Xenoma formation during microsporidial gill disease of salmonids caused by Loma salmonae is affected by host species (Oncorhynchus tshawytscha, O. kisutch, O. mykiss) but not by salinity.

Host species and salinity often affect the development of disease in aquatic species. Eighty chinook salmon Oncorhynchus tshawytscha, 80 coho salmon O. kisutch and 80 rainbow trout O. mykiss were infected with Loma salmonae. Forty of each species were reared in seawater and 40 in freshwater. The mean number of xenomas per gill filament was 8 to 33 times greater in chinook salmon than in rainbow trout (RBT). Coho salmon had a mean xenoma intensity intermediate to that of chinook salmon and RBT. In contrast to the differences between species, salinity had no significant effect on xenoma intensity in any of these host species. The onset of xenoma formation occurred at Week 5 postexposure (PE) for chinook salmon and RBT, and at Week 6 PE for coho salmon. RBT had cleared all visible branchial xenomas by Week 9 PE, whereas xenomas persisted in coho and chinook salmon at Week 9 PE. Histologically, xenomas were visible in the filament arteries of the branchial arch in chinook and coho salmon gills but were absent from RBT gills. Fewer xenomas were seen in the central venous sinusoids of RBT than in chinook and coho salmon. The lower xenoma intensity, shorter duration of infection and pathological characteristics, common to microsporidial gill disease in RBT, suggest a degree of resistance to clinical disease that is not seen in coho and chinook salmon.

Resistance to reinfection in chinook salmon Oncorhynchus tshawytscha to Loma salmonae (Microsporidia).

Chinook salmon Oncorhynchus tshawytscha were experimentally infected per os with Loma salmonae and held in flow-through seawater tanks at 12 to 14 degrees C. The fish exhibited 100% infection when first examined at 7 wk post initial exposure (p.e.), and by 20 wk p.e. they had completely recovered from gill infections. The recovered fish were then re-exposed the following week. All of these fish showed strong protection to new L. salmonae infections, while naïve fish exposed to the same inoculum developed the infection. Most of the re-exposed fish exhibited a few free spores or spores within phagocytes in the kidney interstitium at 20 to 29 wk p.e., but xenomas were not detected in either the gills or visceral organs. The kidney is the primary site of reticulo-endothelial activity, and thus these spores were likely deposited in the kidney by entrapment by fixed macrophages. It is possible that these spores provide immunologic stimuli to reinforce the resistance to new L. salmonae infections.

Efficacy of the fumagillin analog TNP-470 for Nucleospora salmonis and Loma salmonae infections in chinook salmon Oncorhynchus tshawytscha.

Oral treatment with fumagillin is effective for controlling various microsporean and myxosporean infections in fish. We tested a synthetic analog of fumagillin, TNP-470 (Takeda Chemical Industries), for its efficacy against 2 microsporean pathogens of salmon: Loma salmonae and Nucleospora salmonis. Chinook salmon Oncorhynchus tshawytscha were experimentally infected with either L. salmonae (per os) or N. salmonis (intraperitoneal, i.p., injection) and held in fresh water at 15 degrees C. Fish were then divided into 3 replicate groups: untreated or treated orally at 1.0 mg or at 0.1 mg drug kg-1 fish d-1. With L. salmonae, the high dose fish had 0.32 xenomas mm-2 of gill tissue compared to controls at 24.5 xenomas per mm2. With N. salmonis infections, untreated fish exhibited 100% infection, showed prominent clinical signs (e.g. renal swelling, anaemia), and high mortality. In contrast, fish treated at 1.0 mg kg-1 showed no clinical signs, and 16% of those treated at 0.1 mg kg-1 showed only mild gross pathological changes. With the treated groups, over 50% of the fish exhibited extremely light infections, even with high dose treatments, but no mortalities were attributed to N. salmonis infections. Uninfected fish treated at 1.0 mg drug kg-1 fish d-1 for 5 wk appeared clinically normal and showed no reduction in growth. However, about half of these fish exhibited atrophy of the renal interstitial hematopoietic tissue.

Parvicapsula minibicornis n. sp. (Myxozoa, Myxosporea) from the kidney of sockeye salmon (Oncorhynchus nerka) from British Columbia, Canada.

A new species of Myxosporea Parvicapsula minibicornis is described from the kidney of adult sockeye salmon (Oncorhynchus nerka) that had recently returned from the Pacific Ocean to Weaver Creek, a tributary of the Fraser River, British Columbia, Canada. Spores are ovoid, symmetrical, with 2 pyriform polar capsules at the anterior pole. The posterior pole has 2 small, pointed projections. Mean spore dimensions are length 11.0 microm, thickness (perpendicular to suture plane) 6.8 microm, and width (in sutural plane) 7.5 microm. This myxozoan is compared to other described Parvicapsula species and a Parvicapsula sp. from netpen-reared coho salmon (Oncorhynchus kisutch).

14C-labeled microcystin-LR administered to Atlantic salmon via intraperitoneal injection provides in vivo evidence for covalent binding of microcystin-LR in salmon livers.

The tissue distribution and clearance of radiolabeled microcystin-LR administered to Atlantic salmon via i.p. injection has been re-examined using uniformly 14C-labeled toxin. Significant differences were found to exist between these results and those obtained when fish received an i.p. injection of tritium-labeled dihydromicrocystin-LR. In addition, MeOH liver extracts were assayed by both phosphatase assay and 14C counts and the results compared with the total levels of incorporation determined by digestion and subsequent 14C counting of the same live tissues. An attempt to investigate the metabolism and to document the putative products was also undertaken. It was found that microcystin-LR was extensively metabolized to compounds that are more polar than the parent compound.

