Epizootic neoplasia of the lateral line system of lake trout (Salvelinus namaycush) in New York's Finger Lakes.

This article documents an epizootic of inflammation and neoplasia selectively affecting the lateral line system of lake trout (Salvelinus namaycush) in 4 Finger Lakes in New York from 1985 to 1994. We studied more than 100 cases of this disease. Tumors occurred in 8% (5/64) of mature and 21% (3/14) of immature lake trout in the most severely affected lake. Lesions consisted of 1 or more neoplasm(s) in association with lymphocytic inflammation, multifocal erosions, and ulcerations of the epidermis along the lateral line. Lesions progressed from inflammatory to neoplastic, with 2-year-old lake trout showing locally extensive, intense lymphocytic infiltrates; 2- to 3-year-old fish having multiple, variably sized white masses up to 3 mm in diameter; and fish over 5 years old exhibiting 1 or more white, cerebriform masses greater than 1 cm in diameter. Histologic diagnoses of the tumors were predominantly spindle cell sarcomas or benign or malignant peripheral nerve sheath neoplasms, with fewer epitheliomas and carcinomas. Prevalence estimates did not vary significantly between sexes or season. The cause of this epizootic remains unclear. Tumor transmission trials, virus isolation procedures, and ultrastructural study of lesions failed to reveal evidence of a viral etiology. The Finger Lakes in which the disease occurred did not receive substantially more chemical pollution than unaffected lakes in the same chain during the epizootic, making an environmental carcinogen an unlikely primary cause of the epizootic. A hereditary component, however, may have contributed to this syndrome since only fish of the Seneca Lake strain were affected.

Immunohistochemistry and pathology of multiple Great Lakes fish from mortality events associated with viral hemorrhagic septicemia virus type IVb.

A novel viral hemorrhagic septicemia virus (VHSV) (genotype IVb) has been isolated from mortality events in a range of wild freshwater fish from the Great Lakes since 2005. In 2005 and 2006, numerous new freshwater host species (approximately 90 fish from 12 different species) were confirmed to have VHSV by cell culture and reverse transcriptase polymerase chain reaction. A prominent feature observed in infected fish were the petechial and ecchymotic haemorrhages on the body surface and in visceral organs, as well as serosanguinous ascites; however, many fish had few and subtle, gross lesions. Histologically, virtually all fish had a vasculitis and multifocal necrosis of numerous tissues. Excellent correlation was found between the presence of VHSV IVb antigen detected by immunohistochemistry and the pathological changes noted by light microscopy. Intact and degenerate leukocytes, including cells resembling lymphocytes and macrophages, also had cytoplasmic viral antigen. By contrast, renal tubules and gonadal tissues (ovary and testis), were strongly immunopositive for VHSV IVb, but no lesions were noted.

Florfenicol residues in three species of fish after 10-day oral dosing in feed.

Nile tilapia Oreochromis niloticus, walleye Sander vitreus, and hybrid striped bass (female white bass Morone chrysops x male striped bass M. saxatilis) were medicated with florfenicol (AQUAFLOR type A medicated article; Schering-Plough Animal Health, Summit, New Jersey) via a medicated ration of 10 mg florfenicol x kg fish body weight(-1) d(-1) for 10 d to compare the elimination kinetics of the test article. This study was part of a larger effort in support of a species grouping concept that could contribute to the regulatory approval process for therapeutic compounds for cultured fishes. The trials in this study were conducted at the ideal water temperature for each species and at the temperature 5 degrees C lower than the ideal. The test temperatures were 30 degrees C and 25 degrees C for Nile tilapia, 25 degrees C and 20 degrees C for both walleyes and hybrid striped bass. In all cases, the elimination kinetics of florfenicol were more rapid at higher temperatures. The time to reach the tolerance of 1 microg/g in muscle-skin, as set by the U.S. Food and Drug Administration for channel catfish Ictalurus punctatus and salmonids, ranged from 6.1 to 4.1 d for Nile tilapia, from 12.6 to 9.7 d for walleyes, and from 2.6 to 0.7 d for hybrid striped bass at temperatures between 20 degrees C and 30 degrees C.

