SESSION 3. PARASITOLOGY I: Myxozoan Workshop 1
SESSION 7. PARASITOLOGY II: Myxozoan Workshop 2
SESSION 10. PARASITOLOGY III: Myxozoan Workshop 3
SESSION 12. ANTIMICROBIAL USE IN AQUACULTURE: Resistance, Residues and Research





Intrinsic Resistance of Edwardsiella ictaluri to Cationic Antimicrobial Peptides is Mediated by UDP-Glc Dehydrogenase, an Enzyme Required for the O-polysaccharide Synthesis

Santander* and R Curtiss III

Arizona State University, Biodesign Institute, Center for Infectious Diseases and Vaccinology, Tempe, AZ, USA jsantanderm@asu.edu

The genus Edwardsiella comprises a genetically distinct taxon related to other members of the Enterobacteriaceae. It consists of bacteria differing strongly in their biochemical and physiological features, natural habitats, and pathogenic properties. Intrinsic resistance to cyclic cationic antimicrobial peptides (colistin and polymyxin B) is a particular property of the genus Edwardsiella. Here we evaluated the role of the O-polysaccharide (O-PS) in the resistance to cyclic cationic antimicrobial peptides in Edwardsiella ictaluri (Hawke, 1981), the causative agent of fatal systemic infections in channel catfish (Ictalurus punctatus). The putative genes (wibT, galF, gne, and ugd) related to the synthesis of O-PS were systematically inframe deleted by suicide vector technology. Individual deletions of wibT, gne and ugd abolished synthesis of the O-PS, causing auto-agglutination and rough colonies. The wibT and gne mutants conserve their resistance to cyclic antimicrobial peptides. However, deletion of ugd, the gene that encodes the UDP-Glc dehydrogenase enzyme, causes sensitivity to cyclic cationic antimicrobial peptides, without revertants. LPS profile analysis indicates that deletion of ugd affects the lipopolysaccharide (LPS) core. The outer membrane protein profile showed up regulation of a single protein in wibT, gne and ugd mutants. These results indicate that the UDP-Glc dehydrogenase enzyme, and the structure of the LPS core, are essential for the Edwardsiella resistance mechanism to cyclic antimicrobial peptides.




Evaluation of the Edwardsiella ictaluri Type IV Secretion System Role in Virulence

AA Graff1, ML Rogge1 and RL Thune*1,2

1 Department of Pathobiological Sciences, LSU School of Veterinary Medicine, Skip Bertman Drive, Baton Rouge, LA 70803 USA graff.allison@gmail.com, mrogge1@lsu.edu
2 Department of Veterinary Science, Louisiana State University,Baton Rouge, LA USA thune@vetmed.lsu.edu

Type IV secretion systems (T4SS) have roles in the secretion of both DNA and proteins for the purposes of conjugation and virulence, respectively. Edwardsiella ictaluri carries a pathogenicity island encoding genes with homology to T4SSs of other pathogens. In addition to the T4SS, E. ictaluri encodes type III and type VI secretion systems, both of which are important for pathogenesis in the channel catfish (Ictalurus punctatus). To determine if the T4SS is also involved in E. ictaluri pathogenesis, a mutation was constructed in the virD4-like gene, which is putatively involved in coupling secretion substrates to the secretion apparatus. Such a mutation should knock out function of the entire type IV system. Subsequent phenotypic characterization of the mutant included evaluation of invasion and replication in channel catfish cells, as well as in vivo colonization and virulence. No defect in invasion or intracellular replication was observed ex vivo, and the mutant strain colonized the head kidney, persisted, and was virulent. These results demonstrate that virulence of E. ictaluri is not dependent on the T4SS-encoded virD4, but this does not preclude the T4SS from having a role in virulence that is not detectable by the assays conducted. Further characterization is needed to determine if the T4SS components are functional, under what environmental conditions they are expressed, and how they may be involved in virulence.




The Edwardsiella ictaluri Type III and Type VI Secretion Systems Are Co-Regulated and Are Both Required for Virulence in the Channel Catfish


ML Rogge*1 and RL Thune1,2


Departmentof Pathobiological Sciences, LSU School of Veterinary Medicine, Baton Rouge, LA,USA mrogge1@lsu.edu
Departmentof Veterinary Science, Louisiana State University, Baton Rouge, LA, USA thune@vetmed.lsu.edu






Type III and type VI secretion systems (T3SSs and T6SSs) are found in many pathogenic bacteria and mediate the translocation of virulence proteins from the bacterial cytoplasm to the cytoplasm of the host cell. The Edwardsiella ictaluri T3SS is required for intracellular replication and virulence in channel catfish (Ictalurus punctatus). Previous work demonstrated that secretion of a T6SS protein, EvpC, is dependent on the T3SS-encoded regulatory proteins EsrB and EsrC, suggesting the coordinated regulation of two secretion systems by these regulatory proteins. To evaluate the T6SS role in virulence, a mutation was made in EvpC, a putative T6SS translocon protein. Knockout of the translocon protein should prohibit the T6SS from translocating effector proteins. The mutant strain was evaluated for its ability to replicate within channel catfish head kidney-derived macrophages (HKDM), colonize head kidney tissue in vivo, and cause mortality in channel catfish. The T6SS mutant maintained the ability to replicate within HKDM, and was able to successfully colonize the head kidney. Following immersion challenge, however, mortality of catfish exposed to the T6SS mutant was significantly less than with the wild-type, indicating an important virulence function for the T6SS. Further research is required to identify the proteins secreted by the T6SS and determine what their function is in virulence. Additionally, the role of the T3SS-encoded EsrB and EsrC in the regulation of the T6SS and the integrated role of the T3SS and T6SS in the pathogenesis of E. ictaluri requires further study.






Evaluation of the Role of Ammonia and Urea Transporters on the Pathogenesis of Edwardsiella ictaluri


J Beekman and RThune*


Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803 USA thune@vetmed.lsu.edu




Edwardsiella ictaluri is a facultative intracellular pathogen that survives and replicates within channel catfish macrophages.   We previously reported that the E. ictaluri genome contains a urease operon expressing a functional urease that converts urea to ammonia in vitro, and which plays a role in survival of E. ictaluri within cultured head kidney derived macrophages (HKDM).  Taken together, this data suggests that urease contributes to survival within the macrophage by producing ammonia to neutralize the acidic endosome.  Interestingly, the E. ictaluri genome contains two genes in tandem just downstream of the urease operon, one encoding a urea transporter and the second a truncated, but potentially functional, ammonia transporter.   The objective of this study was to evaluate the role these transporters play in the movement of the urease substrate, urea, and the urease product, ammonia, across the E. ictaluri membrane.  Strains of E. ictaluri containing deletion mutations in either the urea or the ammonia transporter were found to be attenuated by 48% and 36%, respectively, compared to wild type in a live catfish challenge.  There was no difference in survival, however, between the mutants and the wild type within cultured HKDM.  RT-PCR of RNA derived from E. ictaluri grown in BHI detected low levels of transporter mRNA in the WT but not the mutants, as measured by qPCR.  The transporter proteins, however, were undetectable by Western blot analysis of E. ictaluri whole-cell lysates.  The live challenge results suggest a role for the urea and ammonia transporters in the pathogenesis of E. ictaluri, but further phenotypic analysis suggests that the effects are subtle and difficult to measure.  Experiments are underway to detect the transporter proteins in isolated bacterial membranes and to directly measure pH changes in HKDM infected with either WT or transporter mutants using fluorescent microscopy and ratio-metric imaging







High Total Ammonia Nitrogen Concentrations Increase Survival of Channel Catfish Experimentally Infected with Flavobacterium columnare


AJ Mitchell* and B Farmer


Harry K. Dupree Stuttgart National Aquaculture Research Center, United States Department of Agriculture/Agricultural Research Stuttgart, AR USA drew.mitchell@ars.usda.gov




