Paul R. Bowser

Effect of soybean protein on serological response, non-specific defense mechanisms, growth, and protein utilization in rainbow trout

Rainbow trout (Oncorhynchus mykiss) were fed chemically and immunologically defined diets containing either a low-temperature fish meal or soybean preparations containing high (SBM) or low levels of the globular antigenic proteins glycinin (G) and beta-conglycinin (BC) for 26 weeks. While trout fed both soybean preparations exhibited poorer growth than those fed fish meal, those fed the soya diet with high levels of G and BC showed the most significant growth diminution, elevated feed conversion and impaired utilization of dietary protein. Changes in intestinal morphology were also observed in fish fed the SBM diet, coinciding with the inferior nutritional performance. Several serologic and non-specific immune defense mechanisms were monitored. Cell counts showed increased leukocyte cell numbers as well as increased concentrations of plasma protein and immunoglobulin in the soya-fed fish. Increased neutrophil, monocyte and macrophage activity, as assessed by several oxidative radical production and phagocytic index assays, were higher in the soya-fed fish, possibly indicating an inflammatory or hypersensitivity response. The results of this study suggest that antigenic soya protein affects non-specific defense mechanisms, growth performance, and protein utilization in rainbow trout. Furthermore, it would appear that dietary sensitivity can be overcome by process removal of this immunologically characterized antigen in soybean protein. Serological and immunological assessments appear to be very useful criteria for evaluating various protein sources for fishes.

Distribution of an Invasive Aquatic Pathogen (Viral Hemorrhagic Septicemia Virus) in the Great Lakes and Its Relationship to Shipping

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus found in fish from oceans of the northern hemisphere and freshwaters of Europe. It has caused extensive losses of cultured and wild fish and has become established in the North American Great Lakes. Large die-offs of wild fish in the Great Lakes due to VHSV have alarmed the public and provoked government attention on the introduction and spread of aquatic animal pathogens in freshwaters. We investigated the relations between VHSV dispersion and shipping and boating activity in the Great Lakes by sampling fish and water at sites that were commercial shipping harbors, recreational boating centers, and open shorelines. Fish and water samples were individually analyzed for VHSV using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and cell culture assays. Of 1,221 fish of 17 species, 55 were VHSV positive with highly varied qRT-PCR titers (1 to 5,950,000 N gene copies). The detections of VHSV in fish and water samples were closely associated and the virus was detected in 21 of 30 sites sampled. The occurrence of VHSV was not related to type of site or shipping related invasion hotspots. Our results indicate that VHSV is widely dispersed in the Great Lakes and is both an enzootic and epizootic pathogen. We demonstrate that pathogen distribution information could be developed quickly and is clearly needed for aquatic ecosystem conservation, management of affected populations, and informed regulation of the worldwide trade of aquatic organisms.

SEASONAL PREVALENCE OF SKIN TUMORS FROM WALLEYE (STIZOSTEDION VITREUM) FROM ONEIDA LAKE, NEW YORK

A seasonal survey of skin tumor prevalence in walleyes (Stizostedion vitreum) was conducted during the ice-free period on Oneida Lake, New York in 1986. During the survey, 1,028 walleyes were collected and examined for the presence of lymphocystis disease, dermal sarcoma, discrete epidermal hyperplasia and diffuse epidermal hyperplasia. Skin growths were high in prevalence in early spring, low in prevalence during the summer, and again high in prevalence in the fall. Lymphocystis disease and dermal sarcoma were more frequently observed than either discrete or diffuse epidermal hyperplasia. Histologically, a moderate to severe inflammatory response was associated with dermal sarcoma in the early spring and late spring but not in the fall. Regardless of the time of year, varying degrees of inflammatory response were seen associated with lymphocystis disease. Samples were inadequate to assess seasonal trends in incidence of discrete and diffuse epidermal hyperplasia.

Comparison of Quantitative RT-PCR with Cell Culture to Detect Viral Hemorrhagic Septicemia Virus (VHSV) IVb Infections in the Great Lakes

