An immune-complex glomerulonephritis of Chinook salmon, Oncorhynchus tshawytscha (Walbaum).

Chinook salmon from New Zealand were shown to have a generalized membranous glomerulonephritis that was most severe in large fish. Marked thickening of the glomerular basement membrane was the most consistent lesion, with the presence of an electron-dense deposit beneath the capillary endothelium.Severely affected glomeruli also had expansion of the mesangium and loss of capillaries,synechiae of the visceral and parietal epithelium and mild fibrosis of Bowmans capsule. Chinook salmon from British Columbia, Canada with bacterial kidney disease caused by Renibacterium salmoninarum had similar histological lesions. They also had thickened glomerular basement membranes that were recognized by rabbit antiserum to rainbow trout immunoglobulin. This was true only when frozen sections of kidney were used and not formalin-fixed tissue. An attempt to experimentally produce a glomerulopathy in rainbow trout by repeated immunization with killed R. salmoninarum was not successful. Case records from the Fish Pathology Laboratory at the University of Guelph over a 10-year period revealed that a range of species were diagnosed with glomerulopathies similar to those seen in Chinook salmon. The majority of these cases were determined to have chronic inflammatory disease. This report has identified the presence of immunoglobulin within thickened basement membranes of Chinook salmon with glomerulonephritis and supports the existence of type III hypersensitivity in fish.

Biochemical, molecular, and virulence characteristics of select Mycobacterium marinum isolates in hybrid striped bass Morone chrysops x M. saxatilis and zebrafish Danio rerio.

A panel of 15 Mycobacterium marinum isolates was characterized by biochemical tests, sequencing the ribosomal DNA intergenic spacer (ITS) region and the heat shock protein 65 gene (hsp65) and pulsed-field gel electrophoresis (PFGE). The biochemical characteristics of all isolates were similar, except for Tween 80 hydrolysis. DNA sequence of hsp65 for a subset of isolates were identical; however, at position 5 of the ITS rDNA, a single nucleotide polymorphism was identified. Isolates possessing a guanine residue at this position (G strains) were unable to hydrolyze Tween 80, while isolates that contained an adenine residue at this position (A strains) were positive for Tween 80 hydrolysis. PFGE successfully discriminated between the G and A strains; all G strains had identical AseI restriction enzyme-cutting patterns while the A strains exhibited a variety of cutting patterns. Eight isolates (4 G and 4 A strains) were further characterized for virulence by experimental infection of hybrid striped bass (HSB) Morone chrysops x M. saxatilis and zebrafish Danio rerio. Seven of the 8 strains produced cumulative mortality ranging from 13.3 to 83.3% in the HSB virulence trial. The M. marinum reference strain ATCC 927T did not produce mortality in HSB. HSB exposed to the G strains had significantly higher cumulative mortality than those exposed to the A strains. When these same isolates were tested in zebrafish, 6 of the 8 strains caused 100% cumulative mortality, with 2 of the A strains being the most pathogenic. In zebrafish, however, ATCC 927T was virulent and produced 28.5% mortality. Collectively, we conclude that the M. marinum G strains are unique and may represent a distinct virulence phenotype in HSB, but this trend was not consistent in zebrafish.

Aquatic Francisella-like bacterium associated with mortality of intensively cultured hybrid striped bass Morone chrysops x M. saxatilis.

The present study identifies an emerging disease associated with an aquatic Francisella-like bacterium that can cause mortality in hybrid striped bass Morone chrysops x M. saxatilis reared intensively in freshwater. Clinically affected fish were lethargic, had scattered haemorrhagic cutaneous lesions and diffuse gill pallor. The head kidney and spleen were markedly swollen and contained numerous interstitial granulomas; histological examination revealed small, pleomorphic Gram-negative coccobacilli within vacuolated cells. The bacterium could not be cultured from head kidney homogenates either with standard or enriched microbiological media or following inoculation of a Chinook salmon embryo (CHSE)-214 cell line. No amplification product was obtained from head kidney DNA by polymerase chain reaction (PCR) assay using Piscirickettsia salmonis-specific primers. PCR analysis of infected head kidney homogenate with primers designed for the eubacterial 16S rRNA produced a single amplicon. Phylogenetic analysis of this DNA sequence demonstrated that the sequence aligned most closely with members of the genus Francisella, identified from tilapia Oreochromis spp. in Taiwan and an aquatic Francisella species that was recently isolated from the three-line grunt Parapristipoma trilineatum in Japan. This Francisella-like disease was transmitted to naive hybrid striped bass fingerlings by intraperitoneal injection of tissue homogenates prepared from a natural outbreak. All fish developed gross and histological lesions identical to those from natural outbreaks. Intracellular Gram-negative bacteria were observed within the cytoplasm of cells (presumably macrophages) within the granulomas, but bacteria were not recovered. The 16S DNA sequence of the bacterium obtained from tissues of experimentally infected fish was identical to that obtained from the fish used as infected donor tissue.

