Rodman G. Getchell

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.

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.

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.

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.

Influence of limnological conditions on Clostridium botulinum type E presence in Eastern Lake Erie Sediments (Great Lakes, USA)

Avian and fish botulism outbreaks have been recorded since 1999 in eastern Lake Erie. These outbreaks are caused by Clostridium botulinum type E, a toxin-producing bacteria that is found in anoxic substrates rich in organic material. We studied the environmental conditions present in eastern Lake Erie during 2002, a year when several botulism outbreaks were observed. We also tested for the presence of C. botulinum type E in lake sediments. Samples were taken at six stations from two sites of different depths in the Dunkirk (New York, USA) area. The depth of the sampling sites influenced physico-chemical and biological processes in the sediments. We used the quantitative polymerase chain reaction (Q-PCR) to quantify the levels of C. botulinum type E in the samples. Sediment samples contained a patchy distribution of type E spore concentrations (from not detectable to 5520 DNA copies/mg). Samples of benthic invertebrates tested positive for C. botulinum type E spores in tissues (Gammarus 2028 DNA copies/mg, oligochaetes 428 DNA copies/mg, chironomids 148 DNA copies/mg and dreissenid mussels 715 DNA copies/mg). Principal components analysis (PCA) from inshore stations indicated that a decrease in dissolved oxygen, pH and redox potential near the sediment was associated to an increase in specific conductance and the type E toxin gene in sediments. We also found that C. botulinum type E spores are present in sediments at different depths and at different times through the ice-free season.

Reproductive Failure of Landlocked Atlantic Salmon from New York's Finger Lakes: Investigations into the Etiology and Epidemiology of the “Cayuga Syndrome”

Abstract We describe a disease syndrome that afflicts larval, landlocked Atlantic salmon Salmo salar from Cayuga Lake, one of central New Yorks Finger Lakes. Mortality associated with the “Cayuga syndrome” is 98–100%. Death usually occurs between 650 and 850 centigrade degreedays after fertilization, approximately 2–4 weeks before yolk resorption is complete. Although there is minor temporal variation in the onset of the Cayuga syndrome in progeny from individual females, all sac fry eventually succumb. Incubation of embryos and sac fry under constant, ambient, or reduced temperature regimens slightly alters the degree-day timing of syndrome onset, but does not improve survival. Based on mortality rate, manifestation of the Cayuga syndrome has not changed in the past 10 years, even though incubation waters of varying chemistry and temperature have been used. Mortality of the negative control stocks used for these studies never exceeded 10% from hatching to first feeding. Findings from reciprocal crossbre...

Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic laboratory setting

Real-time, or quantitative, polymerase chain reaction (qPCR) is quickly supplanting other molecular methods for detecting the nucleic acids of human and other animal pathogens owing to the speed and robustness of the technology. As the aquatic animal health community moves toward implementing national diagnostic testing schemes, it will need to evaluate how qPCR technology should be employed. This review outlines the basic principles of qPCR technology, considerations for assay development, standards and controls, assay performance, diagnostic validation, implementation in the diagnostic laboratory, and quality assurance and control measures. These factors are fundamental for ensuring the validity of qPCR assay results obtained in the diagnostic laboratory setting.

Seasonal Occurrence of Virally Induced Skin Tumors in Wild Fish

Abstract Seasonal trends in the prevalence of virally induced skin tumors are reviewed for a variety of wild fish with emphasis on neoplasms in the walleye Stizostedion vitreum. The relationship of temperature to mechanisms of virally induced tumorigenesis and regression in fish is also summarized. Particular emphasis is placed on teleost physiological parameters such as the immune response and endocrinological levels. Other elements of a multifactorial hypothesis for seasonal tumor fluctuations in fish are also discussed.

Oxytetracycline Residues in Four Species of Fish after 10-Day Oral Dosing in Feed

Abstract Residues of oxytetracycline (OTC) in serum, liver, and muscle–skin were measured in sunshine bass (female white bass Morone chrysops × male striped bass M. saxatilis), Nile tilapia Oreochromis niloticus, summer flounder Paralichthys dentatus, and walleyes Sander vitreus (formerly Stizostedion vitreum). These studies were conducted to compare the time for OTC concentrations to fall below 2.0 μg/g (the current tolerance set by the U.S. Food and Drug Administration (USFDA)) among fish species representing various culture conditions of freshwater and salt water as well as different temperature niches. The sum of mean tissue OTC concentration plus two standard deviations in the edible portion (muscle plus skin) of all fish was below 2.0 μg/g at 11 d postdosing. When possible, depletion data were fitted to a one-compartment pharmacokinetic model. Although temperature niche did not appear to consistently affect residue depletion within species, the warmwater species (sunshine bass and Nile tilapia) ofte...