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Toxicity of acidified chitosan for cultured rainbow trout (Oncorhynchus mykiss)

Chitosan is a deacetylation product of chitin. It is used as a flocculent for sewage and brewery wastes, and as a chelator of heavy metals. In aquaculture, chitosan has been used as an immunostimulant for protection against bacterial diseases in fish, for controlled release of vaccines, and as a diet supplement. Chitosan has generally been considered to be nontoxic to animals, but when it was dissolved in acetic acid and added to a culture system at 1.0 ppm to remove organic solids, we found acute toxicity to rainbow trout ( Oncorhynchus mykiss ). In controlled experiments to determine the extent of toxicity, we found that trout died after several hours exposure to 0.75 ppm and died in 24 h after exposure to 0.075 ppm. Exposure to 0.038 ppm resulted in mortality after 6 days exposure, while exposure to 0.019 ppm resulted in no mortality after 14 days exposure. Histological examination of gills, skin, muscle, and internal organs indicated significant and consistent pathological changes only in gills. Lifting of lamellar epithelium, hypertrophy and hyperplasia of lamellar epithelial cells occurred in trout exposed to 0.019 and 0.038 ppm. In trout exposed to 0.75 or 0.075 ppm chitosan, large areas of lamellar fusion were observed. These results show that soluble acidified chitosan is highly toxic to rainbow trout even at low concentrations.

Aquaculture

Necropsy-based wild fish health assessment

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on fish populations. Adverse effects monitoring, utilizing biomarkers from the organismal to the molecular level, can be used to assess the cumulative effects on fishes and other organisms. Fish health has been used worldwide as an indicator of aquatic ecosystem health. The necropsy-based fish health assessment provides data on visible abnormalities and lesions, parasites, condition and organosomatic indices. These can be compared by site, season and sex, as well as temporally, to document change over time. Severity ratings can be assigned to various observations to calculate a fish health index for more quantitative assessment. A drawback of the necropsy-based assessment is that it is based on visual observations and condition factors, which are not as sensitive as tissue and subcellular biomarkers for sublethal effects. Additionally, it is rarely possible to identify causes or risk factors associated with observed abnormalities. So, for instance a raised lesion or "tumor" on the fins, lips or body surface may be a neoplasm. However, it could also be a response to a parasite, chronic inflammation or hyperplasia of normal cells in response to an irritant. Conversely, neoplasms, certain parasites, other infectious agents and many tissue changes are not visible and so may be underestimated. However, during the necropsy-based assessment, blood (plasma), tissues for histopathology (microscopic pathology), genomics and other molecular analyses, and otoliths for aging can be collected. These downstream analyses, together with geospatial analyses, habitat assessments, water quality and contaminant analyses can all be important in comprehensive ecosystem evaluations.

Journal of Visualized Experiments

Formalin for external protozoan parasites: A report on the prevention and control of Costia necatrix

The smallest and most destructive of the ectoparasitic protozoans infecting salmon and trout, Costia necatrix , has unfortunately been relegated to virtual obscurity during the past few years. Few references to this parasite can be found in the recent literature and, where such things are discussed, one seldom hears a mention of Costia necatrix . This apparent lack of interest in Costia certainly does not result from the infrequency of its appearances nor from any lack of pathogenicity on its part when it does occur. Preserved specimens of ailing trout an dsalmon submitted to the Seattle Pathology Laboratory for diagnosis have yielded very surprising indications concerning the frequency, intensity, and geographic distribution of Costiasis - surprising in that a parasite should be so widespread, so pathogenic, and yet so seldom mentioned. The answer undoubtedly lies in the fact that Costia , being small in size and usually sedentary in its habits, is being overlooked during parasitic examinations. Although Costia is not at all difficult to recognize, even well-trained workers unfamiliar with its appearance almost invariably pass it by.

