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The direct fluorescent antibody technique (FAT) was successfully used to detect the causative agent of bacterial kidney disease (BKD), Renibacterium salmoninarum, in Bouin's solution flexed and paraffinembedded egg and tissue sections. This method is superior to gram stain and may be particularly useful in detecting the BKD organism in fish with low-grade infection.
A hybridoma cell line that continually secretes antibody against infectious hematopoietic necrosis virus was developed and designated M-IHNV-W1. Antibodies in culture fluids were concentrated and purified by Protein A Sepharose CL-4B affinity chromatography. The immunoglobulin was isotyped as IgG 2b with a kappa light chain. The IgG was specific for infectious hematopoietic necrosis virus and had binding activity but low neutralizing activity. Little cross-reactivity occurred with viral hemorrhagic septicemia or infectious pancreatic necrosis viruses.
A cell line from rainbow trout ( Oncorhynchus mykiss ) with phagocytic-like characteristics was established. First we found that it was necessary to inject the fish with antibiotics (penicillinstreptomycin) in order to clear tissue of bacterial contamination. Two days later, the spleen was excised and minced with scissors in MEM10. The large number of erythrocytes in the suspension were removed by low centrifugation, then the tissue fragments were incubated on tissue culture flasks at 18°C for 30 days with complete changes of media every other day. After 30 days in culture, primary explants from spleen were composed of three types of cells. The majority were large mononucleate, plecmorphic cells with thinly spread cytoplasm. These cells were trypsinized and subcultured. Another cell types was simple, round cells, somewhat similar to mature lymphocytes, while the third type appeared to be similar to fibroblastic cells. After 9 months with the culture in 17 to 20 passages, the predominate cell type was showing similarities to phagocytic cell. These cells had a marked reticular cytoplasm and grew individually without parallel orientations. By enzyme-specific stains, the cells were observed to have abundant acid phosphatase and β-glucuronidase activities of the cytoplasm. They were negative for peroxidase. The cells were also found to have highly adherent and phagocytic properties when incubated with latex particles. The morphological, enzymatic, and functional findings suggested the possibility that these cells in culture have some properties similar to the cells which are composed of reticulo-endothelial system.
Genomic RNA was extracted from purified virions of hirame rhabdovirus (HRV), infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV). The full-length RNA was analyzed using formaldehyde agarose gel electrophoresis followed by ethidium bromide staining. Compared with an internal RNA size standard, all three viral genomic RNAs appeared to have identical relative mobilities and were estimated to be approximately 10.7 kilobases in length or about 3.7 megadaltons in molecular mass. Structural protein synthesis of HRV, IHNV, and VHSV was studied using cell cultures treated with actinomycin D. At 2 h intervals, proteins were labeled with 35 S-methionine, extracted, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The five structural proteins of each of the three viruses appeared in the following order : nucleoprotein (N), matrix protein 1 (M1), matrix protein 2 (M2), glycoprotein (G), and polymerase (L) reflecting both the approximate relative abundance of each protein within infected cells and the gene order within the viral genome.
Mortalities of yellowtail Seriola quinqueradiata artificially infected with Lactococcus garvieae and of rainbow trout Oncorhynchus mykiss artificially infected with Vibrio anguillarum were compared with the levels of plasma components measured prior to challenge. The levels of plasma total cholesterol, free cholesterol and phospholipid of fish surviving infection were significantly higher in both yellowtail and rainbow trout than those of fish which died during the challenge test. Mortality of yellowtail with plasma total cholesterol levels lower than 250 mg/100 ml was significantly higher than that of fish which had cholesterol levels higher than 275 mg/100 ml (p < 0.05). Rainbow trout whose cholesterol was lower than 520 mg/100 ml suffered a significantly higher mortality due to vibriosis than fish having cholesterol levels higher than 560 mg/100 ml (p < 0.005). These results indicate that low levels of plasma lipid components may be an indicator of lowered disease resistance in cultured fish.
