Gerald R. Johnson

Uptake, disposition and depuration of domoic acid by blue mussels (Mytilus edulis)

Abstract Domoic acid (DOM), the toxin involved in amnesic shellfish poisoning, was presented to live mussels (at 5°C, 28 0 00 ) in dissolved form (125 nM) and as food encapsulated in liposomes. Less than 1% of dissolved DOM and up to 6% of food-borne DOM was incorporated into mussel tissues. DOM absorbed from solution was most concentrated in gills and kidney, whereas DOM ingested as food was most concentrated in digestive gland and kidney. Gonad, muscle, foot and connective tissues retained the lowest concentrations of toxin. Compared to their proportion of body weight; kidney, digestive gland and gill retained larger than expected proportions of total toxin burden. The concentration of toxin in mussel tissues did not decrease consistently over a depuration period of 48 h, nor did DOM appear to be translocated to any tissue for storage. Small amounts of DOM were eliminated in faeces and larger amounts in dissolved form. Over 80% of intracellular DOM was associated with the TCA (trichloroacetic acid) soluble fraction of digestive gland tissue. DOM in the TCA insoluble fraction was positively correlated with overall DOM concentration.

Haemorrhagic kidney syndrome of Atlantic salmon, Salmo salar L.

This report describes a new syndrome affecting farmed Atlantic salmon on the Canadian east coast that has resulted in increased morbidity and mortality in affected stocks. The major pathological findings are apparent only microscopically and include renal interstitial haemorrhage and acute tubular necrosis and tubular casting. As a result, the disease has become known as haemorrhagic kidney syndrome (HKS). Affected fish are lethargic and anorectic, and lack external lesions. Clinically, HKS fish are anaemic, hypoproteinaemic and hyperosmolalic, with increased serum concentrations of sodium and chloride. At necropsy, internal changes ranged from apparently normal to include one or several of the following: swelling and/or patchy reddening of the kidney, pale gills, exophthalmos, serosanguinous ascites, darkening of the posterior intestine and splenomegaly. Ultrastructurally, viral inclusions were found in the cytoplasm of erythrocytes of HKS fish, and there were unusual electron-dense inclusions within the tips of renal tubular microvilli of HKS fish. The significance and relevance of the ultrastructural findings to HKS are unknown. Virus isolation was attempted using CHSE, RTG-2, FH-10, BB and EPC cell lines; no virus was isolated. Bacteriological analysis failed to reveal significant pathogens. Analysis of tissues for heavy metals and pesticides was negative. Assays for clostridial toxins, lipopolysaccharide and verotoxins were negative. The aetiology of HKS remains unresolved.

Purification and Partial Characterization of a Rainbow Trout Egg Lectin

Abstract A lectin that agglutinates rabbit red blood cells (RBCs) and human type B RBCs was isolated from ova of rainbow trout Oncorhynchus mykiss. Hemagglutination of rabbit RBCs was inhibited completely by 10 mM L-rhamnose but not by certain other sugars, 100 mM EDTA, or 100 mM 2-mercaptoethanol. Partial purification of this hemagglutinating material was achieved by affinity chromatography of an H2O-dialyzed yolk homogenate on rhamnose-linked Sepharose. A polyacrylamide gel electrophoresis (SDS-PAGE) performed on this sample revealed two polypeptides with approximate molecular masses of 19 kilodaltons (kDa) and 30 kDa. By fast-phase liquid chromatography, proteins with a molecular mass of less than 20 kDa were separated from other elements of the affinity-purified hemagglutinating material. These proteins were found to lack hemagglutinating activity. When a western blot with rabbit antilectin antiserum was performed against yolk extract, rainbow trout serum, or yolk from larvae, a 30-kDa polypeptide was...