Florfenicol residues in Nile tilapia after 10-d oral dosing in feed: effect of fish size.

Nile tilapia Oreochromis niloticus were medicated with florfenicol (AQUAFLOR type A medicated article; Schering-Plough Animal Health, Summit, New Jersey) via a medicated ration at 15 mg florfenicol x kg fish body weight(-1) d(-1) for 10 d to compare the elimination kinetics of the test article in different size fish held at 25 degrees C. The groups of fish used in the study had mean weights of approximately 100, 250, and 500 g. In each trial, the fish were provided the medicated ration and 15 fish were processed at each of seven time points postfeeding for determination of the florfenicol concentration in serum and the florfenicol residue in the edible portion (composite muscle and skin). There was a trend toward shorter half-lives of elimination in the smaller fish. The elimination times in muscle-skin (times to reach the established tolerance concentration for channel catfish Ictalurus punctatus and salmonids of 1.0 microg florfenicol residue/g) and half-lives were 9.2 and 1.2 d (100 g), 8.6 and 1.7 d (250 g), and 12.7 and 2.2 d (500 g), respectively.

A survey to determine the presence and distribution of largemouth bass virus in wild freshwater bass in New York State.

During 2004 and 2005 a survey was conducted to investigate the presence and geographic distribution of largemouth bass virus (LMBV) in New York State. This iridovirus is widely distributed across the eastern United States; however, it had not previously been reported in New York State. Two hundred and eighty-three wild largemouth bass Micropterus salmoides and 8 smallmouth bass M. dolomieu were collected from 37 locations across the state. No clinical signs of LMBV or mortalities attributable to the virus were observed in the fish collected. Using a quantitative polymerase chain reaction (QPCR) method, we detected LMBV in 28 fish from 13 locations. Viral cytopathic effect in cell culture was observed in 5 fish from 3 locations. The virus isolated from cell culture was confirmed to be LMBV by an independent PCR method. Statistical analysis of the largemouth bass samples collected during 2005 revealed a wide difference in prevalence between the QPCR results and the cell culture results. Analysis of possible predictors, including age, sex, and month collected, showed no significant associations with the QPCR results. This survey confirms the presence and wide distribution of a potentially pathogenic form of LMBV in multiple water systems across New York State.

Detection of viral hemorrhagic septicemia in round gobies in New York State (USA) waters of Lake Ontario and the St. Lawrence River.

In May 2006 a large mortality of several thousand round gobies Neogobius melanostomus (Pallas, 1814) occurred in New York waters of the St. Lawrence River and Lake Ontario. Necropsies of sampled fish from these areas showed pallor of the liver and gills, and hemorrhagic areas in many organs. Histopathologic examination of affected tissues revealed areas of necrosis and hemorrhage. Inoculations of fathead minnow Pimephales promelas (Rafinesque, 1820) cell cultures with dilutions of tissue samples from the necropsied gobies produced a cytopathic effect within 5 d post-inoculation. Samples of cell culture supernatant were tested using RT-PCR and confirmed the presence of viral hemorrhagic septicemia virus (VHSV). Sequence analysis of the VHSV isolate resulted in its assignment to the type-IVb subgroup. The detection of VHSV in a relatively recent invasive fish species in the Great Lakes and the potential impact of VHSV on the ecology and economy of the area will require further investigation and careful management considerations.

Sulfadimethoxine and ormetoprim residues in three species of fish after oral dosing in feed.

Nile tilapia Oreochromis niloticus, summer flounder Paralichthys dentatus, and walleyes Sander vitreus were treated with Romet-30 (PHARMAQ AS, Oslo, Norway) via a medicated ration at 50 mg Romet-30 kg fish body weight(- 1) d(-1) for 10 d to compare the elimination kinetics of the test substance. This study was part of a larger effort to develop a species grouping concept for the labeling of therapeutic compounds for cultured fishes. The fish tests were conducted at the ideal water temperature for each species and at 5 degrees C lower than the ideal temperature except for summer flounder, which would not feed at the lower temperature of 15 degrees C. Test temperatures were 30 degrees C and 25 degrees C for Nile tilapia, 20 degrees C and 17 degrees C for summer flounder, and 25 degrees C and 20 degrees C for walleyes. Neither component of Romet-30 (sulfadimethoxine and ormetoprim) could be detected in samples of the edible portion of walleyes (muscle plus skin) collected at day 10 posttreatment or thereafter. In studies with summer flounder, only one fish had a detectable concentration of either component on day 21 or thereafter. Elimination of Romet-30 by Nile tilapia was extremely rapid. The limited number of Nile tilapia with detectable sulfadimethoxine or ormetoprim during the posttreatment period prevented the determination of elimination half-life or elimination in this species.