Although it is generally accepted that elevated ammonia levels in the water increase mortalities in Flavobacterium columnare-infected fish, recent observations at our laboratory have indicated otherwise.  Two similar studies were set up to determine the effect of a single high-level treatment of total ammonia nitrogen (TAN: 15 mg/L; un-ionized ammonia: 0.4 mg/L) on the survival of channel catfish experimentally infected with F. columnare (strain LV-359-01 at ~1x108 bacteria/mL of water).  In both studies, channel catfish were stocked at 50 g/L in 10 L of water with four water exchanges/d and were given four replicates of one of the following treatments: no ammonia or bacterial challenge (Treatment 1), ammonia only (Treatment 2), bacterial challenge only (Treatment 3), or both ammonia and bacterial challenge (Treatment 4).  As trial and trial x treatment interaction effects between the two studies were not significantly different, survival data from the two studies were combined in the final determination of differences among treatment means.  There was no significant difference between the survival of fish that received Treatment 1 (95.2±1.2%) versus Treatment 2 (95.6±1.0%).  These two treatments were significantly different from Treatments 3 (8.5±4.5%) and 4 (41.8±12.7%), and fish that received Treatment 4 had a significantly higher survival rate than those that received Treatment 3.  At the TAN levels tested in this study, ammonia itself did not negatively influence survival of the fish but did either limit the survival of F. columnare or otherwise interfere with the infection process.







Ultrastructure of Skin Lesions on Channel Catfish, Ictalurus punctatus Exposed to Flavobacterium columnare– Insights on Skin Discoloration and Epidermal Damage


A McElwain*1, O Olivares-Fuster, CR Arias2 and SA Bullard1


1 Departmentof Fisheries and Allied Aquacultures (FAA), Aquatic Parasitology Laboratory (APL), 203 Swingle Hall, Auburn University (AU), Auburn, AL azm0034@auburn.edu
2 FAA,Aquatic Microbiology Laboratory, crarias@auburn.edu





Flavobacterium columnare, a Gram-negative rod-shaped bacterium, is the causative agent of “columnaris disease” and a serious pathogen of freshwater fishes in aquaculture.  A columnaris infection is typically characterized by the presence of an opaque, dorso-lateral band referred to as “saddleback” lesion.  Although this is a well-known sign of infection, little detailed information is available regarding how this lesion develops in channel catfish, Ictalurus punctatus.  In an effort to better understand columnaris pathogenesis, we used the JCM-21327 strain of F. columnare in an immersion challenge of specific pathogen free channel catfish and compared skin samples from infected and control fish using scanning electron microscopy (SEM).  SEM samples were fixed in 10% neutral buffered formalin for 48 hrs, rinsed in distilled water, dehydrated in a graded ethanol series using an automated tissue processor, immersed in hexamethyldisilazane, and mounted on metal stubs before being sputter coated with gold-palladium.  In our samples, saddleback lesions generally developed dorso-laterally, between the posterior margin of the opercle and the adipose fin.  A ragged epidermal margin consisting of wispy tissue presumed to be epidermal remnants comprised the boundary between infected and uninfected skin.  Infected skin appeared to be devoid of epidermis, and rod-shaped bacteria were present on the exposed dermis.  Bacterial cells on the dermis were 0.20–0.40 mm ´ 8–10 mm and loosely arranged.  The foregoing represents a step towards understanding the development of skin lesions during a columnaris infection.  These results support the notion that saddleback discoloration results from epidermal obliteration in infected catfish.







Expression Profiles of Cobia (Rachycentron canadum) Spleen and Head Kidney Following Vaccination Against Vibrio carchariae, Proteus vulgaris and Streptococcus sp.


J J Feng*1, Z Guo1, Y Su1, L Xu1, J Wang1, Y Hou1,2 and L Mao1,2


1 Department of Fish Disease, South China Sea Fisheries Research Institute, CAFS. Guangzhou, Gangdong, China  janej_feng@msn.com, guozhixun1@163.com, vetsyl@163.com, lwxu@163.com, wjy104@163.com
2 Department of Veterinary Science, South China Agriculture University, Guangdong Province, Guangzhou, 510642, P R China  houyifan920@126.com, maolina2003@163.com







To investigate the response of cobia to vaccination and pathogen exposure, a full-length cDNA library was constructed from head kidney and spleen mRNA collected following vaccination against Vibrio carchariae, Proteus vulgaris and Streptococcus sp. After sequencing 6012 readable sequences were obtained of which 854 clones (14.20%) contained cell/organism defence genes including immunoglobulin, MHC class I and II, interferon consensus sequence binding protein, B-cell receptor-associated protein, early B-cell factor, 10 complement components, heat shock protein 70 and 90, antimicrobial peptides hepcidin type 1 and 2, and CC chemokine (macrophage inflammatory protein-1 beta-like chemokine, MIP-1beta). These results indicate that this cDNA library contains many important genes involved in the immune response, making it an important resource for studying the response of cobia to vaccination or pathogen exposure. At the same time, the characteristic of Ig k and µ chain, MHC II α and β were primarily studied.






Expression Attenuated Francisella asiatica iglC Mutant Induces Protective Immunity to Francisellosis in Tilapia


E Soto*1, J Wiles1, PH Elzer1, 2, KR Macaluso1 and JP Hawke1


1 Departmentof Pathobiological Sciences, LSU-School of Veterinary Medicine, Dr., Baton Rouge, LA, USA esoto1@lsu.edu, jwiles@vetmed.lsu.edu, Kmacaluso@mail.vetmed.lsu.edu jhawke1@lsu.edu
2 LSUAgCenter, Department of Veterinary Science, Baton Rouge, Louisiana,, USA pelzer@agctr.lsu.edu 





Francisella asiatica is a Gram negative, facultative intracellular bacteria that causes fish francisellosis. Fish francisellosis is a severe sub-acute to chronic granulomatous disease with high mortalities and high infectivity rates in cultured and wild fish. To date, there is no approved vaccine for this widespread emergent disease. The goal of this study was to characterize the efficacy of a defined F. asiatica mutant (ΔiglC) as a live attenuated vaccine against subsequent immersion challenge with the wild-type (WT) organism. In previous work, the ΔiglC was found to be attenuated upon intraperitoneal injection and immersion challenges. In vitro, the ΔiglC exhibited reduced growth in tilapia head kidney derived macrophages, and was significantly attenuated (p<0.001) as demonstrated by cytopathogenic and apoptosis assays. In this study, the ΔiglC was tested to determine its ability to protect tilapia against challenge with high doses (lethal dose 80) of WT bacteria. Naive tilapia vaccinated by immersion with a suspension of the ΔiglC and subsequently challenged with WT F. asiatica were protected (90% mean percent survival) from the lethal challenges. F. asiatica-specific antibodies produced in response to immunization with the ΔiglC were subsequently found to protect naive tilapia against high-dose F. asiatica challenge in passive immunization experiments. Significant protection (p<0.05) was obtained when fish were passively immunized and challenged with 104 and 105 CFU/fish of WT F. asiatica; but not when challenged with 106 CFU/fish. This is the first report of a defined live attenuated strain providing protection against F. asiatica in fish.






An Experimental Vaccine Against Aeromonas hydrophila Can Induce Protection in Rainbow Trout


SE LaPatra*1, KP Plant2, WD Shewmaker1, RA Burkhart1 and AW Morton1    

1 Clear Springs Foods, Inc., Research Division, Buhl, ID, USA scottl@clearsprings.com
2 Hagerman Fish Culture Experiment Station, University of Idaho, Hagerman, ID, USA kplant@uidaho.edu




A candidate vaccine against Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss) was developed using a bacterial lysate. To test the strength of protection, A. hydrophila challenge models were compared using injection into both the peritoneal cavity (IP) and the dorsal sinus (DS) with selected doses of live bacteria washed in saline or left untreated. Unlike the IP route, injection into the DS with either saline washed or unwashed cells resulted in consistent cumulative mortality and a clear dose response pattern that could be used to establish a standard challenge having an LD50 of approximately 3 x107 colony forming units per fish. Survivors of the challenge suffered significantly lower mortality upon re-challenge than naïve fish, suggesting a high level of acquired resistance was elicited by infection. Passive immunization using serum from hyper-immunized fish also resulted in significantly reduced mortality indicating protection can be transferred and that some portion of resistance may be antibody mediated. Vaccination of groups of rainbow trout with A. hydrophila lysate resulted in significant protection against a high challenge dose but only when injected along with Freunds complete adjuvant. At a low challenge dose, mortality in all groups was low, but the bacterial lysate alone appeared to offer some protection.







