 48 h. Extracellular products of all cultures contained gelatinase and lecithinase, and most revealed lipase. Congo red absorption and siderophore production was recorded, but not so the suicide phenomenon or slime production. Sodium dodecyl sulphate polyacrylamide gel electrophoresis profile of the outer membrane proteins (OMP) revealed 10–25 bands, of which major bands were seen in the region of 32.5–47.5 and 62–83 kDa. Marked heterogenicity of the OMP and whole cell protein (WCP) profiles within and among the species was observed. Polypeptides of 83–173 kDa were detected in the WCP profile of the cultures, but they were not expressed in OMP fractions.     

The effect of glycyrrhizin on rainbow trout, Oncorhynchus mykiss (Walbaum), leucocyte responses

Abstract. Treatment of rainbow trout macrophages with glycyrrhizin (GL), an aqueous extract of licorice, Glycyrrhiza glabra , enhanced their respiratory burst activity. Maximal effects were seen using concentrations of 10–100 μml⁻¹. GL also modulated trout lymphocytes, increasing proliferation responses to the mitogen phytohaemagglutinin some two-fold over a range of GL concentrations. In addition, GL elicited the release of a macrophage activating factor (MAF) from head kidney leucocytes, as assessed by the ability of generated supernatants to increase respiratory burst activity of target macrophages. MAF activity was most apparent using 100 μg ml⁻¹ GL to induce MAF release and a 48-h incubation period with the target macrophages. Finally, GL was shown to enhance the release of MAF in response to the mitogen concanavalin A. The possible use of GL as a stimulant of fish innate defences is discussed.     

Development of a cell line from primary cultures of rainbow trout, Oncorhynchus mykiss (Walbaum), gills

Abstract. Collagenase was used to prepare primary cell cultures from rainbow trout, Oncorhynchus mykiss (Walbaum), gills. Although difficult to subcultivate, one primary culture led to the development of a cell line, RTgill-W1. RTgill-W1 grew in the basal medium, L-15, supplemented with foetal bovine serum at between 5 and 10%, but not in L-15 alone. The cells have been passaged approximately 50 times over a 4-year period. At confluency, the cell shape was predominantly polygonal or epithelial-like. RTgill-Wl cultures were demonstrated by DNA staining with H33258 and by growth on agar to be contaminated with mycoplasma, but this contaminant was eradicated by treatment with mycoplasma removal agent (MRA) and BM cyclin.     

Factors influencing in vitro respiratory burst assays with head kidney leucocytes from rainbow trout,Oncorhynchus mykiss (Walbaum)

Head kidney leucocytes are central elements in a number of in vivo and in vitro assays elucidating innate and adaptive immune mechanisms in teleosts following stimulation with various antigens. These systems are sensitive to several factors affecting the outcome of the assays. The present work describes the importance of temperature, cell concentration, exposure time and immune-modulatory molecules on the respiratory burst activity (RBA) of rainbow trout head kidney leucocytes in vitro. Some variation in RBA was observed among individual fish. However, use of cells pooled from four individuals produced satisfactory results following exposure to phorbol 12-myristate 13-acetate, zymosan and β-glucan. Temperature was shown to have a significant effect on production of reactive radicals as illustrated by a high activity in cells maintained at 15–20 °C and a reduced activity at temperature extremes (1, 4 and 30 °C). Highest activity was found at a cell concentration of 1 × 10⁷ cells mL⁻¹. Reactivity showed a clear decline when cells were exposed for more than 4 h. Moreover, incubation of cells with inhibitory substances viz., DiMePE2, cortisol and superoxide dismutase decreased the RBA. It is concluded that several biotic and abiotic factors should be taken into account when conducting RBA assays with head kidney leucocytes for elucidation of rainbow trout immune responses.     

Bacterial pathogens in rainbow trout, Oncorhynchus mykiss (Walbaum), reared at Danish freshwater farms

During a 2-year period, bacterial fish pathogens were monitored on five rainbow trout, Oncorhynchus mykiss (Walbaum), freshwater farms in Denmark. A total of 1206 fish were examined and 361 bacterial isolates were identified phenotypically. Enteric redmouth disease, furunculosis and rainbow trout fry syndrome/coldwater disease were recorded. Infections caused by Flavobacterium psychrophilum occurred most frequently, but only one outbreak of enteric redmouth disease caused by Yersinia ruckeri serotype O1 and one of furunculosis caused by Aeromonas salmonicida were recorded during the monitoring period. Flavobacterium psychrophilum was isolated on all farms, both during disease outbreaks and from fish without any signs of disease. Serological investigations of F. psychrophilum showed that serotype Th was the dominant serotype found. The serotypes Th and Fd were involved in disease outbreaks of fry and larger fish. All isolates of F. psychrophilum showed proteolytic activities; however, a few isolates, belonging to serotype FpT did not degrade elastin and were not associated with mortality. Increasing resistance problems to oxytetracycline were demonstrated. More than half of the F. psychrophilum isolates showed resistance to oxolinic acid and oxytetracycline. No antibiotic resistant isolates were found among Y. ruckeri and A. salmonicida .     

Indirect estimation of stomach volume of rainbow trout Oncorhynchus mykiss (Walbaum)

The main hypothesis of this study was that if stomach volume is correlated with food intake it can be estimated without laborious and destructive direct measurement. Rainbow trout, Oncorhynchus mykiss (Walbaum), ca. 500–1300 g, were starved for 1, 4, 8 or 16 days at 15°C after which they were fed in excess with dry pellets containing known amounts of X‐ray‐dense markers. Immediately after feeding the fish were killed, X rayed and weighed. Then the stomach was dissected, its contents removed and weighed, and stomach volume was measured. X‐ray plates were developed and feed intake was estimated based on the amount of marker. All measured variables correlated positively with stomach volume. The best fit for linear regression models was obtained for fish starved for 4 days, where stomach content (dry mass) explained 94%, food intake (based on X‐ray measurement) 77% and fish mass 62% of the variation in stomach volume. However, as stomach content measurement can be a lethal, or at least very stressful, event for the fish, the accuracy of food intake measurement (X‐ray) could be increased using multiple regression. In multiple linear regressions, R²‐values varied between 0.79 (16‐day starvation) and 0.91 (1‐day starvation) with food intake and fish mass as explanatory variables for stomach volume. These results indicate that the stomach volume in rainbow trout can be estimated satisfactorily using indirect methods, which are not detrimental to the fish, although feeding history may affect the accuracy of the estimates.     

Seasonal production of rainbow trout, Oncorhynchus mykiss (Walbaum), in ponds using different feeding practices

Abstract. In temperate regions of North America, the culture of rainbow trout, Oncorhynchus mykiss (Walbaum), in ponds is constrained to the period mid-November to early April by water temperature. Feeding practice may influence production time. The effects of three feeding practices on growth and body composition of rainbow trout raised in ponds during a 142-day winter growth period were investigated. Feeding practices were (1) hand-feeding to satiation, (2) feeding according to a fish size/water temperature chart, and (3) feeding by demand feeder.
Fish fed to satiation consumed 66% more diet than fish fed according to a feeding chart and 163% more than fish fed by demand feeders. Fish fed to satiation had significantly higher (P < 0·05) weight gain, harvest weight, specific growth rate, and protein gain with no significant increase (P > 0·05) in percentage body fat or feed conversion ratio (FCR) compared to the other two treatments. Survival percentage did not differ significantly (P > 0·05) between treatments. These data indicate that by using satiation feeding, marketable size rainbow trout can be produced in temperate region ponds without increases in percentage body fat or FCR.