Evidence for a covalently bound form of microcystin-LR in salmon liver and Dungeness crab larvae.

The chemically unique nature of the C20 beta-amino acid (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6- dienoic acid (Adda) portion of the microcystins has been exploited to develop a strategy to analyze for the total microcystin-LR (1; see Figure 1) burden in salmon liver and crab larvae tissues. Lemieux oxidation of microcystin-LR (1) gives 2-methyl-3-methoxy-4-phenylbutanoic acid (2), a unique marker for the presence of microcystins. The butanoic acid 2 can be isolated and detected by GC/MS from the livers of Atlantic salmon that received an ip injection of microcystin-LR (1) and from tissues of wild-caught crab larvae. The Lemieux oxidation-GC/MS results are compared with those from MeOH extraction-PPase analysis. Only approximately 24% of the total microcystin-LR (1) burden in salmon liver tissue is found to be extractable with MeOH. Similarly, the Lemieux oxidation-GC/MS method detected 10,000-fold greater microcystin concentrations in Cypress Island Dungeness crab larvae than did the MeOH extraction-PPase method. The disparity in microcystin concentrations measured by the two methods is taken as direct evidence for the existence of covalently bound microcystins in vivo.

Bioaccumulation and clearance of microcystins from salt water mussels, Mytilus edulis, and in vivo evidence for covalently bound microcystins in mussel tissues.

Over a period of 3 days saltwater mussels, Mytilus edulis, were fed a cyanobacteria, Microcystis aeruginosa, that contained a high concentration of microcystins. The mussels were killed on a periodic basis over the course of 2 months. Mussels were also collected at two sites were high levels of microcystins in tissues had been noted. A strategy based on the chemically unique nature of the C20 beta-amino acid, (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6- dienoic acid (Adda), portion of the microcystins was used in conjunction with a protein phosphatase (PPase) assay to analyse for both covalently bound microcystins and free microcystins in the mussel tissues. The mussel PPase assay results were compared with the Lemieux oxidation gas chromatography-mass spectrometry (GCMS) analysis. Less than 0.1% of the total microcystin burden in the mussel tissue was found to be extractable with MeOH. Thus, direct evidence was provided for the existence of covalently bound microcystins in mussel tissues in vivo. The mussels rapidly cleared the covalently bound microcystins when transferred to untreated seawater. Within 4 days the total microcystin burden dropped from a high of 336.9 (+/- 45.8) micrograms/g wet tissue to 11.3 (+/- 2.6) micrograms/g. After 4 days postexposure until completion of the experiment the total levels remained below the detection limits of the GCMS method. The levels of free microcystins, extracted with MeOH and detected by the PPase assay, fell from 204 ng/g wet tissue to a residual 14 ng/g over a 53 day postexposure period. Presumably the bound microcystin present in the mussel tissue exists as a covalent complex with the PP-1 and PP-2A enzymes. We conclude that in any shellfish monitoring program it is the total tissue microcystin burden that needs to be considered.

Transmission of Loma salmonae (Microsporea) to chinook salmon in sea water.

Transmission studies were conducted to determine if Loma salmonae was transmissible in sea water. Transmission of L. salmonae to chinook salmon (Oncorhynchus tshawytscha) held in sea water was achieved by exposing fish to macerated, infected gill tissue. Fish were exposed in seawater in a flow-through aquarium, and the infection was detected as soon as 5 wk after exposure. Heavily infected fish exhibited numerous xenomas in the branchial arteries, central venous sinusoids, and within the blood channels of the lamellae. The pathological changes were similar to those seen in pen-reared salmon with L. salmonae infections. The infection was not observed in Atlantic salmon (Salmo salar), Pacific herring (Clupea pallasi, family Clupeidae), or shiner perch (Cymatogaster aggregata, family Embiotocidae), experimentally exposed using identical methods. This study suggests that L. salmonae is transmissible to chinook salmon in seawater netpens. Fish farmers and fish health specialists should consider this possibility when developing and implementing strategies to control the infection.

Experimental transmission of a plasmacytoid leukemia of chinook salmon, Oncorhynchus tshawytscha.

A plasmacytoid leukemia of chinook salmon, Oncorhynchus tshawytscha, has recently been recognized in seawater netpens in British Columbia, Canada. The disease has occurred at several sites and has caused high mortality. Plasmacytoid leukemia is characterized by a generalized invasion of visceral tissues and the orbit of the eye by plasmacytoid cells. The disease was experimentally transmitted to healthy chinook salmon by i.p. injection of kidney tissue homogenates, but transmission with a cell-free filtrate was equivocal. In another experiment, chinook salmon, coho salmon, O. kisutch, sockeye salmon, O. nerka, rainbow trout, O. mykiss (or Salmo gairdneri), and Atlantic salmon, Salmo salar, were given injections of a tissue homogenate from affected chinook salmon. Ten wk after exposure, plasmacytoid leukemia was observed in all of the sockeye salmon and chinook salmon, one of ten Atlantic salmon, and none of the rainbow trout. Seven of the ten coho salmon examined at 10 wk had lesions suggestive of early development or a mild form of the disease. Multifocal areas of proliferating cells resembling plasmablasts were observed in the visceral fat, and the kidneys exhibited mild to moderate hyperplasia of the hematopoietic interstitium. Our studies support the hypothesis of an infectious etiology for plasmacytoid leukemia, but the agent, perhaps an oncogenic virus, has yet to be detected.