Confirmation of Safety and Efficacy for a Live Attenuated Flavobacterium psychrophilum Vaccine


TJ Johnson*1, N Parvez1, DR Call2, BR LaFrentz3, T Miura4 and KD Cain1


1 Departmentof Fish and Wildlife Resources, University of Idaho,  Moscow, USA john3002@vandals.uidaho.edu, nparvez@uidaho.edu, kcain@uidaho.edu  
2 DepartmentofVeterinary Microbiology and Pathology, Washington State University, Pullman, WA  USA drcall@vetmed.wsu.edu
3 USDA-ARSAAHRU, Auburn, AL USA benjamin.lafrentz@ars.usda.gov
4 Departmentof Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID USA tmiura@uidaho.edu  










Bacterial coldwater disease (CWD) caused by Flavobacterium psychrophilum causes severe economic losses in both private (food fish) and public aquaculture facilities.  A live attenuated F. psychrophilum strain has recently developed and shows promise as a CWD vaccine.  This strain, 259-93B.17 (B.17) was further evaluated for safety and efficacy.  The B.17 strain was passed 5X in rainbow trout (Oncorhynchus mykiss) via immersion and injection at approximately 10X the reported commercialization dose.  No mortality was observed during the trials and fish did not develop clinical signs of disease, indicating that there is no risk of reversion to virulence.  The B.17 strain was re-isolated in the immersion group at the 4th and 5th passage and in the injection group at the 3rd, 4th, and 5th passage showing that it was able to survive in fish for a period of time.  Evaluation of the efficacy of the B.17 strain was expanded from earlier studies to include feed delivery as an additional mass vaccination strategy.  Immunization groups included injection, immersion (with and without adipose fin clip), and oral delivery of B.17 on feed in fish oil.  Rainbow trout were immunized at 0.7 g and boosted at 4 and 6 weeks post initial vaccination before challenging fish at 8 weeks.  Serum samples were collected before the first booster vaccination and at the time of challenge to determine the antibody (Ab) response.  Results showed the highest protection and Ab titers (1707) in fish injection immunized with a relative percent survival (RPS) of 91%. Fish immunized by immersion or feed delivery had RPS values between 20% and 23%.  







Development, Characterization and Early Evaluation of New Modified Live Vaccines Against Columnaris Disease


CR Arias* and O Olivares-Fuster


Departmentof Fisheries and Allied Aquacultures, Auburn University, AL USA ariascr@auburn.edu



Columnaris disease, caused by Flavobacterium columnare, has been described as a globally-distributed, acute to chronic, bacterial infection of freshwater and brackish water fish. Many commercially important species are affected by columnaris disease, including channel catfish (Ictalurus punctatus Rafinesque), common carp (Cyprinus carpio Linnaeus), rainbow trout (Oncorhynchus mykiss Walbaum) and other salmonids, Japanese eel (Anguilla japonica Temminck & Schlegel), tilapia (Oreochromis spp.) and a variety of ornamental species such as goldfish (Carassius auratus Linnaeus). In aquaculture settings, columnaris outbreaks are often related to environmental stress associated with high stocking densities, increased feeding rates, elevated organic loads and high temperatures. Given the ubiquity of F. columnare in aquatic environments, it is unrealistic to consider eradication as an option, but prevention through vaccination has the potential to lower the impact of columnaris disease. This study aimed at developing new avirulent mutants from highly virulent strains of F. columnare, which could be used as modified live vaccines against columnaris disease. Thirteen rifampin-resistant mutants from F. columnare genomovar II were obtained and characterized. Mutants were confirmed to be stable, and avirulent for channel catfish. Genetic and phenotypic differences between parent and mutant strains were identified and found to be mutant-specific. Vaccination experiments of channel catfish showed that all tested mutants offered significant protection against columnaris disease with relative percent survivals between 26% to 50%. Two of the new mutants showed greater protection than the commercial vaccine, AQUAVAC-COLÓ.







Initial Evaluation of an Edwardsiella ictaluri Type III Secretion System Mutant as a Vaccine Candidate Against Enteric Septicemia of Catfish


ML Rogge*1 and RL Thune1,2


1 Departmentof Pathobiological Sciences, LSU School of Veterinary Medicine, Skip Bertman Drive, Baton  Rouge, LA 70803 USA mrogge1@lsu.edu
2 DepartmentofVeterinary Science, Louisiana State University, 111 Dalrymple Building, Baton Rouge, LA 70803 USA thune@vetmed.lsu.ed






Although Edwardsiella ictaluri encodes a type III secretion system (T3SS) that is required for virulence in channel catfish (Ictalurus punctatus) and for intracellular replication within channel catfish cells, the mechanisms for these capabilities are not yet known. In this study, a putative T3SS regulatory gene, esrC, was mutated and analyzed for its ability to replicate in channel catfish head kidney-derived macrophages (HKDM), persist in channel catfish tissues, and provide protection against wild-type E. ictaluri challenge. The esrC mutant strain replicated as well as the wild type strain in HKDM, and while colonization of the channel catfish head kidney was similar to that of the wild-type, there were significantly fewer mutant bacteria present in the head kidney three days post-infection than for the wild-type, with mutant persistence extending to six days post-infection. In spite of intra-macrophage replication and in vivo persistence for six days, the esrC mutant caused no mortality in channel catfish compared to 64% mortality by the wild-type. Subsequent challenge of the esrC mutant survivors with wild-type E. ictaluri 28 days after the initial challenge resulted in a relative percent survival of 81%. The results of these data demonstrate the potential of an esrC mutant as a vaccine candidate for enteric septicemia of catfish.


SESSION 3. PARASITOLOGY I: Myxozoan Workshop 1  [TOP]




An Overview of Myxozoan Parasites Found in Smallmouth Bass (Micropterus dolomieu) from Three Tributary Rivers of the Chesapeake Bay, USA


HL Ellery*, VS Blazer, LR Iwanowicz and DD Iwanowicz


United States Geological Survey, National Fish Health Research Laboratory, Kearneysville, WV, USA hellery@usgs.gov, vblazer@usgs.gov, liwanowicz@usgs.gov, diwanowicz@usgs.gov

The health of aquatic organisms is commonly used as a biological indicator of environmental pollution.  Over the past eight years, the Shenandoah and Potomac Rivers have experienced serious kills/skin lesions of adult fishes, indicating that the health of these river systems is in some way being compromised.   The Susquehanna River in Pennsylvania has also dealt with the mortality of young of the year bass.  Many theories on the cause of these fish kills have included low dissolved oxygen, fluctuations in pH, contaminants, agricultural runoff, bacteria and parasites.  The occurrence of some helminth parasites in wild fishes is normal, but a high intensity in tissues may cause debilitation and contribute to mortality. Smallmouth bass (Micropterus dolomieu) were collected from selected sites and a full health examination was conducted using chemical, bacterial, histopathological and parasite analyses.  The existence and abundance of parasite infestation was reviewed with histological techniques, morphological identification, and DNA sequencing.  One type of parasite that was often identified in the smallmouth bass was myxozoans parasites.  Myxozoan parasites were observed in macrophage aggregates, kidney tubules, gills, skin, and to a lesser extent in the bile duct of the liver.  As with many types of parasites, low intensity infections of myxozoans may have minimal effect on the health of a fish, but when infections become extreme, mortality can occur.  Histological results indicate heavy infections of the myxozoan in the gills and bile ducts may have had an impact on the health of the adult bass, while a third species may contribute to lesions and mortality of the young.  The use of DNA sequencing has revealed that at least one of the myxozoans is possibly a new species.  Seasonal and site comparisons have been made in an effort to better understand the dynamic between the fish kills and myxozoan parasites. 