Progressive Fish-Culturist

Histopathology of kidney disease in fish

Kidney disease is one of the most puzzling fish diseases known to exist in the United States. In less than Io years it has invaded the Pacific Northwest, exacting a heavy toll of hatchery salmon. Its first appearance apparently was in Massachusetts where Belding and Merrill' described a disease similar to that now seen on the Pacific Coast. In I946 it was diagnosed in Washington2 and since that time has been observed in an ever increasing number of hatcheries. There are unpublished reports of the same or similar diseases in both California and Washington in the early I93o's.3 The latest outbreaks occurred in the Federal hatcheries at Berlin, New Hampshire, and Cortland, New York, in brook, brown, and rainbow trout.4 There is evidence to indicate that the disease may be much more widely spread in New York State.5 The disease is especially dangerous since little is known of the origin or source of the causative agent. Indeed, the classification of the diplobacillus associated with kidney disease is still uncertain. Thus, with our present knowledge, it is difficult or impossible to eradicate the malady from an infected hatchery. Histopathologic studies were undertaken to clarify the pathology of the disease and to compare the eastern form with the western form.

American Journal of Pathology

Ceroid in fish

Since the original description of ceroid in rats, many papers have appeared on the etiology and characteristics of this pigment. It was first seen as a yellow, granular pigment in hematoxylin and eosin sections of the cirrhotic livers of choline deficient rats. The pigment was more fully characterized by Endicott and Lillie, and additional stainging reactions were summarized recently by Lillie. The pigment is sudanophilic in paraffin sections, acid-fast, basophilic, isotropic, iron negative, and highly resistant to solution in water, alcohol, fat solvents, and dilute aqueous acids and alkalis. It is stained by Mallory's hemofuscin stain and Weigert's myelin stain. It reduces osmium tetraoxide and diamine silver carbonate but not ferric ferricyanide. The Gmelin reactions for bile pigments is negative. It has a greenish yellow fluorescence at 3650-3660 Å. It is Schiff positive with or without antecedent diastase digestion after performic or periodic acid oxidation.

The American Journal of Pathology

Aquatic bird disease and mortality as an indicator of changing ecosystem health

We analyzed data from pathologic investigations in the United States, collected by the USGS National Wildlife Health Center between 1971 and 2005, into aquatic bird mortality events. A total of 3619 mortality events was documented for aquatic birds, involving at least 633 708 dead birds from 158 species belonging to 23 families. Environmental causes accounted for the largest proportion of mortality events (1737 or 48%) and dead birds (437 258 or 69%); these numbers increased between 1971 and 2000, with biotoxin mortalities due to botulinum intoxication (Types C and E) being the leading cause of death. Infectious diseases were the second leading cause of mortality events (20%) and dead birds (20%), with both viral diseases, including duck plague ( Herpes virus ), paramyxovirus of cormorants ( Paramyxovirus PMV1) and West Nile virus ( Flavivirus ), and bacterial diseases, including avian cholera ( Pasteurella multocida ), chlamydiosis ( Chalmydia psittici ), and salmonellosis ( Salmonella sp.), contributing. Pelagic, coastal marine birds and species that use marine and freshwater habitats were impacted most frequently by environmental causes of death, with biotoxin exposure, primarily botulinum toxin, resulting in mortalities of both coastal and freshwater species. Pelagic birds were impacted most severely by emaciation and starvation, which may reflect increased anthropogenic pressure on the marine habitat from over-fishing, pollution, and other factors. Our study provides important information on broad trends in aquatic bird mortality and highlights how long-term wildlife disease studies can be used to identify anthropogenic threats to wildlife conservation and ecosystem health. In particular, mortality data for the past 30 yr suggest that biotoxins, viral, and bacterial diseases could have impacted >5 million aquatic birds.

Marine Ecology Progress Series

Occupancy modeling for improved accuracy and understanding of pathogen prevalence and dynamics

Most pathogen detection tests are imperfect, with a sensitivity < 100%, thereby resulting in the potential for a false negative, where a pathogen is present but not detected. False negatives in a sample inflate the number of non-detections, negatively biasing estimates of pathogen prevalence. Histological examination of tissues as a diagnostic test can be advantageous as multiple pathogens can be examined and providing important information on associated pathological changes to the host. However, it is usually less sensitive than molecular or microbiological tests for specific pathogens. Our study objectives were to 1) develop a hierarchical occupancy model to examine pathogen prevalence in spring Chinook salmon Oncorhynchus tshawytscha and their distribution among host tissues 2) use the model to estimate pathogen-specific test sensitivities and infection rates, and 3) illustrate the effect of using replicate within host sampling on sample sizes required to detect a pathogen. We examined histological sections of replicate tissue samples from spring Chinook salmon O. tshawytscha collected after spawning for common pathogens seen in this population: Apophallus/ echinostome metacercariae, Parvicapsula minibicornis, Nanophyetus salmincola/ metacercariae, and Renibacterium salmoninarum . A hierarchical occupancy model was developed to estimate pathogen and tissue-specific test sensitivities and unbiased estimation of host- and organ-level infection rates. Model estimated sensitivities and host- and organ-level infections rates varied among pathogens and model estimated infection rate was higher than prevalence unadjusted for test sensitivity, confirming that prevalence unadjusted for test sensitivity was negatively biased. The modeling approach provided an analytical approach for using hierarchically structured pathogen detection data from lower sensitivity diagnostic tests, such as histology, to obtain unbiased pathogen prevalence estimates with associated uncertainties. Accounting for test sensitivity using within host replicate samples also required fewer individual fish to be sampled. This approach is useful for evaluating pathogen or microbe community dynamics when test sensitivity is <100%.