The Charles River Project provided an opportunity to simultaneously deploy a combination of biomonitoring techniques routinely used by the U.S. Geological Survey National Water Quality Assessment Program, the Biomonitoring of Environmental Status and Trends Project, and the Contaminant Biology Program at an urban site suspected to be contaminated with polycyclic aromatic hydrocarbons. In addition to these standardized methods, additional techniques were used to further elucidate contaminant exposure and potential impacts of exposure on biota. The purpose of the study was to generate a comprehensive, multi-metric data set to support assessment of contaminant exposure and effects at the site. Furthermore, the data set could be assessed to determine the relative performance of the standardized method suites typically used by the National Water Quality Assessment Program and the Biomonitoring of Environmental Status and Trends Project, as well as the additional biomonitoring methods used in the study to demonstrate ecological effects of contaminant exposure. The Contaminant Effects Workgroup, an advisory committee of the U.S. Geological Survey/Contaminant Biology Program, identified polycyclic aromatic hydrocarbons as the contaminant class of greatest concern in urban streams of all sizes. The reach of the Charles River near Watertown, Massachusetts, was selected as the site for this study based on the suspected presence of polycyclic aromatic hydrocarbon contamination and the presence of common carp ( Cyprinus carpio ), largemouth bass ( Micropterus salmoides ), and white sucker ( Catostomus commersoni ). All of these fish have extensive contaminant-exposure profiles related to polycyclic aromatic hydrocarbons and other environmental contaminants. This project represented a collaboration of universities, Department of the Interior bureaus including multiple components of the USGS (Biological Resources Discipline and Water Resources Discipline Science Centers, the Contaminant Biology Program, and the Status and Trends of Biological Resources Program), and the U.S. Fish and Wildlife Service. Samples for analyzing water chemistry, sediment chemistry and toxicity, fish community structure, tissue chemistry, and fish (20 carp, 20 bass, and 40 white sucker) and invertebrate pathology were collected in late August, 2005. This report provides results from the analyses of fish pathology, biomarkers of exposure and effects (reproductive, carcinogenic, genotoxic, and immunologic), sediment chemistry, toxicity, and fish and invertebrate community structure.
No abstract available at this time
In complementary studies of the chronic effects of heptachlor on bluegills, four similar earthen ponds were treated once with different concentrations of heptachlor, and the fish in six plastic pools were routinely fed different levels of heptachlor. A fifth pond and two plastic pools were used as controls. Higher dosages resulted in higher concentrations of heptachlor residues, more pronounced tissue pathology, and higher mortality in the test fish. Heptachlor reached a maximum in the pond fish about 3 days after treatment, and could no longer be found in the fish or mud after 56 days. The fish fed continuous levels of heptachlor developed their highest residues at 56 days. Invertebrate populations in the two higher treatment level ponds were eliminated. Lesser effects appeared in the two lower concentrations. At the termination of the experiment, invertebrate populations in the treated ponds and the control were not significantly different, and had achieved or surpassed pretreatment levels. Degenerative liver lesions became pronounced in fish from the two higher exposure ponds, but a heavy infestation of trematode metacercariae in the fish of all the test ponds, except the control, clouded further interpretation of histopathological examinations. Pathology results from the fish in the feeding test, although inconclusive, tended to support the trends which were developing in the contact test. There was an inverse proportional relationship between growth of fish in the feeding test and the amount of heptachlor introduced. The control group showed the highest growth rate, and the groups with progressively higher intake of heptachlor had progressively lower growth rates.
Fish are critically important to the welfare of this planet and its occupants, the health of both wild and captive fish populations paramount to our survival. This book presents the gross pathology of the most commonly encountered diseases and syndromes of fish in an organ system-based approach. It provides an overview of the diseases and disorders of tropical, ornamental, bait and food fish from freshwater, brackish and marine environments. Readers will gain a broader understanding of the basic biology of infectious and non-infectious diseases in fish, as well as novel diagnostic techniques and innovative disease control methods.
We describe a new myxozoan, Henneguya akule n. sp., infecting the carangid fish Selar crumenophthalmus in Hawaii. Spores were found only in the aortic bulb, characterized by elliptical capsule with 2 tails, and pyriform polar capsules that angled toward the anterior end of the spore. Polar filaments had 3–4 coils. Parasites were present in apparently healthy fishes and caused no evident gross pathology. On microscopy, parasites evinced a mild inflammatory response in the host characterized by accumulations of eosinophilic fibrillar material around spores and a mononuclear infiltrate in the adventitia of the bulbus arteriosus. Overall prevalence was 20%, and prevalence between 2001 and 2006 ranged from 12 to 27%, but did not differ significantly between years. In contrast, prevalence of infection was highest in south-central Oahu. There was no relationship between infection status and body condition or gender of fish, and infection was absent in the smallest and largest fishes. Phylogenetically, H. akule n. sp. is most closely related to other Henneguya species infecting the heart of marine fishes based on ribosomal DNA analysis. This is the first documentation of a myxozoan parasite in marine fishes from Hawaii.