Sphaeromyxa sp.  (Myxosporea:  Sphaeromyxidae), a Parasite of Lined Seahorses, Hippocampus erectus, from the Gulf of Mexico


B F Sears*1, P Anderson2 and E C Greiner3


1 Department of Integrative Biology, University of South Florida, Tampa, USA bsears@mail.usf.edu
2 The Florida Aquarium, Inc., Tampa, Florida, USA panderson@flaquarium.org
3 College of Veterinary Medicine, University of Florida, Gainesville, FL  USA greinere@vetmed.ufl.edu  





A Sphaeromyxa species is described from the bile ducts of aquaria-maintained lined seahorses (Hippocampus erectus) from the Gulf of Mexico. Spores are linear, 17-18 μm long and 5-6 μm wide, with flattened tips; polar capsules measure 4 x 3 μm. The morphmetrics appear distinct from previously described species of Sphaeromyxa. Necropsies of healthy H. erectus following euthanasia revealed liver pathology and myxozoan parasites in the bile ducts of 11 of the 40 animals sampled (27.5%). The presence of Sphaeromyxa sp. in an aquaculture setting should prompt keepers to carefully quarantine new animals and exclude annelid fauna, a potential intermediate host of myxozoans, from aquaria. A manuscript describing this parasite, and proposing to erect a new species for it, has been submitted for publication.






Henneguya zschokkei s.l. (Myxozoa) in Whitefish (Coregonus lavaretus) in SW Finland, and Possible Interaction between Wild and Farmed Fish


H-P Fagerholm*1, B Szostakowska2 and T Wiklund3


1 Laboratory of Aquatic Pathobiology, Department of biosciences, Åbo Akademi University, Åbo/Turku, Finland hafagerh@abo.fi, twiklund@abo.fi
2 Medical University of Gdańsk, Department of Tropical Parasitology, Interfaculty Institute of Maritime and Tropical Medicine, Gdynia, Poland bszost@gumed.edu







The myxozoans seriously threatening whitefish farming in Finland has been studied. Our preliminary results suggests the presence of two genetically isolated parasite types, one from brackish water, and one from freshwater sites. The parasites form cysts in the muscles the fish host. Both appear different from forms previously described, and are noted here as Henneguya zschockei sensu lato. Based on knowledge of the life-history of the parasite (where the invertebrate annelid recently has been isolated by Saarikoski et al., 2010) it is of considerable importance to find control strategies in specific cases. In a project aimed at establishing the identity, extent of infection, and strategies for preventing the infection of Henneguya zschokkei sensu lato in farmed whitefish Coregonus lavaretus, both fresh and brackish water fish farms in Finland were studied from 2008 - 2010. The prevalence of the infection ranged from 0% to 55%. In one case, the origin of the infection most probably stemmed from a specific fresh water area from which the fish had been brought for further growth in a brackish water site. By defining the likely origin of the infection, control of the infection can be considered.  Development of control methods is important because of the recent increase in farming of whitefish. Infected fish cannot be used for human consumption because of hygienic and aesthetic reasons. The infection results in heavy economic losses for the farmers and the fish industry.  






Application of a Real-time PCR Assay for the Detection of Henneguya ictaluri in Channel Catfish Ponds


MJ Griffin*1, LM Pote2, AC Camus3, MJ Mauel1, TE Greenway1 and DJ Wise1


1 Thad Cochran National Warmwater Aquaculture Center, Mississippi State University, Stoneville, MS  USA
2 Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS USA
3 Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia USA







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 PCR (QPCR) assay was developed to determine parasite levels in commercial channel catfish ponds and evaluate the risk of losing fish when ponds are initially stocked or when ponds are restocked following PGD epizootics. Water samples collected on 2 separate days with a minimum of 1 week between sampling, not only determine the approximate concentrations of actinospores in the pond but also lends insight into whether these parasite levels are increasing, decreasing or staying the same.  Increases in mean actinospore concentrations correlated with increased mortality in sentinel fish (r = 0.63; p = 0.003).  Direct detection and quantification of the organism in pond water is more rapid than current protocols for evaluating the PGD status of a catfish pond, which rely on the use of sentinel fish.  This work identified actinospore levels that correlate to both high and low risk of losing fish to PGD.  This assay also provides a method of determining the overall prevalence of H. ictaluri throughout the industry, significantly reducing the labor associated with sentinel fish trials.  Current research regarding the year-round prevalence of Henneguya ictaluri in catfish ponds will also be discussed.   





Predicting Salmon Mortality from Ceratomyxa shasta by Measuring Parasite Densities in Water Samples


SL Hallett*, RA Ray, RA Holt, SD Atkinson and JL Bartholomew


Department of Microbiology, Nash Hall 220, Oregon State University, Corvallis, OR USA halletts@science.oregonstate.edu


The myxozoan parasite, Ceratomyxa shasta, is responsible for losses among both wild and hatchery-reared juvenile salmonids.  It is a significant pathogen in the Klamath River, where it has been identified as a key factor limiting salmon recovery.  Monitoring studies involving sentinel fish exposures, polychaete sampling, and water analysis have increased our understanding of the temporal and spatial distribution of the parasite and have moved us closer to reaching management goals of reducing mortality to below 40% in native stocks.  Water sampling in particular has proven to be a simple, high-resolution tool to determine and map total parasite density (number of spores per liter) in river water.  We have now used combined water sampling and fish exposures to address the following question: how does parasite density relate to population effects in each salmon species?  In other words, can we identify threshold dose levels?  We exposed different salmon species in cages at five lower Klamath River index sites in the spring, summer, and fall from 2005 through 2009.  In parallel, we collected triplicate 1-L water samples on the first and final days of the three-day exposures.  Fish were then held at the Salmon Disease Laboratory (OSU) for up to 90 days, and mortality due to C. shasta and mean day to death were recorded. Water samples were filtered; the captured DNA, extracted; and C. shasta, quantified by TaqMan qPCR.  We are now correlating the two data sets to discover patterns of fish mortality, parasite density, parasite genotype, water temperature, and flow.




Evaluation of the Risk of Myxobolus cerebralis Introduction as a Result of Fish Passage


CM Zielinski, SL Hallett and JL Bartholomew*


Department of Microbiology, Oregon State University, Corvallis, OR Halletts@science.oregonstate.edu, bartholj@science.oregonstate.edu




Dams along the Deschutes River (DR) in central Oregon, USA, have blocked fish migration for over 40 years.  Re-establishment of anadromous fish runs above the dams, as part of a fish passage plan, may introduce fish pathogens such as Myxobolus cerebralis.  This myxozoan parasite, which causes salmonid whirling disease, is carried into the DR basin by adult salmon that stray during their return to enzootic areas of the upper Columbia River basin, and we have confirmed that it has become established in at least one lower DR tributary.  To determine the likelihood of parasite establishment above the dams, we conducted benthic sediment surveys between 1998 and 2007.  We found that T. tubifex, an oligochaete worm that is one of two obligate hosts for M. cerebralis, had a patchy distribution and low relative abundance.  Mitochondrial 16S rDNA gene analysis indicated that two lineages of T. tubifex, III and VI, were present both above and below the dams.  Laboratory susceptibility studies, designed to characterize differences in infection prevalence and parasite production between nine T. tubifex populations, revealed that production varied considerably among exposed groups and was proportional to the number of lineage III worms present.  Our results suggest that M. cerebralis could become established above the dams if infected fish are allowed passage.  Not all areas of the Deschutes River basin have the same likelihood for parasite establishment, however; thus the potential impact will be location-dependent.





