Erythromycin and florfenicol treatment of rainbow trout Oncorhynchus mykiss (Walbaum) experimentally infected with Flavobacterium psychrophilum

Flavobacterium psychrophilum is responsible for significant economic losses in rainbow trout aquaculture. Antimicrobial treatment remains the primary means of control; however, there are limited choices available for use. The objectives of the study were therefore to determine the minimum inhibitory concentrations for erythromycin and florfenicol in selected F. psychrophilum isolates and to evaluate their clinical treatment efficacy in experimentally infected rainbow trout. All isolates tested had moderate susceptibility to florfenicol and erythromycin except one isolate, which had low susceptibility to erythromycin. Two isolates (one with moderate and one with low susceptibility to erythromycin) were used in an experimental infection trial. Rainbow trout juveniles were injected intraperitoneally with 10⁸ cfu/fish and after mortality had begun, fish were given erythromycin‐ and florfenicol‐medicated feed at a rate of 75 mg kg^?1 day^?1 and 10 mg kg^?1 day^?1 fish body weight, respectively, for 10 consecutive days. The splenic F. psychrophilum load was determined using an rpoC quantitative PCR throughout the 30‐day trial. Relative to antibiotic‐free controls, erythromycin treatment significantly (p < 0.05) reduced mortality of rainbow trout juveniles infected with FPG101, even when treatment was initiated after clinical signs developed.     

Pathology and immunohistochemistry in three species of salmonids after experimental infection with Flavobacterium psychrophilum

Rainbow trout, Oncorhynchus mykiss (Walbaum), sea trout, Salmo trutta L., and Atlantic salmon, Salmo salar L., were experimentally infected with Flavobacterium psychrophilum in order to evaluate any species differences in susceptibility to the bacterium. Furthermore, differences in pathological changes and distribution of the bacteria in internal organs were studied. The bacteria were injected intraperitoneally in two doses, high dose (Hd) 1 x 10(7) colony forming units (CFU) fish(-1) and low dose (Ld) 1 x 10(6) CFU fish(-1). The mortalities in the Ld groups varied between 0 and 7.5% and in the Hd groups between 55-70%. No significant differences in mortality between the species were recorded. Clinical signs and pathological findings were similar in the three species and in accordance with those of rainbow trout fry syndrome. Rainbow trout showed more pronounced lesions in the spleen compared with the other species. Necrosis of renal tubular epithelium and haematopoietic tissue was most prominent in rainbow trout and Atlantic salmon. Intracellular eosinophilic droplets in the kidney tubular epithelium were a prominent finding in rainbow trout and sea trout surviving the infection. The distribution of the bacteria in internal organs was similar in the three species, as studied with immunohistochemistry.     

Inhibition of the attachment of Flavobacterium branchiophilum to the gills of rainbow trout, Oncorhynchus mykiss (Walbaum)

Preliminary experiments are described which aimed to identify compounds that could inhibit the attachment of Flavobacterium branchiophilum strains LAB4a and ATCC 35035 to the gills of rainbow trout. Total inhibition was never achieved, regardless of the compound tested. Formalin-killed or acetone-killed F. branchiophilum cells retained at least some of their adherent nature, relative to untreated (live) cells. Adherence was reduced by 22–33% following immersion of fish in one litre of water containing 0.21 mg of a homologous crude fimbrial extract. When fish were immersed in water containing hyperimmune rainbow trout antiLAB4a serum, a dose-dependent decrease in attachment (a reduction of 15% to 63%) of LAB4a to the gills was observed. Rainbow trout anti-LAB4a serum also reduced the attachment of ATCC 35035 to the gills, but this reduction was not significant. Adherence of LAB4a was not inhibited following exposure of fish to group 1 carbohydrates (arabinose, mannose and xylose), group 2 carbohydrates (dextrose, galactose and lactose), group 3 carbohydrates (galactosamine, glucosamine and fucose) or group 4 carbohydrates (N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetyl-neuraminic acid and the globoceramide glycolipid from human erythrocytes). In contrast, when rainbow trout erythrocytes were incubated with the bacteria prior to bath challenge, this resulted in an 87% and 53% reduction in gill-associated LAB4a and ATCC 35035 antigen, respectively, following immersion of rainbow trout in this suspension.     