PLoS ONE

Molecular characterization of a novel orthomyxovirus from rainbow and steelhead trout ( Oncorhynchus mykiss )

A novel virus, rainbow trout orthomyxovirus (RbtOV), was isolated in 1997 and again in 2000 from commercially-reared rainbow trout ( Oncorhynchus mykiss ) in Idaho, USA. The virus grew optimally in the CHSE-214 cell line at 15°C producing a diffuse cytopathic effect; however, juvenile rainbow trout exposed to cell culture-grown virus showed no mortality or gross pathology. Electron microscopy of preparations from infected cell cultures revealed the presence of typical orthomyxovirus particles. The complete genome of RbtOV is comprised of eight linear segments of single-stranded, negative-sense RNA having highly conserved 5′ and 3′-terminal nucleotide sequences. Another virus isolated in 2014 from steelhead trout (also O. mykiss ) in Wisconsin, USA, and designated SttOV was found to have eight genome segments with high amino acid sequence identities (89–99%) to the corresponding genes of RbtOV, suggesting these new viruses are isolates of the same virus species and may be more widespread than currently realized. The new isolates had the same genome segment order and the closest pairwise amino acid sequence identities of 16–42% with Infectious salmon anemia virus (ISAV), the type species and currently only member of the genus Isavirus in the family Orthomyxoviridae . However, pairwise comparisons of the predicted amino acid sequences of the 10 RbtOV and SttOV proteins with orthologs from representatives of the established orthomyxoviral genera and a phylogenetic analysis using the PB1 protein showed that while RbtOV and SttOV clustered most closely with ISAV, they diverged sufficiently to merit consideration as representatives of a novel genus. A set of PCR primers was designed using conserved regions of the PB1 gene to produce amplicons that may be sequenced for identification of similar fish orthomyxoviruses in the future.