Procedures are described for processing fish for examination of external and internal anomalies and pathologies indicative of exposure to environmental contaminants and other peturbations. For the procedures described here, fish are captured (preferably by electrofishing) and held alive until processing (generally < 1 h). Fish are weighed, measured, and necropsied, and a scale sample is obtained from for age determination. Information is given for the collection and preservation of tissue samples for histopathological analysis. Photographs of most abnormalities are included along with normal conditions for easier identification of external (oral, head, eye, gill, opercula, and fin) and internal (liver, spleen, gonad, and kidney) anomalies. The report also includes recommendations for record keeping, sample labeling, and shipping records, equipment, supplies,and samples. A list of suggested equipment and supplies for field processing is included as are instructions for cleaning equipment.
Understanding the effects of anthropogenic changes on fish disease is vital for supporting mitigative actions for fisheries population recovery. The Lake Washington watershed in the Pacific Northwestern United States is influenced by high urban development and climate change, compromising the sustainability of anadromous salmon. We investigated Ceratonova shasta , a myxozoan parasite, in Chinook Salmon Oncorhynchus tshawytscha to determine the impacts of C. shasta -induced enteronecrosis during salmon freshwater migrations within this watershed. All sampled adult Chinook Salmon returning to an enhancement hatchery to spawn were infected with genotype I C. shasta with a high prevalence of severe enteritis and enteronecrosis. Parasite loads and associated pathology were more severe in adult fish succumbing to prespawn mortality than in fish that spawned. Natural prespawn mortality of Chinook Salmon in the Cedar River was also associated with high C. shasta loads. Sentinel exposure studies with Chinook Salmon juveniles in the spring and fall demonstrated high levels of C. shasta genotype II actinospores in the Lake Washington Ship Canal, a human engineered canal that salmon must traverse in their freshwater migrations from the Puget Sound. The sentinel juvenile Chinook Salmon succumbed to clinical disease and high mortality from genotype II C. shasta , suggesting that these stocks have high susceptibility to this genotype. The results show that both genotypes I and II pose disease risks to adult and juvenile stages of Chinook Salmon in the watershed, and that the Lake Washington Ship Canal could be a C. shasta ‘hot spot’ for genotype II that may increase disease risk for juveniles during smolt emigration.
To determine the earliest time after in vivo immunisation that the spleen could be excised and held in vitro to detect an immune response, lake trout ( Salvelinus namaycush ) were exposed to DNP-Ficoll or Yersinia ruckeri O antigen administered by intraperitoneal injection or by bath. The spleens were excised from the fish at selected times after immunisation, placed in vitro and held in tissue culture media at 14°C until 10 days after the in vivo immunisation. They then were analysed for an immune response by counting the numbers of plaqueforming cells (PFC). PFC were first detected in samples taken 3 days after injection with either antigen. With bath immunisation, however, PFC were first seen 6 days post-immunisation when using the Y. ruckeri O antigen and no PFC above background levels appeared in fish bathed in the DNP-Ficoll solution. On day 10 after immunisation, the spleens taken directly from immunised fish always produced more PFC than when the spleens were taken through in vitro culture. A unique feature of this method, is that an immune response to an antigen may be detected by excising the spleen and holding it in in vitro culture without necessarily holding the fish for long periods of time after antigen injection or bath. This technique also adds more information on how rapidly the bacterins or antigens are processed by fish to initiate an immune response.
The alewife ( Alosa pseudoharengus) is an anadromous herring that inhabits waters of northeastern North America. This prey species is a critical forage for piscivorous birds, mammals, and fishes in estuarine and oceanic ecosystems. During a discovery project tailored to identify potentially emerging pathogens of this species, we obtained the full genome of a novel hepadnavirus (ApHBV) from clinically normal alewives collected from the Maurice River, Great Egg Harbor River, and Delaware River in New Jersey, USA during 2015–2018. This previously undescribed hepadnavirus contained a circular DNA genome of 3146 nucleotides. Phylogenetic analysis of the polymerase protein placed this virus in the clade of metahepadnaviruses (family: Hepadnaviridae ; genus: Metahepadnavirus ). There was no evidence of pathology in the internal organs of infected fish and virions were not observed in liver tissues by electron microscopy. We developed a Taqman-based quantitative (qPCR) assay and screened 182 individuals collected between 2015 and 2018 and detected additional qPCR positives (n = 6). An additional complete genome was obtained in 2018 and it has 99.4% genome nucleotide identity to the first virus. Single-nucleotide polymorphisms were observed between the two genomes, including 7/9 and 12/8 synonymous vs nonsynonymous mutations across the polymerase and surface proteins, respectively. While there was no evidence that this virus was associated with disease in this species, alewives are migratory interjurisdictional fishes of management concern. Identification of microbial agents using de novo sequencing and other advanced technologies is a critical aspect of understanding disease ecology for informed population management.
