Alvin C. Camus

The ancient evolutionary history of polyomaviruses

Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae.

Nonlesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies: a guide for investigators, authors, reviewers, and readers.

Differentiating salient histopathologic changes from normal anatomic features or tissue artifacts can be decidedly challenging, especially for the novice fish pathologist. As a consequence, findings of questionable accuracy may be reported inadvertently, and the potential negative impacts of publishing inaccurate histopathologic interpretations are not always fully appreciated. The objectives of this article are to illustrate a number of specific morphologic findings in commonly examined fish tissues (e.g., gills, liver, kidney, and gonads) that are frequently either misdiagnosed or underdiagnosed, and to address related issues involving the interpretation of histopathologic data. To enhance the utility of this article as a guide, photomicrographs of normal and abnormal specimens are presented. General recommendations for generating and publishing results from histopathology studies are additionally provided. It is hoped that the furnished information will be a useful resource for manuscript generation, by helping authors, reviewers, and readers to critically assess fish histopathologic data.

15 Health management

Publisher Summary Channel catfish health management begins with creating and maintaining a proper living environment for the fish. Good management also includes the design and operation of a production facility that minimizes the impact of stress on the fish. To promote health and minimize stress, fish should be provided with their basic needs: sufficient space, good water quality, a nutritionally complete diet, limited physical disturbance, protection from predators, and prudent handling. Once these needs are met, it is important to limit contact with disease-causing agents and to prevent environmental deterioration. Even in well-designed and properly managed facilities, it is not uncommon for diseases to occur. Once a disease outbreak ensues, effective health management requires three basic steps: problem identification, diagnosis, and corrective management—all of which must be performed in a timely manner to avoid further losses. Whenever multiple factors contribute to the disease process, it makes the diagnosis more difficult and often complicates corrective management. Many fish health problems, such as oxygen depletions, can be recognized and corrected by the fish culturist on the pond bank. This chapter discusses factors affecting the disease process in catfish and steps that can be taken to diagnose disease problems. The chapter provides an overview of the various options for managing infectious diseases. Several specific management procedures for the important catfish diseases are also discussed.

Expression of CYP1C1 and CYP1A in Fundulus heteroclitus during PAH-induced carcinogenesis

CYP1C1 is a relatively newly identified member of the cytochrome P450 family 1 in teleost fish. However, CYP1C1s expression and physiological roles relative to the more recognized CYP1A in polycyclic aromatic hydrocarbons (PAHs) induced toxicities are unclear. Fundulus heteroclitus fry were exposed at 6-8 days post-hatch (dph) and again at 13-15dph for 6h to dimethyl sulfoxide (DMSO) control, 5mg/L benzo[a]pyrene (BaP), or 5mg/L dimethylbenzanthracene (DMBA). Fry were euthanized at 0, 6, 18, 24 and 30h after the second exposure. In these groups, both CYP1A and CYP1C1 protein expression were induced within 6h after the second exposure. Immunohistochemistry (IHC) results from fry revealed strongest CYP1C1 expression in renal tubular and intestinal epithelial cells. Additional fish were examined for liver lesions 8 months after initial exposure. Gross lesions were observed in 20% of the BaP and 35% of the DMBA-treated fish livers. Histopathologic findings included foci of cellular alteration and neoplasms, including hepatocellular adenoma, hepatocellular carcinoma and cholangioma. Strong CYP1A immunostaining was detected diffusely in altered cell foci and on the invading margin of hepatocelluar carcinomas. Lower CYP1A expression was seen in central regions of the neoplasms. In contrast, CYP1C1 was only detectable and highly expressed in proliferated bile duct epithelial cells. Our CYP1C1 results suggest the potential for tissue specific CYP1C1-mediated PAH metabolism but not a more chronic role in progression to liver hepatocellular carcinoma.

Application of a real-time PCR assay for the detection of Henneguya ictaluri in commercial channel catfish ponds

Proliferative gill disease (PGD) in channel catfish Ictalurus punctatus is caused by the myxozoan parasite Henneguya ictaluri. Prolonged exposure of channel catfish to the actinospore stage of the parasite results in extensive gill damage, leading to reduced production and significant mortality in commercial operations. A H. ictaluri-specific real-time (Q)PCR assay was used to determine parasite levels in commercial channel catfish ponds and evaluate the risk of losing fish newly stocked into the system. Previous research has shown the H. ictaluri actinospore to be infective for approximately 24 h; therefore, determining the parasite load (ratio of parasite DNA to host DNA) in sentinel fish exposed for 2 separate 24 h periods with a minimum of 1 wk between sampling indirectly represents the rate at which infective actinospores are being released by the oligochaete host and if that rate is changing over time. Alternatively, QPCR analysis of pond water samples eliminates the need for sentinel fish. Water samples collected on 2 separate days, with a minimum of 1 wk between sampling, not only determines the approximate concentrations of actinospores in the pond but if these concentrations are remaining stable. Increases in parasite load (r = 0.69, p = 0.054) correlated with percent mortality in sentinel fish, as did increases in mean actinospore concentrations (r = 0.63, p = 0.003). Both applications are more rapid than current protocols for evaluating the PGD status of a catfish pond and identified actinospore levels that correlate to both high and low risk of fish loss.