Virus Research

Book review: Proceedings of the First International Snakehead Symposium

Snakehead fishes (family Channidae) are among the most maligned aquatic invasive species in the USA and some other countries where they have been introduced outside of their native range in Asia and Africa. Nevertheless, snakeheads continue to be widely exploited in the live‐food trade in aquaculture and wild‐capture fisheries, are highly sought by anglers, and are also popular in the aquarium trade (Courtenay and Williams 2004). The Northern Snakehead Channa argus is the most widespread of the three channid species that are currently naturalized in the USA. This species has generated much concern and controversy, a situation that is partly fueled by sensational media coverage and B‐grade science fiction horror films, such as “Frankenfish,” “Snakehead Terror,” and “Snakehead Swamp.” Media reports of snakehead introductions are often replete with provocative terms, such as “vicious,” “villain,” “voracious,” “monster,” “diabolical,” and even “ecological Armageddon.” When snakeheads first appeared in natural waters of the USA, fisheries professionals became increasingly interested in their status. Established populations rapidly expanded in the mid‐Atlantic region and Arkansas, with scattered reports of introduced snakeheads from isolated locations in Hawaii, California, North Carolina, Florida, the Upper Midwest, and New England. In 2002, snakeheads were added to the list of injurious fishes under the Lacey Act, thereby prohibiting their importation or transport across state lines without a permit. This symposium was conceived by the editors and other concerned fisheries professionals of the Mississippi River Basin Panel on Aquatic Invasive Species. The mission of the symposium, held in Alexandria, Virginia, in July 2018, was to bring together experts on snakehead biology and ecology and to synthesize existing information into summary papers. In this book, 35 authors contributed to 15 peer‐reviewed articles that detail the current state of knowledge about snakehead introductions in the USA. Additionally, 16 abstracts are included from meeting presentations that were not accompanied by full‐length manuscripts. Also included is a summary of a facilitated symposium panel discussion featuring eight experts representing state and federal natural resource agencies and private fishing organizations. The book is organized into six sections. In the first section (Distribution), three papers provide an overview of the Channa species introduced into the USA and historical accounts of occurrence and dispersal of the Northern Snakehead in the mid‐Atlantic region and Arkansas. The second section (Biology/Ecology) consists of two articles that examine growth and energetics of Northern Snakehead populations and two papers that investigate diet, diel feeding activity, and movement of this species in the Potomac River drainage. The third section (Monitoring/Response) includes a paper that models range expansion of the Northern Snakehead in the southeastern USA based on occurrence data and environmental conditions. Also included in this section is a paper summarizing an environmental DNA study to assess the status and range of the Bullseye Snakehead C. marulius in southern Florida. The fourth section (Management/Control) is comprised of four papers that address harvest, age and growth, and development of a stock–recruitment model to inform management decisions regarding control and mitigation for Northern Snakehead populations in the greater Chesapeake Bay area. The fifth section (Perspectives) includes a paper on the history of snakehead introductions in Japan and a thought‐provoking social commentary on the human dimensions of Northern Snakehead management. Abstracts in the final section provide brief summaries of a diversity of snakehead studies, including aspects of distribution, ecology, behavior, control and monitoring efforts, public outreach, and pathology. The summary of the panel discussion is an engaging dialogue about the challenges of snakehead management in the context of conflicts regarding snakeheads as injurious versus their value as game and food species. Most of this book is focused on the Northern Snakehead. Much has been done to document snakehead distributions and certain aspects of snakehead biology, such as diets, age, and growth. Less research has been devoted to understanding the ecological impacts of snakeheads to native aquatic communities and ecosystems. This book would have benefited from a chapter summarizing the current systematics and diversity of the Channidae to inform fisheries biologists about the morphological characteristics of the family, approximate numbers of genera and species, and taxonomic instability. Exemplifying the latter, recent molecular and morphological evidence indicates uncertainty regarding identification of the feral snakehead population in Florida (Adamson and Britz 2019). Those authors suggest that this population may have originated from western Thailand, a possibility that could have implications for understanding historical pathways of snakehead introductions into the USA. In comparison with many published AFS symposia, this volume is relatively narrow in scope and lacks cohesive integration. It will primarily be of interest to those fisheries professionals engaged in the study of snakeheads as well as other nonnative species for which there are contrasting social values regarding their management: whether to monitor and attempt control or eradication efforts or to maintain populations for harvest as game or food species. The book should serve to identify information gaps and guide future research.

Transactions of the American Fisheries Society

Propagation of the myxozoan parasite Myxobolus cerebralis by different geographic and genetic populations of Tubifex tubifex: An Oregon perspective

Tubifex tubifex are obligate invertebrate hosts in the life cycle of Myxobolus cerebralis, the myxozoan parasite that causes whirling disease in salmonid fishes. This exotic parasite is established to varying degrees across Oregon's Columbia River system (Pacific Northwest, USA) and characteristics of local T. tubifex populations likely play a role in the pattern of disease occurrence. To better understand these patterns, we collected T. tubifex from three Oregon river basins (Willamette, Deschutes, and Grande Ronde), determined their genotype (mitochondrial 16S rDNA lineage and RAPD genotype) and exposed 10 different populations to M. cerebralis in the laboratory. Four mt lineages were identified: I, III, V and VI. Lineage III was found in all river basins but dominated both central and eastern sites. The RAPD assay further divided these lineages into geographic sub-populations; no RAPD genotype was common to all basins. There was a significant difference in prevalence of infection and level of parasite production among the populations we exposed to M. cerebralis that was attributed to genotypic composition. Only lineage III worms released actinospores and only populations dominated by this lineage amplified the parasite. These populations had the lowest survival, however, the lineage dominant before exposure remained dominant despite the high prevalence of infection. The distribution and infection dynamics of susceptible T. tubifex throughout Oregon may contribute to the differences in M. cerebralis occurrence; our studies further support the influence of oligochaete genotypes on the manifestation of whirling disease in salmonid populations. ?? 2009 Elsevier Inc. All rights reserved.

Journal of Invertebrate Pathology