Multiple whole bacterial antigens in polyvalent vaccine may result in inhibition of specific responses in rainbow trout (Oncorhynchus mykiss).

The goal of fish vaccination today is to protect fish against multiple bacterial fish pathogens simultaneously using polyvalent vaccines. However, many immunological processes such as antigenic cross-reaction, antigenic competition, affinity maturation and antigen-induced suppression may affect the specificity, avidity and level of antibodies. Consequently, the biological function of antibodies may be markedly different from that predicted by conventional serologic tests. Here, we investigated the effects of vaccination and composition of vaccine on the plasma antibody levels, biological function of antibodies in opsonophagocytosis as well as the effects of vaccination on the blood leucocyte counts. Rainbow trout were vaccinated with saline or with two different polyvalent, mineral oil-adjuvanted vaccines. Vaccine 1 contained Aeromonas salmonicida, Listonella anguillarum and both Th and Fd serotypes of Flavobacterium psychrophilum antigens and vaccine 2 contained A. salmonicida, L. anguillarum and only Fd serotype of Fl. psychrophilum. The antibody-mediated opsonophagocytosis was determined as the respiratory burst (RB) activity of blood monocytes and granulocytes against the tested bacterial antigens. Three weeks after vaccination both vaccine groups and the control group showed increased RB activity against all bacterial strains. However, the increase in RB activities was non-specific and originated from the increased number of circulating granulocytes and monocytes. On the other hand, at 6 weeks post-vaccination both specific antibodies and antibody-dependent opsonophagocytosis appeared in both vaccine groups. However, the composition of the vaccine had a marked effect on the magnitude of specific responses. The Fd+Th vaccine enhanced the target specific opsonophagocytosis, to a lesser extent than the Fd vaccine. Both polyvalent vaccines appeared to mainly affect the numbers of circulating monocytes and our results suggest that the monocytes play a more significant role than the granulocytes in antibody-dependent opsonophagocytosis. Our results also suggest that the presented opsonophagocytic assay is an advantageous method to predict vaccine efficiency and that the number, and properties, of bacterial antigens in polyvalent vaccines should be carefully selected in order to avoid inhibitory effects of antigens on the specific response of fish.

Characterisation of atypical Aeromonas salmonicida infection in Arctic charr Salvelinus alpinus and European grayling Thymallus thymallus.

Cultured stocks of Arctic charr Salvelinus alpinus and European grayling Thymallus thymallus are vulnerable to infection by achromogenic atypical Aeromonas salmonicida (AAS). In Finland, natural stocks of both fish species have to be supported by restocking, and AAS infection poses a threat to successful restocking because no preventive means are available. In this study, we analysed AAS isolates from Arctic charr and European grayling and from other sources genetically, and characterised the signs and pathology of AAS infection in Arctic charr and European grayling both under farming conditions and after experimental challenge. AAS outbreaks were recorded in 1 fish farm over an 8 yr period. Among various salmonid fishes under farming conditions, only Arctic charr and European grayling were susceptible to AAS infection. The disease caused by AAS could be reproduced in both species using the same AAS strain in an experimental challenge. The course of the disease and pathology of natural and experimental AAS infection differed between the 2 species, even though only 1 strain was used for challenge. Isolates of AAS from Arctic charr and European grayling were genetically identical within a single river water basin. However, genetic heterogeneity was observed among the isolates from different water basins. In both species, AAS caused systemic infection. The results suggest that the same AAS strain could be used to develop a vaccine to protect both Arctic charr and European grayling from AAS infection.

Survival and virulence of Flavobacterium psychrophilum in water microcosms.

Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome and cold water disease in salmonids, causes serious disease outbreaks in fish farms worldwide. The aim of the present study was to examine the survival capacity of F. psychrophilum in laboratory microcosms containing sterilised water under different environmental conditions and to examine the virulence of starving F. psychrophilum cells. The results showed that F. psychrophilum survived for very long time in sterilised fresh water at 15 degrees C and the cells were still culturable after starvation for 300 days. A high salinity of the water (30 per thousand) drastically reduced the number of culturable cells below detection limit after incubation for 1 day. A water salinity of approximately 6 per thousand initially reduced the number of culturable cells below the detection limit, but cells were again recovered on agar plates at the end of the experiment. The presence of sediment containing nutrients in the experimental water microcosms increased the survival of F. psychrophilum. The challenge experiments indicated that the virulence of starving F. psychrophilum is maintained for at least seven days after the transfer of the bacterial cells to fresh water.

Detection of the fish pathogen Flavobacterium psychrophilum in water from fish farms.

Rainbow trout fry syndrome and cold-water disease are serious diseases in farmed salmonid fish. In the present study, three methods were compared, for the detection of the causative pathogen, Flavobacterium psychrophilum in water. The methods included traditional agar plate cultivation on tryptone yeast extract salts (TYES) agar, immunofluorescence antibody technique (IFAT) and nested PCR. The three methods were subsequently used for the detection of F. psychrophilum from fish farm environments. The nested PCR was the most sensitive method used for a detection of F. psychrophilum. As low as 3 CFU estimated by agar plate cultivation or 41 cells estimated by IFAT of F. psychrophilum per ml of non-sterile well water were needed for a detection using the nested PCR method. The obtained detection limits for the agar plate cultivation and the IFAT was 32 CFU/ml and 410 cells/ml, respectively. Using IFAT and nested PCR F. psychrophilum was detected most frequently in water samples from fish farms, but the pathogen was isolated from only a few samples using agar plate cultivation. In the present study IFAT and nested PCR proved to be rapid, specific and sensitive methods compared to traditional agar plate cultivation for the detection of F. psychrophilum from environmental samples. It is suggested that IFAT and nested PCR provide effective tools for the examination of F. psychrophilum in the environment.

Occurrence of Flavobacterium psychrophilum in fish-farming environments.

Occurrence of Flavobacterium psychrophilum in fish farms and fish-farming environments was studied using agar plate cultivation, the immunoflourescence antibody technique (IFAT) and nested PCR. Characteristics of 64 F. psychrophilum isolates from rainbow trout Oncorhynchus mykiss, fish farm rearing water, ovarian fluid and wild fish were serotyped, ribotyped and compared biochemically. Virulence of F. psychrophilum isolates from different sources was compared by injection into rainbow trout. Additionally, the number of F. psychrophilum cells shed by naturally infected rainbow trout was determined. F. psychrophilum was detected and isolated from skin mucus, skin lesions and internal organs of diseased rainbow trout and from fish without clinical disease. The pathogen was also present in wild perch Perca fluviatilis, roach Rutilus rutilus, and ovarian fluids of farmed rainbow trout brood fish. Isolates were biochemically homogenous, excluding the capability to degrade elastin. Five different agglutination patterns with different antisera against F. psychrophilum were found among the isolates studied. Although several different ribopatterns were found (ClaI: 12 ribopatterns and HaeIII: 9 ribopatterns), ribotype A was the most dominant. Farmed rainbow trout brood fish carried a broad-spectrum of serologically and genetically different F. psychrophilum in ovarian fluids. Virulence of the tested isolates in rainbow trout varied and naturally infected rainbow trout shed 10(4) to 10(8) cells fish(-1) h(-1) of F. psychrophilum into the surrounding water.