Francisella noatunensis subsp. orientalis infection in Indo-Pacific reef fish entering the United States through the ornamental fish trade

Infections caused by Piscirickettsia-like organisms (PLO) appeared in Taiwanese tilapia, Oreochromis spp., culture in the early 1990s (Chen et al. 1994; Chern & Chao 1994). Additional reports quickly appeared making it evident that PLO posed an emerging threat to a range of fish species in both fresh and salt water (Mauel & Miller 2002). Early reports were based largely on gross and histologic features of the disease and failure of the organisms to react serologically or in PCR assays for Piscirickettsia salmonis. Improved culture and molecular detection methods determined later PLO cases to be caused by Francisella spp. bacteria capable of survival and growth within macrophages (Ottem et al. 2009; Birkbeck, Feist & Verner-Jeffreys 2011; Colquhoun & Duodu 2011). Taxonomic clarification of the pleomorphic 0.5to 1.7-lm bacteria indicates a single species, Francisella noatunensis, for all fish isolates. Two subspecies are recognized, F. noatunensis subsp. noatunensis (Fnn) and F. noatunensis subsp. orientalis (Fno), with apparent predilections for cold and warmwater fish, respectively (Ottem et al. 2009). Regardless of the bacterial subspecies involved, infections share clinical and pathologic features, including acute to chronic mortalities accompanied by non-specific signs. Most notable grossly is splenomegaly and renomegaly, with whitish-tan nodules corresponding histologically to granulomas typified by vacuolated macrophages containing the bacteria (Birkbeck et al. 2011; Colquhoun & Duodu 2011). Some granulomas develop cuffs of lymphocytes (Mauel et al. 2007) and with time may undergo central necrosis and fibrous encapsulation (Hsieh et al. 2006; Birkbeck et al. 2011). The spleen and kidney are most severely affected, although lesions have been described in all organ systems (Birkbeck et al. 2011; Colquhoun & Duodu 2011). Evidence for a PLO induced disease, suggestive of francisellosis, in the continental United States was reported from California in 1998 involving white seabass, Atractoscion nobilis (Ayres) (Chen et al. 2000). To the best of our knowledge, however, francisellosis has not emerged in the marine tropical fish trade in this country, although movements of live fish are likely involved in dissemination of the agent, and it has been predicted that the number of affected species will continue to rise (Birkbeck et al. 2011). This report describes Fno infections diagnosed in Indo-Pacific reef fish following international shipment to and distribution in the United States from Asian waters. Correspondence E Soto, Ross University, School of Veterinary Medicine, Department of Pathobiology, P.O. Box 334, Basseterre, St. Kitts, West Indies (e-mail: esoto@rossvet.edu.kn)

B cell memory following infection and challenge of channel catfish with Ichthyophthirius multifiliis

B cell responses in channel catfish to infection with the parasitic ciliate Ichthyophthirius multifiliis were followed for 3 years. High titers of serum IgM antibodies recognizing I. multifiliis immobilization antigens were present 5weeks after immunizing infection, but by 1 year titers were at low or undetectable levels. Two to three years after infection the numbers of antibody secreting cells recognizing immobilization antigens in skin and head kidney of immune fish had decreased to the level found in uninfected controls. Challenge of immune fish showed they remained immune and that the numbers of antibody secreting cells recognizing immobilization antigens increased in skin but not head kidney. This suggests that antigen-specific memory B cells persisted for 3 years after infection and upon challenge differentiated into antibody secreting cells that localized in skin. Our results suggest that humoral immunity in channel catfish is maintained through IgM(+) memory B cells.

Investigation of shell disease in map turtles (Graptemys spp.).