A study of dichlorvos (Nuvan; 2,2 dichloroethenyl dimethyl phosphate), a therapeutic agent for the treatment of salmonids infected with sea lice (Lepeophtheirus salmonis)

Abstract Larval lobsters ( Homarus americanus ), juvenile lobsters, adult lobsters ( H. americanus ), species of zooplankton, species of phytoplankton, mussels ( Mytilus edulis ) and periwinkles ( Littorina littorina ) were exposed to dichlorvos at various concentrations in the laboratory and mortalities were recorded. Dichlorvos was also released from an ocean field site and at a fish farm while stage IV larval or juvenile lobsters ( H. americanus ) were suspended in the water column in areas adjacent to the release site to determine the effects of dichlorvos on their survival. Results indicate that dichlorvos is not toxic to mussels or periwinkles at 1.0 ppm, for 1 h exposure, but is toxic to larval lobsters, adult lobsters, zooplankton and phytoplankton. In field trials larval and juvenile lobsters housed adiacent to sea-cage operations were not killed following treatment with dichlorvos.

Correlation of virus replication in tissues with histologic lesions in Atlantic salmon experimentally infected with infectious salmon anemia virus.

We have studied the replication of virus in tissues and development of lesions associated with infectious salmon anemia virus (ISAV) infection in Atlantic salmon using in situ hybridization (ISH) with a riboprobe targeting ISAV RNA segment 7 messenger RNA. Fish were infected with three ISAV isolates (U55751, RPC-01-0593-1, Norway 810/9/99) and then euthanatized sequentially at 3, 6, 10, and 13 days postinoculation (dpi) and thereafter once a week for 8 weeks. Severe histopathologic lesions were observed in tissues from all groups beginning at the onset of mortality. The severe histopathologic lesions correlated with maximum intensity and frequency of ISH signals (P < 0.001). There was a strong association between the hybridization signals and severity of lesions in the liver, kidney, and heart (R = 0.81, 0.70, and 0.78, respectively; P < 0.001). The distribution of ISH signals indicated the presence of a viremia because signals were observed predominantly in individual blood cells and endothelial cells, and possibly hematopoietic cells of head kidney, but not in the necrotic hepatocytes and renal epithelium. Of the organs sampled, the heart was the first and last to show ISH signals, possibly because of increased activity of the endocardial endothelial cells and the underlining macrophages, which continuously trap and remove circulating virus, and therefore represents the best tissue sample for screening of suspected infected fish. On the basis of mortality, severity of lesions, and intensity and frequency of ISH signals, ISAV isolate Norway 810/9/99 was the most virulent and U5575-1 the least virulent isolate studied.

In vitro study of domoic acid uptake by gland tissue of blue mussel (Mytilus L.)

Abstract Domoic acid is a neurotoxic amino acid responsible for an outbreak of human food poisoning in late 1987 following consumption of contaminated blue mussels ( Mytilus edulis L.) from eastern Prince Edward Island, Canada. In vitro techniques were used to examine the uptake of domoic acid by digestive gland tissue of the blue mussel. Uptake of domoic acid over different periods of time by digestive gland tissue was compared with that of structurally related amino acids, glutamic add and kainic acid. Domoic acid uptake was observed to be a function of time and concentration. Uptake of glutamic acid, which is a physiological amino acid, was greater than that of domoic acid. Kainic add, a non-protein, rare amino acid, was least absorbed. In a series of experiments to determine the inhibitory actions of chemically similar and dissimilar amino acids, kainic acid, glutamic acid and proline inhibited the uptake of domoic add by 42, 38 and 34%, respectively, indicating competition for the same carrier site. Glycine, which showed least inhibition is likely to have a separate pathway. A marginal enhancing effect of ATP and slight inhibitory effects of metabolic inhibitors (NaCN and maleic acid) suggested that uptake of domoic add by digestive gland tissue is not totally an energy-dependent process.

Fish Disease and Biosecurity: Attitudes, Beliefs, and Perceptions of Managers and Owners of Commercial Finfish Recirculating Facilities in the United States and Canada

Abstract In recirculation finfish facilities in the United States and Canada, biosecurity utilization is neither consistent nor uniform. Seeking reasons for this situation, we examined the beliefs, perceptions, and attitudes of managers and owners of such facilities about fish disease and biosecurity utilization. A questionnaire was mailed in the fall of 2001 to the managers and owners of 152 finfish-rearing recirculation facilities in the United States and Canada to gather information on their attitudes, beliefs, and perceptions regarding fish disease and biosecurity. The response rate to the survey was 86%. This paper reports on a subset of the overall responses, the responses of managers and owners who identified their facility as a business operation. Although respondents had a positive attitude towards biosecurity utilization, they had different beliefs about which disease types were of greatest concern for their farm. Respondents also had differing perceptions on the risk of disease transmission ass...