Myxobolus cerebralis in Fishes in Reservoirs of Kamchatka


TV Gavruseva*


Fish and Shellfish Disease Laboratory, Kamchatka Research Institute of Fishery and Oceanography (KamchatNIRO), 18 Naberezhnaya Street, Petropavlovsk-Kamchatski, 683000 Russia  gavrt2004@mail.ru




The myxosporidian Myxobolus (syn. Myxosoma) cerebralis, the agent of whirling disease, developing within the cartilage of farmed salmonids including trout, can cause skeletal deformities, whirling behaviour and mortality. There is little known about the occurrence of this parasite in wild fish in the Kamchatka region of Russia. Previously M. cerebralis was found in reared fingerlings of Oncorhynchus keta and Oncorhynchus gorbuscha (Ushkovskii hatchery, Kamchatka) (Bogdanova, 1970). Then for many years (1989 to 2006) this pathogenic agent was not recorded in the reservoirs of Kamchatka, nor in salmonids in the Far East (Boutorina, 2008). However, in 2000 to 2010, histological and histochemical examinations of 2,750 wild and cultured young salmonids (O. keta, Oncorhynchus kisutch, Oncorhyncus nerka, Oncorhynchus tschawytscha) and trout (Salvelinus malma) from Kamchatka during revealed M. cerebralis in wild fingerlings of sockeye salmon, with pathological signs only from Lake Nachikinskoe (western sea coast of Kamchatka) in August 2006. In August 2009, spores of M. cerebralis were determined in the dead nine-spined stickleback Pungitius pungitius from Lake Nalychevskoe (eastern sea coast of Kamchatka). Pungitius pungitius is a typical hydrobiont of Kamchatka rivers and lakes. Histological examination of sockeye salmon showed that spores and developing stages of the parasite caused local necrosis and lysis of the chondrocytes. There was marked formation of epithelial granuloma around the affected area of a tissue. The cartilaginous tissue of a spinal column of nine-spined stickleback has been completely replaced with mature spores of the parasite without granuloma formation. We suggest that P. pungitius is natural carrier and source of M. cerebralis for salmonid fish.











The National Wild Fish Health Survey: Selected Findings and Limitations


SL Mumford*


Olympia Fish Health Center, US Fish and Wildlife Service, Olympia, WA 98506 USA Sonia_mumford@fws


Since 1996, the USFWS with the help of partnering organizations has been collecting wild fish and testing for specific fish pathogens.  The pathogens selected have been important in cultured fishes, and additional pathogens are added as needed.  The nine USFWS fish health centers use standardized methods for assays, which makes comparisons between species, areas, and watersheds possible.  The major goal of the National Wild Fish Health Survey (NWFHS) is to determine the geographical distribution of certain pathogens in wild fish. With a better understanding of pathogen distribution, managers can better assess the risks and benefits of stocking and fish transport activities.  Data collected from the National Wild Fish Health Survey has been or is currently being used for: management decisions such as broodstock selection and egg distribution for restoration programs, assessing interactions between wild and hatchery fishes, monitoring dam removal projects (ie Elwha, and White Salmon River), and surveillance for exotic pathogens (ie Spring Viremia or Carp Virus (SVCV)) along with other potentially devastating viruses (ie Viral Hemorrhagic Septicemia Virus).  Other useful information from the NWFHS includes unexpected findings such detection of pathogens in species not previously thought to be susceptible (ie SVCV in Bluegill and Largemouth bass). While much information can be gained from the NWFHS, there are also limitations.  The data provides a snapshot in time at a specific location in the species of fish that are collected.  Since only limited funds are available for collection of fish, selection of sampling sites have largely been opportunistic, and fish numbers are not always ideal.  Through the support of our partners, there are some sites that provide statistically significant data, while other sites can only supply supportive data.  Due to sampling biases (difficulty in collecting clinically ill wild fish), differences in number of fish collected, and numbers of sampling locations over time, one should not assume the data shown reflects prevalence data or an emergence of a given pathogen.






Demonstration of New Web Interface for the National Wild Fish Health Survey


KK Peters*1 and JL Bradley2


1 US Fish and Wildlife Service, Bozeman Fish Health Center, Bozeman, MT, USA ken_peters@fws.gov
2 US Fish and Wildlife Service, Division of Fisheries and Aquatic Resource Conservation, Idaho Fish Health Center, Orofino, ID USA





Beginning in 1996, the U. S. Fish and Wildlife Service (Service) has been working with aquatic resource partners, under auspices of the National Wild Fish Health Survey (Survey), to better understand the occurrence of important fish pathogens in free-ranging fish.  During this time, nearly 220,000 fish have been sampled from some 4,600 distinct sample sites, representing 2,560 bodies of water.  Samples are tested using standardized protocols and procedures for the presence or absence of fish pathogens.  Data generated by these tests are managed in regional databases by the Service’s network of nine fish health laboratories.  Additionally, spatial information including state, county, hydrologic unit code, latitude and longitude, and name of water body are recorded for each sample site.  Records from regional laboratories are periodically uploaded to a centralized national database residing on a dedicated server in Denver, Colorado.  In June 2010, the Service released a new, public, web-based interface for searching and displaying information from the national database.  The interface is designed to display search options, tabular search results, and an interactive map simultaneously in a web-browser.  Users can download case reports and create custom maps of search results.  Search results may also be downloaded for use in spreadsheet applications (CSV) or “earth-browsers” such as Google Earth® (KML).  The web interface is accessed by navigating to the Survey homepage at [http://www.fws.gov/wildfishsurvey] and then clicking on the “National Wild Fish Health Survey Database” button, followed by “Start the Database” button.




Results of Fish Health Assessments of Glass Eels, Anguilla rostrata, from Canadian Maritime Rivers from 2006-2010


D Groman*1, R Threader2, D Wadowska3, T Maynard2 and L Blimke4


1 Aquatic Diagnostic Services, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Canada. groman@upei.ca
2 Ontario Power Generation, Renfrew, ON Canada.  liisa.blimke@opg.com  ron.threader@opg.com
3 Electron Microscopy Laboratory, Atlantic Veterinary College, University of Prince Edward Island  Charlottetown, Canada  wadowska@groupwise.upei.ca
4 Kleinschimidt Associates, Essex, CT 06426 USA.  Tracy.Maynard@kleinschmidtusa.com









This paper reports on the results of fish health assessments for newly captured glass eels (Anguilla rostrata) from Nova Scotia and New Brunswick in Atlantic Canada, which were part of a regional re-stocking program for Lake Ontario from 2006-2010.  Glass eel populations were segregated by watershed or river of capture, held in quarantine and subsequently assessed for selected virology, bacteriology and parasitology.  Results of virus isolation and molecular assays of pooled samples from all years were negative for all viruses of interest.  Bacteriological culture of the 2009-10 pooled samples revealed one lot, held on surface water prior to sampling, was harboring Yersinia ruckeri.  Gross parasitological examination revealed sporadic regions of integumental hyperplasia, that were histologically determined to be trophozoite stages of the ciliate Ichthyophthirius multifiliis.  Histopathological examination of individuals from all lots revealed necrotizing hepatitis and associated intranuclear microsporidian, morphologically consistent with a Nucleospora sp.  One lot of older elvers from the estuary of the St. Mary’s River in Guysbourgh County, Nova Scotia, was found to be harboring larval and pre-adult nematodes in association with the wall and lumen of the gas bladder.  These nematodes were morphologically consistent with Anquillicoloides crassus. In addition, this lot was infected with an as yet identified Myxosporidian infection of the urethra and urinary bladder.  The significance of these results will be discussed.