Virulence of Flavobacterium psychrophilum isolates in rainbow trout Oncorhynchus mykiss (Walbaum)

Flavobacterium psychrophilum, the causative agent of bacterial cold‐water disease (BCWD) in freshwater‐reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 10⁸ cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 10⁶, 10⁷ or 10⁸ cfu/fish of F. psychrophilum FPG 101, the 10⁸ cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R² = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety‐seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log₁₀ gene/copies and >3.0 log₁₀ gene copies/reaction, respectively, following infection with 10⁸ cfu/fish.     

Effects of hydrogen peroxide on clearance of formalin-killed Flavobacterium branchiophilum from the gills of rainbow trout, Oncorhynchus mykiss (Walbaum)

The effect of hydrogen peroxide (H₂O₂) treatment on the clearance of formalin-killed Flavobacterium branchiophilum from the gills of rainbow trout was examined. In untreated control fish, clearance was characterized by a rapid initial phase, with 50% reduction in bacterial antigen in the first 12 h after exposure. The bacteria then cleared more gradually, with total clearance being achieved by 40 h. Treatment of fish with 100 p.p.m. H₂O₂ did not influence bacterial clearance compared to untreated controls. Exposure for 1 h to H₂O₂ concentrations ≤ 100 p.p.m. caused no detectable clinical signs or evidence of ultrastructural damage to the respiratory epithelium. However, levels in excess of this caused significant gill damage.     

Host-parasite interface of Neoechinorhynchus rutili (Eoacanthocephala) in naturally infected salmonids*

Abstract. The fine structure of the presomal tegument of Neoechinorhynchus rutili is described from worms attached to the intestinal wall of salmonid fishes. The host's cellular reaction at the worms’ point of attachment is described from the same hosts. The acanthocephalans’ presomal surface coat had a liquid lipid appearance. It seemed to be fed by droplets of lipid from necrotic host cells, by the contents of the granules of the hosts' coarsely granulated granulocytes and by excretions of the parasite. The crypts of the worms’ presomal outer limiting membrane were unbranched while those of the metasoma branched up in the striped layer. The striped layer of the proboscis hooks was more sclerotized than that of the surrounding (presomal) tegument. Inward from the hooks’ striped layer the crypts formed a mesh. The contents of the crypts were usually continuous with the presomal surface coat. The host's intestinal tissue in the region of the worms’ proboscides was heavily inflamed, especially around the hooks, where numerous granulocytes accumulated and degranulated. The acanthocephalans did not seem to be adversely affected by the inflammation and survived for at least 6 months in fish kept in the laboratory. The inflammatory tissue around the proboscides of worms exceeding a length of about 5 mm was enriched with fibroblasts and collagen fibres forming a capsule. Generally, the worms showed a shallow attachment. They were never found in extra-intestinal positions.     

Diversity of Flavobacterium psychrophilum and the potential use of its phages for protection against bacterial cold water disease in salmonids

Flavobacterium psychrophilum causes rainbow trout fry syndrome (RTFS) and cold water disease (CWD) in salmonid aquaculture. We report characterization of F. psychrophilum strains and their bacteriophages isolated in Chilean salmonid aquaculture. Results suggest that under laboratory conditions phages can decrease mortality of salmonids from infection by their F. psychrophilum host strain. Twelve F. psychrophilum isolates were characterized, with DNA restriction patterns showing low diversity between strains despite their being obtained from different salmonid production sites and from different tissues. We isolated 15 bacteriophages able to infect some of the F. psychrophilum isolates and characterized six of them in detail. DNA genome sizes were close to 50 Kbp and corresponded to the Siphoviridae and Podoviridae families. One isolate, 6H, probably contains lipids as an essential virion component, based on its chloroform sensitivity and low buoyant density in CsCl. Each phage isolate rarely infected F. psychrophilum strains other than the strain used for its enrichment and isolation. Some bacteriophages could decrease mortality from intraperitoneal injection of its host strain when added together with the bacteria in a ratio of 10 plaque-forming units per colony-forming unit. While we recognize the artificial laboratory conditions used for these protection assays, this work is the first to demonstrate that phages might be able protect salmonids from RTFS or CWD.     