Viral hemorrhagic septicemia virus (VHSV) is an important pathogen of cultured and wild fish in marine and freshwater environments. A new genotype, VHSV IVb, was isolated from a fish collected from the Great Lakes in 2003. Since the first isolation, VHSV IVb has been confirmed in 28 species, signaling the early invasion and continued spread of this Office International des Epizooties-reportable agent. For surveillance of this virus in both wild and experimental settings, we have developed a rapid and sensitive one-step quantitative real-time polymerase chain reaction (qRT-PCR) assay that amplifies a 100-base-pair conserved segment from both the genomic negative strand and the mRNA positive strand of the nucleoprotein (N) gene of VHSV IVb. This assay is linear over seven orders of magnitude, with an analytical capability of detecting a single copy of viral RNA and reproducibility at 100 copies. The assay is approximately linear with RNA input from 50 to 1000 ng per assay and works equally well with RNA prepared from a column-based or phenol-chloroform-based method. In wild-caught fish, 97% of the cases were found to be more than three orders of magnitude more sensitive using qRT-PCR than using cell culture. Of the 1,428 fish from the Great Lakes region tested in 2006 and 2007, 24% were positive by qRT-PCR whereas only 5% were positive by cell culture. All of the fish that were positive by cell culture were also positive by qRT-PCR. Importantly, qRT-PCR sensitivity is comparable to that of cell culture detection when comparing VHSV viral RNA levels with viral titer stocks, confirming that the high qRT-PCR signals obtained with diagnostic samples are due to the accumulation of N gene mRNA by transcriptional attenuation. The qRT-PCR assay is particularly valuable for rapid and high-throughput prescreening of fish before confirmatory testing by cell culture or sequencing tissue-derived amplicons and especially in detecting infection in fish that do not show clinical signs of VHS.

Blood chemistry of healthy, nephrocalcinosis-affected and ozone-treated tilapia in a recirculation system, with application of discriminant analysis

Blood chemistry parameters of Nile tilapia (pure strain Oreochromis niloticus) raised in a recirculation system were studied. Plasma samples were collected from 30 healthy tilapia every 3 months during a 1-year period. An additional 30 tilapia affected with nephrocalcinosis, and 30 fish from a system treated with ozone were also examined. Significant differences among healthy groups were observed in all parameters except for plasma magnesium, iron, albumin-to-globulin ratio, glucose, cholesterol, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphotase (AP) and creatine kinase (CK) activities. Nephrocalcinosis-affected tilapia differed significantly from healthy fish in most electrolyte and metabolite concentrations, as well as in ALT, AST and AP activities. Ozone-treated fish showed significant differences in electrolyte concentrations and ALT, AST and AP activities. Discriminant analysis revealed good separation of these groups. Evaluation of the data set indicated that the number of blood chemistry parameters could be reduced from 23 to 9 without losing the ability to separate the groups (90% and 92% correct, respectively). Most of the incorrect prediction came from less satisfactory separation of ozone-treated fish from healthy fish, due to the lack of apparent adverse impact on health of this treatment. Thirty tilapia from the same facility that were experiencing a low mortality rate were also used to test the model; individuals with abnormal blood chemistry profiles were identified.

Effects of Thiamine on Reproduction of Atlantic Salmon and a New Hypothesis for Their Extirpation in Lake Ontario

Abstract Previous researchers demonstrated that a mortality in fry (called Cayuga syndrome) of Atlantic salmon Salmo salar from Cayuga Lake (New York) was associated with low levels of thiamine. They reduced the mortality of fry by bathing or injecting fry with thiamine. We injected four to six gravid female Atlantic salmon with either physiological saline (PS) or PS plus thiamine (7 mg/kg weight) 14–23 d before eggs were stripped, fertilized, and incubated in individual lots. Chemical analyses showed that eggs from control and treated salmon contained 1.1 and 1.6 nmol thiamine/g, respectively. Thiamine injections had no significant effect on the percentage of eggs that hatched. Between 700 and 800 Celius degree-days postfertilization, control fry (saline) showed signs of Cayuga syndrome and a 45% incidence of mortality; in contrast, mortality was only 1.9% for fry that received thiamine. By 1,078 degree-days postfertilization, mean mortality of control fry was 98.6%, whereas that for thiamine-injected sa...

Emergence of viral hemorrhagic septicemia virus in the North American Great Lakes region is associated with low viral genetic diversity

Viral hemorrhagic septicemia virus (VHSV) is a fish rhabdovirus that causes disease in a broad range of marine and freshwater hosts. The known geographic range includes the Northern Atlantic and Pacific Oceans, and recently it has invaded the Great Lakes region of North America. The goal of this work was to characterize genetic diversity of Great Lakes VHSV isolates at the early stage of this viral emergence by comparing a partial glycoprotein (G) gene sequence (669 nt) of 108 isolates collected from 2003 to 2009 from 31 species and at 37 sites. Phylogenetic analysis showed that all isolates fell into sub-lineage IVb within the major VHSV genetic group IV. Among these 108 isolates, genetic diversity was low, with a maximum of 1.05% within the 669 nt region. There were 11 unique sequences, designated vcG001 to vcG011. Two dominant sequence types, vcG001 and vcG002, accounted for 90% (97 of 108) of the isolates. The vcG001 isolates were most widespread. We saw no apparent association of sequence type with host or year of isolation, but we did note a spatial pattern, in which vcG002 isolates were more prevalent in the easternmost sub-regions, including inland New York state and the St. Lawrence Seaway. Different sequence types were found among isolates from single disease outbreaks, and mixtures of types were evident within 2 isolates from individual fish. Overall, the genetic diversity of VHSV in the Great Lakes region was found to be extremely low, consistent with an introduction of a new virus into a geographic region with previously naive host populations.