Cutthroat Trout Virus Surveys in New Mexico and Arizona


TD Lewis*, EM Rodarte, and DD Hampton


Dexter Fish Health Center, USFWS Region 2, P. O. Box 219, Dexter NM  88230 USA teresa_lewis@fws.gov, marlene_rodarte@fws.gov, dave_hampton@fws.gov





Although cutthroat trout virus (CTV) has been characterized as a benign virus in common trout species, there is a lack of knowledge relating to the susceptibility and transmission potential of this virus in listed trout species, specifically Apache, Gila, and Rio Grande cutthroat trout in the southwest United States.  Enhanced CTV surveillance has been ongoing in federal hatcheries rearing trout in New Mexico and Arizona since 2008.  Tissue culture diagnostics conducted throughout 2009 did not identify any viral cytopathic effect (CPE).  In 2010, ovarian fluid from trout at federal and state hatcheries in Arizona and New Mexico developed CPE characteristic of CTV and was confirmed positive for the virus using RT-PCR.  An experiment using archived Apache trout ovarian fluid that was negative by cell culture tested CTV positive using RT-PCR.  Since that time, the USFWS and partners have been conducting wild fish health surveys of native trout and surrogates (brown trout, hybrid cutthroat trout, others) to establish a baseline for CTV in the southwest.  Cell culture and RT-PCR methods have been used in tandem for this baseline.   While diagnosis of CTV continues to rely upon isolation in cell culture there is mounting evidence that viral cDNA may be amplified in samples that do not evidence CPE in culture.  Despite the apparent avirulent nature of CTV, concerns linger over its potential impacts in ways that do not clearly result in morbidity or mortality (i.e., effects on reproduction, stress responses, immunocompetence), especially in threatened or endangered species. 







Epidemics and Control Strategies for Diseases of Farmed Salmonids: A Parameter Study


ART Jonkers1,2, KJ Sharkey3, M Thrush4, JF Turnbull5 and K Morgan*6


1 Institute for Geophysics, University of Münster, Corrensstrasse 24, Münster 48149, Germany
2 Department of Earth & Ocean Sciences, University of Liverpool, 4 Brownlow Street, Liverpool, L69 3GP, UK
3 Department of Mathematical Sciences, University of Liverpool, Liverpool, L69 7ZL, UK
4 Centre for Environment Fisheries and Aquaculture Science (Cefas), Barrack Road, Weymouth DT4 8UB, UK
5 Institute of Aquaculture, University of Stirling, Stirling, Stirlingshire, FK9 4LA, UK
6 Department of Veterinary Clinical Science, University of Liverpool, Leahurst, Neston, CH64 7TE, UK







Simulated contact networks can help us understand properties of epidemics including dynamics and risk, as well as their sensitivity to changes in likelihood of transmission delays, and mitigation measures. When such simulations closely reflect observed reality, inferences may be drawn regarding the relative importance of specific parameters that have direct implications for biosecurity and disease control policy decisions. Here we present a large-scale simulation study of the English and Welsh fish farming and fisheries industry, comprising 2,090 sites as recorded in 2004. We explore this network’s resilience for different conditions and mitigation strategies. A survey of stream flow speeds demonstrated that transmission via rivers takes less than two days on average. Directed river and transport contacts in this network causes the risk of outward transmissions to be geographically more confined than that of incoming transmissions.  Balanced analysis of variance shows that detection delay is the most important outbreak-limiting factor, followed by laboratory capacity and culling delay. Furthermore, a reactive policy with some additional proactive dangerous-contact tracing, performs better than either strategy on its own. Finally, we distinguish fish farm sites from fisheries in terms of specific biosecurity weaknesses, and derive some general, quantified policy guidelines to mitigate future epidemics on this network.









Development of a U.S. National Fish Health Laboratory Network

AE Goodwin*1, JB Rolland2 and KR Snekvik3

1 University of Arkansas at Pine Bluff, Aquaculture/Fisheries Center, Pine Bluff, AR USA agoodwin@uaex.edu
2 USDA/APHIS/VS/ASEP, Riverdale, MD USA Jill.B.Rolland@aphis.usda.gov
3 Washington Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Washington State University, Pullman, WA, USA ksnek@vetmed.wsu.edu







An important part of the new U.S. National Aquatic Animal Health Plan (NAAHP) is the development of a national network of fish health laboratories that will work together to protect the health of wild and cultured fish and shellfish, provide quality inspections in support of interstate and international trade, and meet challenges associated with implementation of the NAAHP.  Development of the network has been a joint project undertaken by the U.S. Animal Health Association, the American Association of Veterinary Laboratory Diagnosticians, and the three federal co-competent authorities for fish health: the U.S. Department of Agriculture's Animal and Plant Health Inspection Service, the National Oceanic and Atmospheric Administration, and the U.S. Fish and Wildlife Service.  Implementation of the network is directed by a steering committee composed of these three federal agencies, aquatic animal diagnostic laboratories, and the aquaculture industry.  The network is founded on the following principles: 1) membership in the network may include all qualified state, federal, academic, extension, and private laboratories; 2) laboratories will use a single standardized protocol; 3) there will be quality assurance systems in place; 4) there will be a reporting system in place; 5) results from the laboratories will be recognized by the federal authorities as appropriate for export inspections; and 6) labs will only remain eligible for participation in the network if they meet training and proficiency requirements.  The network will begin with a trial period that will include only a protocol for the detection of VHSV IVb in cell culture with an RT-PCR confirmatory test. 







ICHV-2 (the Other Catfish Herpesvirus): Detection by qPCR


AE Goodwin1*, E Marecaux1 and LA Hanson2


1 University of Arkansas at Pine Bluff, Aquaculture/Fisheries Center, Pine Bluff, AR USA agoodwin@uaex.edu
2 College of Veterinary Medicine, Mississippi State, MS USA hanson@cvm.msstate.edu





Channel catfish herpesvirus (Ictalurid herpesvirus-1 or IcHV-1) is a common and cosmopolitan pathogen that causes sporadic mortality in young-of-the-year channel catfish during hot summer months in the Southeast USA.  Less well know is Ictalurid herpesvirus-2 (IcHV-2), a pathogen of cultured black bullhead (Ameiurus melas) that produces high mortality in experimental exposures to channel catfish (Ictalurus punctatus).  During acute infections, the virus grows readily in cell cultures but produces a cytopathic effect (CPE) similar to that of IcHV-1 and the channel catfish reovirus.  We have developed a quantitative PCR assay that can be used to detect IcHV-2 in fish tissues and cell-culture supernatants.  The assay does not amplify other fish herpesviruses tested, or host DNA.  It is quantitative over a range of eight logs, and the limit of detection is less than 10 copies per reaction.  In replicate assays carried out on different days, the coefficient of variability was 10%.  Our assay detected acute IcHV-2 infections best in the spleen and kidney.  We have used it to screen a large collection of U.S. catfish herpesvirus isolates, and have confirmed them all to be IcHV-1.   This assay should be useful for the diagnosis of IcHV-2 disease, the identification of syncytial CPEs in cell cultures, and the detection of latent infections in carrier fish.




Use of Diagnostic Assays to Screen Rainbow Trout (Oncorhynchus mykiss) Broodstock for Flavobacterium psychrophilum


A Long*1, DR Call2 and KD Cain1


1 Department of Fish and Wildlife Resources, University of Idaho, Moscow, ID USA long8524@vandals.uidaho.edu;  kcain@uidaho.edu
2 Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA USA drcall@vetmed.wsu.edu






Flavobacterium psychrophilum, the causative agent of Bacterial Coldwater Disease (BCWD), affects several economically important species of fish in the Pacific Northwest.  F. psychrophilum has been shown to survive both on the exterior of eggs and in fluid from within the egg indicating it may be vertically transmitted.  To further evaluate this, our lab has developed an ELISA and membrane filtration fluorescent antibody test (MF-FAT) using a monoclonal antibody specific to F. psychrophilum.  Evaluation of these diagnostic assays as tools for managing BCWD is underway.  Sixty rainbow trout broodstock were screened for F. psychrophilum using various diagnostic assays including ELISA and MF-FAT.  Five families showing F. psychrophilum infection levels ranging from below detection limits to 106 CFU ml-1 were selected for further experiments.  Eyed eggs were shipped to UI and fry are currently being reared on pathogen-free water and sampled for F. psychrophilum on a weekly basis.  After five weeks of sampling, disinfected egg and fry homogenates from each family have tested positive by nested PCR at least once.  To determine if progeny from infected broodstock have a greater risk of developing BCWD, fish will be exposed to chronic gas supersaturation, a stressor that has been linked to disease outbreaks in hatcheries, and monitored for BCWD outbreaks.  We will also test if progeny from heavily infected broodstock are more susceptible to F. psychrophilum than progeny from uninfected broodstock when directly challenged with the bacterium.  Results will provide insight on the use of these assays to set threshold levels for screening broodstock.