Infectious salmon anemia virus RNA in fish cell cultures and in tissue sections of Atlantic salmon experimentally infected with infectious salmon anemia virus

Current understanding of the etiopathogenesis of infectious salmon anemia (ISA) virus (ISAV) infection in fish comes mostly from virus detection in homogenized tissues taken from ISA-suspected mortalities. This study combined in situ hybridization (ISH) and histology to demonstrate viral RNA transcripts in different fish cell lines infected with ISAV and in tissues collected during the clinical phase of ISAV infection in Atlantic salmon. For this, a riboprobe to mRNA transcripts of ISAV RNA segment 8 was shown to detect viral mRNA in ISAV-infected TO, CHSE-214, and SHK-1 cell cultures. Specific hybridization was initially detected exclusively in the nuclei of infected cells, which is consistent with the nuclear transcription of orthomyxoviruses. For use of the riboprobe on fish tissues fixed in paraformaldehyde or formalin, the conditions used to permeabilize tissues before ISH (Proteinase K or Tween 20) were first optimized. Tissues were collected 15—20 days after challenge from 7 fresh mortalities of Atlantic salmon parr (∼20 g) showing severe gross and microscopic lesions, consistent with ISAV infection. Reverse transcription-polymerase chain reaction on tissue pools confirmed the presence of ISAV in each of the 7 fish. Of the tissues examined in each fish, the heart and liver consistently showed the strongest hybridization signal and, therefore, the most in situ virus, which was located in the endothelium of small blood vessels and in macrophage-like cells.

Effects of a Prophylactic Chloramine-T Treatment on Growth Performance and Condition Indices of Rainbow Trout

Abstract Chloramine-T has been commonly used as a therapeutic agent for the treatment of bacterial gill disease and related phenomena in the salmonid aquaculture industry, and it is now commonly used as a prophylactic agent to prevent diseases in commercial salmonid hatcheries. Rainbow trout Oncorhynchus mykiss (average weight 98 g) were exposed to chloramine-T at 10 mg/L for 1 h twice weekly (a commonly used regime in the aquaculture industry) for 11 weeks. Fish were fed ad libitum without feed wastage throughout the 11 -week trial. Body weight, specific growth rate, feed conversion index, and appetite were assessed at the end of weeks 3, 6, 9, and 11. Chloramine-T treatment was not associated with either clinical disease or mortality. However, by the end of the trial, growth (based on body weight) of treated fish was significantly suppressed (δ = 7.3%) compared with control fish. Growth suppression was attributed to a significant reduction (δ = 9.3%) of feed conversion efficiency in treated fish. Based ...

A comparative study of uptake and release of glutamic acid and kainic acid by blue mussel (Mytilus edulis L.)

Abstract Glutamic acid (OA) and kainic acid (KA) are structurally related to domoic acid (DOM), a neurotoxic amino acid that caused human food poisoning in 1987, following consumption of toxic mussels from Prince Edward Island, Canada. To study the difference in response of mussels to a physiological amino acid (GA) and a non-physiological amino acid (KA), GA and KA were presented to mussels in dissolved form and as a food encapsulated in liposomes. Kainic acid was absorbed from solution to a lesser degree (0.4%), while GA was taken up more readily (1.1%). Both amino acids were absorbed across the gastrointestinal epithelium. Kainic acid was concentrated in the kidney, whereas glutamic acid showed equal distribution in all visceral tissues. Glutamic acid was steadily released from mussel tissues, whereas KA resisted depuration. Over 80% of intracellular KA was associated with the TCA soluble fraction of digestive gland tissue, while about 42% of GA was associated with the TCA insoluble fraction. Although KA is structurally related to GA, it was handled differently by mussels, but in the same way as DOM, suggesting that non-protein amino acids are recognized as undesirable compounds.