Nineteen map turtles (Graptemys spp.) maintained under natural conditions were investigated because of chronic shell abnormalities. Animals were evaluated using a novel shell scoring system that divided the 54 scutes into six regions, with each region scored for lesion extent and severity, and summated to produce a total shell disease score (TSDS). Complete blood counts and various biochemistry analytes (total protein, albumin, globulin, urea, uric acid, 25-hydroxycholecalciferol, phosphorus, and ionized and total calcium) were measured. Under ketamine-medetomidine-morphine anesthesia, cytology tape strips and full thickness shell biopsies were collected aseptically for microbiologic, histologic (including scoring of biopsy quality), and ultrastructural evaluations. The TSDSs were low and ranged from 4 to 22 (median=9) out of a possible score of 54. There were no correlations between TSDS and any hematologic or biochemistry parameter. The histologic quality of shell biopsies was good, and normal shell structure, by both light and electron microscopy, is described. Small clefts and pitting lesions were noted in 8/19 sections. There was no evidence of erosion, ulceration, inflammation, or infectious agents, but algae and diatoms were observed. Six biopsies yielded aerobic isolates (Chryseobacterium indologenes, Aeromonas hydrophila, Ralstonia pickettii, and Morganella morganii), whereas 11 shell samples grew various clostridial anerobes. No fungal organisms were cultured. Although the etiology of the lesions described remains unknown, the use of a scoring system in conjunction with full thickness biopsies is suggested to help standardize investigations into chelonian shell disease in the future.

Effect of Sublethal Exposure to the Trematode Bolbophorus spp. on the Severity of Enteric Septicemia of Catfish inChannel Catfish Fingerlings

Abstract The digenetic trematode Bolbophorus damnificus has been implicated as a cause of mortalities and reduced production in farmed channel catfish Ictalurus punctatus in northwestern Mississippi. Field observations indicate that infection can exacerbate losses from common bacterial diseases, such as enteric septicemia of catfish (ESC), which is caused by Edwardsiella ictaluri. The interaction of concurrent infections with Bolbophorus spp. and E. ictaluri was investigated using three replicates of four experimental treatments. Trematode infections were established by placing fingerlings for 24 h in triplicate static tanks containing marsh rams-horn snails Planorbella trivolvis shedding Bolbophorus cercariae at a rate of 770 ± 82 (mean ± SE) per 24-h period. Unexposed fish were maintained in three tanks under similar conditions. From each tank, trematode-infected or noninfected fish were transferred to six aquaria (30 fish/aquarium). Three aquaria from each replicate treatment tank received 7.5 × 105 co...

Distinct Viral Lineages from Fish and Amphibians Reveal the Complex Evolutionary History of Hepadnaviruses

ABSTRACT Hepadnaviruses (hepatitis B viruses [HBVs]) are the only animal viruses that replicate their DNA by reverse transcription of an RNA intermediate. Until recently, the known host range of hepadnaviruses was limited to mammals and birds. We obtained and analyzed the first amphibian HBV genome, as well as several prototype fish HBVs, which allow the first comprehensive comparative genomic analysis of hepadnaviruses from four classes of vertebrates. Bluegill hepadnavirus (BGHBV) was characterized from in-house viral metagenomic sequencing. The African cichlid hepadnavirus (ACHBV) and the Tibetan frog hepadnavirus (TFHBV) were discovered using in silico analyses of the whole-genome shotgun and transcriptome shotgun assembly databases. Residues in the hydrophobic base of the capsid (core) proteins, designated motifs I, II, and III, are highly conserved, suggesting that structural constraints for proper capsid folding are key to capsid protein evolution. Surface proteins in all vertebrate HBVs contain similar predicted membrane topologies, characterized by three transmembrane domains. Most striking was the fact that BGHBV, ACHBV, and the previously described white sucker hepadnavirus did not form a fish-specific monophyletic group in the phylogenetic analysis of all three hepadnaviral genes. Notably, BGHBV was more closely related to the mammalian hepadnaviruses, indicating that cross-species transmission events have played a major role in viral evolution. Evidence of cross-species transmission was also observed with TFHBV. Hence, these data indicate that the evolutionary history of the hepadnaviruses is more complex than previously realized and combines both virus-host codivergence over millions of years and host species jumping. IMPORTANCE Hepadnaviruses are responsible for significant disease in humans (hepatitis B virus) and have been reported from a diverse range of vertebrates as both exogenous and endogenous viruses. We report the full-length genome of a novel hepadnavirus from a fish and the first hepadnavirus genome from an amphibian. The novel fish hepadnavirus, sampled from bluegills, was more closely related to mammalian hepadnaviruses than to other fish viruses. This phylogenetic pattern reveals that, although hepadnaviruses have likely been associated with vertebrates for hundreds of millions of years, they have also been characterized by species jumping across wide phylogenetic distances.