Experimental Transmission of a Dermal Sarcoma in Fingerling Walleyes (Stizostedion vitreum vitreum)

Dermal sarcoma is a benign skin tumor of adult walleyes (Stizostedion vitreum vitreum) with a suspected viral etiology. A laboratory study was initiated to determine if the tumor could be experimentally transmitted by inoculating young walleyes with materials prepared from tumors from adult fish. Eighty walleye fingerlings were divided into four groups of 20 fish each. Two groups were inoculated intramuscularly at 4 months of age either with live tumor cells or with cell-free filtrates of sonicated tumor cells. The two other groups were used as controls and were inoculated either with cultured cells from normal walleye fry or with tissue culture media. Neoplasms, similar to the dermal sarcoma affecting adult walleyes, were observed after 4 months only in fingerlings inoculated with cell-free filtrates of sonicated tumor cells. Like the tumor affecting wild adult walleyes, the transmitted tumors were restricted to the dermis and originated from the superficial surface of scales. They never invaded locally and never metastasized. The transmitted tumors differed from tumors of adult walleyes in their severity and the absence of osteoid. The multicentric origin of transmitted walleye dermal sarcoma suggests that the virus spreads systemically and that tumor cells are polyclonal. This successful transmission of the lesion, along with the presence of C-type virus particles budding from tumor cells in two of seven tumor-bearing fingerlings, supports a retroviral etiology.

Histologic and Ultrastructural Studies of Dermal Sarcoma of Walleye (Pisces: Stizostedion vitreum)

Sixty-seven adult walleye fish were examined by light and transmission electron microscopy. The fish were affected by a mesenchymal tumor previously termed Walleye Dermal Sarcoma that commonly affects up to 27% of the population seasonally. Biopsies from 24 fish were collected, and complete postmortem examinations were performed on 43 fish. Grossly, the tumors had the appearance of randomly distributed, often clustered, spherical nodules, 2-5 mm in diameter with a smooth and often ulcerated surface. The tumors arose from the superficial surface of scales and consisted of fibroblast-like cells separated by a moderate amount of collagen (43/67) or osteoid material (24/67). Lymphocytic infiltration (28/67) associated with vacuolar degeneration of tumor cells (28/67) and centrally located coagulation necrosis (30/67) were observed. Although tumor cells were often highly anaplastic, no local invasions or metastases were present. In contrast with previous descriptions of this tumor, no viral particles could be observed electron microscopically. The variably anaplastic appearance of the tumor, its biological behavior, and its restriction to dermis are features in common with canine cutaneous histiocytoma and equine sarcoid. The multicentric origin, the restriction to the dermis, and the absence of invasion or metastases of Walleye Dermal Sarcoma differ from retrovirus-induced avian and murine sarcomas that arise locally, that invade, and that often metastasize.

An Unusual Koi Herpesvirus Associated with a Mortality Event of Common Carp Cyprinus carpio in New York State, USA

Koi herpesvirus (KHV), a highly contagious and lethal virus that affects both koi (Cyprinus carpio koi) and common carp (Cyprinus carpio), was isolated in 1998 from two outbreaks of koi suffering mass mortality in New York State, USA, and in Israel. The disease had been described as early as 1996 in Europe. In July 2004, this virus was found associated with a mass mortality event in wild common carp in the Chadakoin River, New York, USA (42°07′N, 79°W). Affected fish typically showed marked hyperplasia of gill tissues, abdominal adhesions, and severe multifocal to diffuse external hemorrhages. The virus isolated in this outbreak was somewhat unusual in that it initially replicated well in fathead minnow cell cultures, which is typical of spring viremia of carp virus. Testing at the National Veterinary Services Laboratories, Ames, Iowa, USA, confirmed the viruss identity to be KHV. Koi herpesvirus is not currently on the OIE (World Organisation for Animal Health) list of notifiable diseases; however, it is capable of causing mass mortality in susceptible fish at permissive temperatures.