Particle clearance from the gills of rainbow trout (Oncorhynchus mykiss).

The rate of particle clearance from the gills was assessed in healthy rainbow trout (Oncorhynchus mykiss), challenged with the formalin-killed bacterium Flavobacterium branchiophilum, as well as in fish with altered ventilation levels produced by varying the concentrations of dissolved oxygen (DO) in the water. The clearance of F. branchiophilum from the gills was quantified by means of an enzyme-linked immunosorbent assay. Fish held under normoxic conditions (DO = 9.5 mg/l) showed an initial rapid reduction in bacterial antigen, with 50% of the bacteria being cleared in the first 12 h after exposure, followed by slower clearance for the remaining bacteria; total elimination was achieved by 40 h. Fish with reduced ventilation rates (hyperoxia; DO = 25 mg/l) and elevated ventilation rates (hypoxia; DO = 4.5 mg/l) had significantly impaired particle clearance (r < 0.05), achieving only 60 and 20% reduction, respectively, at 72 h after challenge.

Blood chemistry and acid-base balance in rainbow trout Oncorhynchus mykiss with experimentally-induced acute bacterial gill disease.

Rainbow trout were experimentally infected with the causative agent of bacterial gill disease (BGD) (Flavobacterium branchiophilum) via bath challenge. All fish were cannulated with dorsal aortic catheters, had nasogastric tubes sutured in place for feeding, and were maintained individually, in plexiglass boxes with a flow-through water system. Fish were either fed, or unfed during the trial. Acute changes in blood gas, serum biochemistry and clinical parameters were monitored. By 24h post-challenge, BGD-infected trout that had been fed had significant hypoxemia, hypercapnia, increased blood ammonia, hypoosmolality, hyponatremia, hypochloremia, and increased cough and respiratory rates when compared to control levels. Unfed BGD-infected trout had similar, but less severe blood gas and clinical changes, and no electrolyte disturbances. The BGD-induced hypoxemia is likely exacerbated by increased oxygen demands brought on by feeding. It is not known what association feeding has with the development of low serum ion levels in BGD-infected trout. This is the first study to report the use of fed fish, as opposed to unfed or starved trout, in obtaining blood chemistry values from indisturbed and cannulated animals.

Pathology associated with meningoencephalitis during bacterial kidney disease of salmonids.

The neural pathology associated with spontaneous cases of bacterial kidney disease (BKD), in five species of commercially reared salmonids, was investigated histopathologically and with immunofluorescence. Patterns of localisation of the causative organism of BKD within the central nervous system suggest that haematogenous spread to the meninges, particularly the tela choroidea posterior, the tela choroidea and vascularised capsule of the saccus dorsalis and epiphysis of the epithalamus, and the saccus vasculosus of the hypophysis, appears to be a frequent route by which the central nervous system becomes infected. Retrograde extension from the posterior uvea to the floor of the diencephalon along the epineurium and perineurium of the optic nerve also may be a mechanism of neural invasion. Extension appeared to occur from these sites into adjacent areas of the meninges, the neural parenchyma and ventricles. Demonstration of bacteria within salmonid ependymal cells, as well as the apparent ability of salmonid ependymal cells to respond metaplastically suggest a similarity to mammalian type III ependymal cells (tanycytes). Based on this study, it is apparent that teleosts can survive protracted severe brain damage. This, combined with the apparent similarities of neural response to infection between the salmonids used in this study and higher vertebrates, suggests that teleosts may be a useful lower vertebrate model for studying the pathogenesis and sequelae of bacterial meningitis.

Responses of mucus-producing cells in gill disease of rainbow trout (Oncorhynchus mykiss).

This paper documents the responses of mucus-producing cells in the gills of rainbow trout (Oncorhynchus mykiss) throughout a naturally occurring outbreak of bacterial gill disease (BGD) and following exposure to experimentally induced high concentrations of ammonia and suspended solids. The responses were examined at three sites on the gill filament with three histochemical stains selected to identify the main types of mucous glycoproteins; these were periodic acid-Schiff (PAS), alcian blue pH 2.5 (AB2) and alcian blue pH 1.0 (AB1). In the BGD-infected fish, there was an increase in the numbers of PAS-positive and AB2-positive mucous cells and a corresponding decrease in AB1-positive cells. The greatest increase in mucus-producing cells occurred at the tips of the filaments, but the greatest relative change occurred at the mid-filamental (inter-lamellar) position. Fish exposed to high ammonia concentrations also had elevated numbers of mucus-producing cells, but there was no statistically significant change in fish exposed to high amounts of kaolin. The possible implications of these findings are discussed.