Quantitative polymerase chain reaction assay for largemouth bass virus.

The use of quantitative polymerase chain reaction (QPCR) to test for largemouth bass virus (LMBV) was evaluated during a challenge experiment in which largemouth bass Micropterus salmoides were immersed in the type strain of LMBV. The real-time PCR and cell culture methods were both used to measure LMBV present in the inoculum. Additional samples tested by QPCR included gill, gonad, kidney, liver, mucus, spleen, and swim bladder. A plasmid clone containing a 248-base pair (bp) fragment of the major capsid protein gene (MCP*) was serially diluted and used as a standard to quantify the number of LMBV DNA copies present in the samples tested. A 62-bp fragment of DNA located in MCP* was amplified in the real-time PCR assay. This work has demonstrated the value of the QPCR assay in LMBV surveys.

Dose Titration of Walleye Dermal Sarcoma (WDS) Tumor Filtrate.

Walleyes Stizostedion vitreum were challenged with a topical application of a dilution series of cell-free dermal sarcoma tumor filtrates to determine the minimum dose of virus needed to induce these walleye tumors. A series of six 10-fold dilutions of the filtrate were applied to the side of the fish, which were allowed to develop grossly visible tumors at 15°C for 20 weeks. Quantification of the virus in the filtrates was accomplished by quantitative (real-time) reverse transcriptase-polymerase chain reaction. We determined that there are approximately 10(10) viral RNA copies in 100 µL of walleye dermal sarcoma inoculum. The minimum dose of walleye dermal sarcoma virus that could induce tumors by the topical challenge method was the 1,000-fold dilution of this 10(10) inoculum, or approximately 10(7) viral RNA copies.

Dose Titration of Walleye Dermal Sarcoma (WDS) Tumor Filtrate.

Walleyes Stizostedion vitreum were challenged with a topical application of a dilution series of cell-free dermal sarcoma tumor filtrates to determine the minimum dose of virus needed to induce these walleye tumors. A series of six 10-fold dilutions of the filtrate were applied to the side of the fish, which were allowed to develop grossly visible tumors at 15°C for 20 weeks. Quantification of the virus in the filtrates was accomplished by quantitative (real-time) reverse transcriptase-polymerase chain reaction. We determined that there are approximately 1010 viral RNA copies in 100 µL of walleye dermal sarcoma inoculum. The minimum dose of walleye dermal sarcoma virus that could induce tumors by the topical challenge method was the 1,000-fold dilution of this 1010 inoculum, or approximately 107 viral RNA copies.

Reporter gene expression in fish following cutaneous infection with pantropic retroviral vectors.

A central issue in gene delivery systems is choosing promoters that will direct defined and sustainable levels of gene expression. Pantropic retroviral vectors provide a means to insert genes into either somatic or germline cells. In this study, we focused on somatic cell infection by evaluating the activity of 3 promoters inserted by vectors into fish cell lines and fish skin using pantropic retroviruses. In bluegill and zebrafish cell lines, the highest levels of luciferase expression were observed from the 5' murine leukemia virus long terminal repeat of the retroviral vector. The Rous sarcoma virus long terminal repeat and cytomegalovirus early promoter, as internal promoters, generated lower levels of luciferase. Luciferase reporter vectors infected zebrafish skin, as measured by the presence of viral DNA, and expressed luciferase. We infected developing walleye dermal sarcomas with retroviral vectors to provide an environment with enhanced cell proliferation, a condition necessary for integration of the provirus into the host genome. We demonstrated a 4-fold to 7-fold increase in luciferase gene expression in tumor tissue over infections in normal walleye skin.