Development of Immunochromatography Assay Kit for Ranavirus Determination


RK Young1, A Bayley2, KD Thompson3, A Adams3, T Aoki1 and TS Jung*1


1 Aquatic Biotechnology Center of WCU project, College of Veterinary Medicine, Gyeongsang National University, Jinju, 660-701, Korea  yl0808@lycos.co.kr; aoki@kaiyodai.ac.jp; jungts@gnu.ac.kr
2 Cefas, Weymouth laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, UK amanda.bayley@cefas.co.uk
3 Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland, UK k.d.thompson@stir.ac.uk; alexandra.adams@stir.ac.uk








Ranaviruses are serious pathogens of fish, amphibians and reptiles and cause disease problems worldwide. In the present study, we isolated ranavirus from both diseased tadpoles and frogs (Rana plancyi chosenica) and identified the virus involved as Korean ranavirus-1 (KRV-1). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to characterise the virus and the results were compared to the profile obtained with frog virus-3 (FV-3). Several bands were observed between 35 to 49 kDa for isolates of both FV-3 and KRV-1 and these were identified as the major capsid proteins (MCPs) of the virus by MALDI-TOF MASS analysis. The antigenic proteins of KRV-1 were identified using sera from frogs infected with KRV-1. Protein A-HRP was used to recognize the frog antibodies in the Western blot analysis with antigen bands observed at 34 and 93 kDa. These bands were identified as MCP and neurofilament triplet H1-like protein (NF-H1) by MALDI-TOF/TOF. Monoclonal antibodies (MAbs) were produced against ranavirus KRV-1 for the development of an immunochromatography assay kit. The antigenic proteins recognised by these MAbs were also identified by MALDI-TOF/TOF and found to be MCP and NF-H1. The sensitivity of the kit was similar to that of PCR, with a detection limit of 10 TCID50ml-1.






Development of Two Novel Quantitative Polymerase Chain Reaction (qPCR) Assays to Measure Alphaherpesviral Load in Bottlenose Dolphins (Tursiops truncatus)


HTD Maness*1, S Venn-Watson2, LL Archer3, R Rivera4, ED Jensen5 and HH Nollens6


1 Aquatic Animal Health Program and the Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL USA htdaniel@ufl.edu
2 National Marine Mammal Foundation, San Diego, CA USA stephanie@epitracker.com
3 Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida,  Gainesville, FL USA archerl@ufl.edu
4 Hubbs-SeaWorld Research Institute, San Diego, CA USA rrivera@hswri.org
5 U.S. Navy Marine Mammal Program, Space and Naval Warfare Systems Center Pacific, San Diego, CA USA eric.d.jensen@navy.mil
6 SeaWorld San Diego, San Diego, CA USA hendrik.nollens@seaworld.com











During viral surveillance of a collection of bottlenose dolphins (Tursiops truncatus), two distinct alphaherpesviruses, tentatively named Delphinid herpesvirus 2 (DeHV-2) and Delphinid herpesvirus 8 (DeHV-8), were detected using conventional polymerase chain reaction (PCR) in the white blood cell (WBC) fraction of a 22-year-old female with a history of persistent lymphocytosis. Alphaherpesviruses are generally considered to persist in the sensory nerve ganglion, thus, the presence of herpesvirus in circulating blood cells was presumed to be indicative of infection with active virus replication. Real-time quantitative PCR (qPCR) assays targeting the DNA-dependent DNA polymerase gene were developed to measure circulating DeHV-2 and DeHV-8 loads among healthy dolphins and a series of samples from the case dolphin. Both qPCR assays were found to be more sensitive (detection limit: 10 virus copies/sample), and faster, than conventional PCR. Circulating viral load of DeHV-2 in the case dolphin (N = 16) ranged from 0 to 1.3 virons per ng of DNA during a 30 month period. A cross-sectional survey of the WBC fraction among clinically healthy bottlenose dolphins (N = 55) revealed a slightly higher prevalence for DeHV-2 (N =2) than for DeHV-8 (N = 0). We conclude that the qPCR assays are capable of detecting DeHV-2 and DeHV-8 in bottlenose dolphin blood. Further studies are needed for a clearer understanding of the clinical relevance of these two viruses in dolphin populations.










Sinking or Swimming in the OIE Aquatic Animal Health Code


MD Remmenga* and LL Gustafson


National Surveillance Unit, USDA, APHIS, VS, Centers for Epidemiology & Animal Health, Fort Collins CO, USA marta.d.remmenga@aphis.usda.gov, marta.d.remmenga@aphis.usda.gov




The OIE Aquatic Animal Health Code provides flexible guidelines for designing surveillance systems to demonstrate disease freedom.  This flexibility does introduce a level of complexity that can lead to confusion when developing a new system.  Because there are multiple paths to achieving disease freedom under the OIE guidelines, it may not be immediately evident whether a facility or State surveillance program that claims to follow the guidelines is truly adequate.  Unfortunately, prescribed systems (e.g., AFS Blue Book) may not be sufficient in all situations, and can lead to under- or over-sampling depending on the risk of disease in the sampled population.  Understanding the role of three different types of evidence for demonstrating disease freedom can remove some confusion and frustration with the OIE guidelines.  First, sampling and testing can be used to establish the current disease status of the population. Second, appropriately documented historical evidence can reduce the sampling required.  Finally, knowledge about the risk of disease introduction and about how the bio-security measures reduce risk informs how frequently retesting is required. The flexibility in the OIE guidelines provides an opportunity to create sustainable surveillance systems that are also cost effective. Example scenarios will be provided.




Perspectives on Global Preparedness for Aquatic Animal Diseases


KH Hartman*


USDA, Animal and Plant Health Inspection Service, Veterinary Services, Ruskin, FL USA kathleen.h.hartman@aphis.usda.gov




Aquatic animal health authorities, state and federal, as well as aquaculture facilities must be prepared to efficiently and effectively respond to outbreaks of aquatic animal diseases. Being prepared encompasses specific threat identification, response plans, identification of resources, training resources for responders, and conducting drills and exercises to test the procedures and the plan.  Implementing the incident command system (ICS) is an important way to functionally organize the response to a disaster.  ICS implementation in association with specific disaster types and required operations remains a continuing challenge, and will be discussed.





Developing, Implementing, Auditing and Certifying Veterinary Biosecurity Programs


AD Scarfe*1, D Palić2 and CI Walster3


1 American Veterinary Medical Association, Schaumburg, IL USA.  dscarfe@avma.org
2 College of Veterinary Medicine, Iowa State University, Ames, IA USA.  dulep@iastate.edu
3 The Island Veterinary Associates, Staffordshire, UK.  chris.walster@onlinevets.co.uk





One outcome of a 2009 International Aquaculture Biosecurity Conference in Norway was a standardized approach developed by international experts, for applying core elements necessary for practical and effective biosecurity programs.  When correctly applied to epidemiological units (individual tanks or ponds, whole farms, compartments, zones or watersheds, or geopolitical regions) they are viable for any type of aquaculture operation and for any infectious and contagious disease of concern, and may meet legislative or regulatory requirements, including OIE standards.  In order for biosecurity programs to be functional, effective and justifiable they need to involve and integrate several formal processes including: hazard and risk analysis (hazard identification and prioritization, risk assessment/evaluation, risk management/mitigation and risk communication); analysis and remediation of critical control points (including evaluation and mitigation plans for correcting practices where disease could enter or leave the epidemiological unit); applied epidemiological practices (including disease diagnostics, surveillance, monitoring and determination of disease status or freedom); emergency preparedness (contingency protocols for disease control and eradication); auditing of procedures and records; and certification (providing assurance of disease freedom).  Optimally, for biosecurity programs to be effective, useful and justifiable to all stakeholders (i.e, producers, industries, service providers and government agencies) programs need to include: (1) documented disease prevention, control and eradication practices; (2) a sound science basis; (3) easily understood practical approaches; (4) economical viability; and, (5) strong incentives to encourage compliance – whether voluntarily initiated at farm or industry level, or mandated through government legislation and regulations.