There is an increasing emphasis on effects-based monitoring to document responses associated with exposure to complex mixtures of chemicals, climate change, pathogens, parasites and other environmental stressors in fish populations. For decades aquatic monitoring programs have included the collection of tissues preserved for microscopic pathology. Consequently, formalin-fixed, paraffin-embedded (FFPE) tissue can be an important reservoir of nucleic acids as technologies emerge that utilize molecular endpoints. Despite the cross-linking effects of formalin, its impact on nucleic acid quality and concentration, amplification, and sequencing are not well described. While fresh-frozen tissue is optimal for working with nucleic acids, FFPE samples have been shown to be conducive for molecular studies. Laser capture microdissection (LCM) is one technology which allows for collection of specific regions or cell populations from fresh or preserved specimens with pathological alterations, pathogens, or parasites. In this study, smallmouth bass (Micropterus dolomieu) liver was preserved in three different fixatives, including 10% neutral buffered formalin (NBF), Z-Fix® (ZF), and PAXgene® (PG) for four time periods (24 hr, 48 hr, seven days, and 14 days). Controls consisted of pieces of liver preserved in RNALater® or 95% ethanol. Smallmouth bass were chosen as they are an economically important sportfish and have been utilized as indicators of exposure to endocrine disruptors and other environmental stressors. Small liver sections were cut out with laser microdissection and DNA and RNA were purified and analyzed for nucleic acid concentration and quality. Sanger sequencing and the NanoString nCounter® technology were used to assess the suitability of these samples in downstream molecular techniques. The results revealed that of the formalin fixatives, NBF samples fixed for 24 and 48 hr were superior to ZF samples for both Sanger sequencing and the Nanostring nCounter®. The non-formalin PAXgene® samples were equally successful and they showed greater stability in nucleic acid quality and concentration over longer fixation times. This study demonstrated that small quantities of preserved tissue from smallmouth bass can be utilized in downstream molecular techniques; however, future studies will need to optimize the methods presented here for different tissue types, fish species, and pathological conditions.
This document describes procedures used to collect information, tissues, and fluids for documenting the exposure of fish to environmental contaminants. For the procedures described here, fish are captured (preferably by electrofishing) and held alive until processing (generally <1 h). Fish are weighed, measured, and examined for grossly visible external lesions and pathologies. A blood sample is collected by caudal veinipuncture using a needle and syringe. The fish is subdued and it's abdominal cavity opened. The internal organs are dissected from the fish for examination. The sex of the fish is determined by direct observation of its gonads. The liver is weighed (most species) and cut into small cubes and flash-frozen in cryogenic vials, which are stored and shipped in dry ice or liquid nitrogen. Additional liver cubes plus all grossly visible anomalies are preserved for histopathology. The gonads and spleen are weighed, and samples are preserved for histopathology. The kidneys are examined, and histopathology samples collected. A gill sample is also collected and preserved. All remaining tissues are returned to the carcass, which is wrapped in foil, labeled for chemical analysis, and chilled. Individual fish carcasses are composited by station, species, and gender; frozen; and shipped to the analytical laboratory. Procedures are also described for record keeping; processing blood to obtain serum and plasma; flash-freezing samples; cleaning equipment; and preventing the transport of living organisms among waterways. A list of necessary equipment and supplies is also provided.
Cutthroat trout ( Salmo clarki ) were exposed for 90 days in the laboratory to a refined oil collected from the North Platte River at a seepage site below the American Oil Company refinery at Casper, Wyoming. Fish were exposed to five concentrations and a control, and seven biological responses (survival, growth, gill pathology, liver pathology, caudal fin erosion, caudal fin pathology and swimming performance) were correlated with water concentration and tissue accumulation of petroleum hydrocarbons. Fish in the highest water concentration, 183 μ g/L total oil, accumulated tissue concentrations of 4.6 μ g/g total naphthalenes and responded adversely to all seven biological measurements. Cutthroat trout in the lowest water concentration, 24 μ g/L total oil, had a mean tissue concentration of 1.2 μ g/g total naphthalenes, but their response was similar to that of the control fish to all seven biological responses. Fish exposed to 39 μ g/L water concentration accumulated tissue concentrations of 2.7 μ g/g and responded negatively to 2 of the 7 biological measurements. Therefore, the maximum safe limit for this oil and cutthroat trout is between 24 and 39 μ g/L.