Real-time RT-PCR detection of betanodavirus in naturally and experimentally infected fish from Spain

Infections with betanodavirus affect a wide range of wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to develop and validate real-time RT-PCR assays for sensitive and specific detection of nodavirus in diseased or carrier fish. The new detection assay was used to study the transmission and development of nodavirus infection in juvenile sea bass, Dicentrarchus labrax (L.), challenged by different routes, and also to screen for nodavirus in various farmed fish species. On average, the sensitivity was 10-100 times higher than a standard RT-PCR, and the assay was able to detect asymptomatic carrier fish that otherwise could have been classified as free of infection. Clinical signs of nodavirus infection were reproduced in fish infected following bath exposure or intramuscular injection, demonstrating horizontal transmission of the disease. Nodavirus was always detected in the brain of diseased fish but also in many recovered fish. The new assay enables us to confirm the presence of the virus at an early phase in the production cycle and may represent a useful tool to prevent or slow down the spread of nodavirus to new locations.     

Farming of salmonids in Russia

Abstract. Salmonids of the genera Salmo and Oncorhynchus are the main aquaculture Interest of Russia. Culture of salmonids, as well as other species, is determined by two factors: vulnerability of coastal areas to storms and high temperatures in the south of Russia and long periods of icing and low temperatures in the north. Adaptation of salmonids to these conditions therefore is an important aspect of research in Russia.
The culture of salmonids in Russia can be divided into three categories:
(1) traditional hatchery rearing and release of fingerlings into natural populations;
(2) acclimatization of Pacific salmon through transoceanic transportation; and
(3) commercial production in sea cages.     

Development of a new antibody for detecting natural killer enhancing factor (NKEF)-like protein in infected salmonids

The main cellular responses of innate immunity are phagocytic activity and the respiratory burst, which produces a high amount of reactive oxygen species. Natural killer enhancing factor (NKEF) belongs to the peroxiredoxin family that has an antioxidant function and enhances cytotoxic cell activity. This molecule may play a key role in macrophage and cytotoxic cell communication during the innate immune response of fish against pathogens. In fish, the NKEF gene has been characterized in some species as showing an up-regulation in infected fish, suggesting a trigger effect upon NK-like cells. To detect and localize this molecule in salmonids at protein level, a monospecific polyclonal antibody was generated. A probable NKEF-like protein epitope region was identified and characterized using bioinformatic tools, and the sequence was chemically synthesized using Fmoc strategy, analysed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was immunized and antibodies from ascitic fluid were obtained. The resulting antibody is a versatile tool for detecting NKEF by different immune techniques such as ELISA, Western blotting and immunohistochemistry. Analysis of NKEF-like protein is a useful method for characterizing immune properties of this molecule in fish during response to pathogens.     

Bacterial kidney disease in salmonids

Bacterial kidney disease (BKD) is the most important bacterial infection in salmonid fish. Clinics, pathomorphology, distribution and possibility of therapy of BKD are discussed regarding results of literature as well as own experiences.     

Virulence assay of rhizoid and non‐rhizoid morphotypes of Flavobacterium columnare in red tilapia,Oreochromis sp., fry

Numerous isolates of Flavobacterium columnare were previously recovered from red tilapia, Oreochromis sp., exhibiting columnaris‐like disease in Thai farms, and the phenotypic and genetic characteristics were described. The objective of this study was to determine the virulence of two morphotypes (rhizoid and non‐rhizoid colonies) of F. columnare and to determine their ability to adhere to and persist in red tilapia fry. The results showed that the typical rhizoid isolate (CUVET1214) was a highly virulent isolate and caused 100% mortality within 24 h following bath challenge of red tilapia with three different doses. The non‐rhizoid isolate (CUVET1201) was avirulent to red tilapia fry. Both morphotypes adhered to and persisted in tilapia similarly at 0.5 and 6 h post‐challenge as determined by whole fish bacterial loads. At 24 and 48 h post‐challenge, fry challenged with the rhizoid morphotype exhibited significantly higher bacterial loads than the non‐rhizoid morphotype. The results suggested that an inability of the non‐rhizoid morphotype to persist in tilapia fry may explain lack of virulence.