Emerging Diseases of Important Mollusks

R Smolowitz*

Aquatic Diagnostic Laboratory, Roger Williams University, Bristol, RI USA, rsmolowitz@rwu.edu



The types of mollusks cultured for food worldwide include various types of bivalves (oysters, clams, mussels, etc.) as well as gastropods (abalone).  The effects of disease on the aquaculture of this important food source can be devastating and not only destroy the availability of this food animal, but also cause significant local and region economic impacts.  Many emerging diseases in molluscan animals are a result of transference of the disease agent to another susceptible host or transferring the infected host to a different environment resulting in significant disease.   Perkinus marinus has caused significant mortality of both cultured and wild eastern oysters (Crassostrea virginica) along the entire eastern and gulf coastlines of the U.S. since the 1940’s.  Genetic evidence suggests that disease occurred when the Pacific oysters containing the agent (but no disease) were transferred to the east coast of the U.S.    Perkinus olseni, a close relative of P. marinus, is the cause of significant disease in several types of molluscan animals in the Asia-Pacific area.  It is listed by O.I.E. as a disease of concern and trade of infected animals between countries is not recommended.  Recently, examination of a group of Tridacna sp., giant clams, which are imported routinely to the U.S. as part of the aquarium trade, showed infection and resulting disease caused by P. olseni.  Other examples of emerging diseases include Herpes virus infections of Pacific oysters in Europe and ganglioneuritis of abalone in Australia.







Pet Industry Efforts to Control Chytridiomycosis in Amphibians


N Saint-Erne*


PetSmart, Inc., Phoenix, Arizona USA nsainterne@ssg.petsmart.com



Chytridiomycosis (Batrachochytrium dendrobatidis) is a fungal infection of amphibians that has been spreading across the globe and is linked to the decimation of wild frog populations.  The fungus was first identified in southern Africa as a stable endemic infection years before any positive specimen was found outside Africa.  The global transportation of frogs and other amphibians for scientific research and the pet trade brings with it the risk of spreading this fungal infection.  African dwarf frogs (Hymenochirus curtipes) are popular aquarium pets, but they can be carriers of this devastating fungal infection. These frogs sold in pet stores can be treated for the fungus with a variety of topical antifungal medications (Betadine, Benzalkonium chloride), as well as through heating the water to a temperature that kills the fungus but is safe for the frogs. Preventive treatments using one of these methods will reduce the risk of spreading this serious fungal infection, and reduce the losses associated with the fungus in aquatic frogs kept as pets. The lesions associated with chytridiomycosis include hyperplasia of the epidermis and hyperkeratosis of the stratum corneum. Typical clinical signs in frogs infected with chytridiomycosis include lethargy, inappetence, skin discoloration, presence of excessive sloughed skin, and sitting unprotected during the day with their hind legs held loosely to the body. Frogs become moribund in the terminal stages with loss of their righting reflex, and death usually occurs a few days after the onset of clinical signs. Cause of death is associated with electrolyte loss through the damaged epidermis.






Aquarium and Aquaculture Facility Emergency Preparedness and Recovery


BR Whitaker*


National Aquarium - Baltimore, Baltimore, Maryland USA bwhitaker@aqua.org



Disasters in aquarium and aquaculture facilities can be avoided or minimized by careful planning and risk management. A disaster can be defined as any tragic event with great loss stemming from events such as earthquakes, floods, catastrophic accidents, fires, or explosions [http://en.wikipedia.org/wiki/Disaster]. Emergency situations are likely to occur within the lifespan of a zoo, aquarium, aquaculture facility, research laboratory or other aquatic animal facility. Direct damage to the facility or disruption of supporting services can lead to a loss of animal life and production. It can also cause injury or even death to staff attempting to control or mitigate the impacts of the emergency. Preparing for such an event, referred to as “emergency preparedness”, will minimize the risk of loss or injury to both animals and humans. A solid plan is easier to prepare in advance, provides staff with knowledge and confidence to manage a situation when it occurs, and will result in avoiding or minimizing the disaster and facilitating recovery. Following every event is the opportunity to improve your emergency preparedness plan. Once life returns to normal, take the time to review the event, the actions taken during the event, and the final outcome of the emergency. This is your opportunity to learn and make improvements to your plan that will change the outcome of future events.  




SESSION 7. PARASITOLOGY II: Myxozoan Workshop 2  [TOP]






Spatial, Temporal and Host Factors Structure the Ceratomyxa shasta (Myxozoa) Population in the Klamath River Basin


 SD Atkinson*1,2 and JL Bartholomew2


1 School of Chemistry and Molecular Biosciences, The University of Queensland, Australia stephen@2am.com.au
2 Department of Microbiology, Oregon State University, Corvallis, Oregon, USA





The myxozoan parasite Ceratomyxa shasta is a virulent pathogen of salmonid fish in the Klamath River, Oregon/California, USA. We previously defined four principal genotypes of the parasite (O, I, II, III) based on a trinucleotide repeat (ATC)0-3 in Internal Transcribed Spacer region 1 sequences. Genotypes occur in sympatry and show marked host preference: I in Chinook salmon (Oncorhynchus tschawytscha) and II in non-native rainbow trout (O. mykiss). In the present study, we sequenced the parasite from river water samples collected in May, June and September at three localities below, above and between the Klamath’s five dams. We also sampled adult and juvenile coho salmon (O. kisutch), steelhead trout (O. mykiss, anadromous form) and native redband rainbow trout (O. mykiss, freshwater form) and additional Chinook salmon and non-native rainbow trout. We found that the C. shasta population was highly structured spatially, temporally and with respect to fish host species. Genotype O was present in water throughout the basin but detected almost exclusively in steelhead and native rainbow trout. Genotype I was in water only below the dams and detected only in Chinook salmon. Genotype II was detected in coho salmon below the dams, and in non-native rainbow trout exposed both above and below the dams. The same genotypes were detected in adult and juvenile fish of the same species. These findings have major implications for the design of effective surveillance and control programs for this economically and ecologically important fish parasite.






Host-specific Virulence and Transmission of Ceratomyxa shasta Genotypes I and II


CN Hurst* and JL Bartholomew


Department of Microbiology, Oregon State University, Corvallis, OR USA, hurstch@onid.orst.edu, bartholj@science.oregonstate.edu




The myxozoan Ceratomyxa shasta is endemic to the Klamath River (KR) basin in the Pacific Northwest region of the United States. The parasite is dependent upon both a polychaete (Manayunkia speciosa) and a salmonid to complete its life cycle and is established throughout the main-stem KR. Chinook salmon (Oncorhynchus tshawytscha) were extirpated from upper KR basin with the construction of Copco dam in 1917, and the severe effects of the parasite on Chinook in the lower KR raises questions about the outcome of reintroducing these fish into the upper basin. Recent studies demonstrate that C. shasta is comprised of at least four genotypes with different salmonid host specificities. To support these findings, we  seeded populations of polychaetes with parasite genotypes I and II and exposed three species of salmonids present in the KR basin; Chinook and coho (Oncorhynchus kisutch) salmon and rainbow trout (Oncorhynchus mykiss). Genetic analyses of laboratory exposed fish support the hypothesis of host-specific virulence. We found that genotype I caused mortality in Chinook, while genotype II was responsible for rainbow trout mortality. To determine if host-specific transmission occurs, Chinook and rainbow trout were exposed to genotypes I and II in the field. We analyzed blood samples taken within the first two weeks post exposure and results demonstrate that genotypes I and II are transmitted to both fish species, but the genotype associated with mortality is dominant.






The Dynamics of Mixed Myxozoan Infections and Potential Mechanisms of Within-Host Interactions


AS Holzer*1,2, R Wootten3 and C Sommerville3


1 Institute for Specific Prophylaxis and Tropical Medicine, Medical University Vienna, Kinderspitalgasse 15, 1090 Vienna, Austria
2 Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, P.O. Box 22085, 46071 Valencia, Spain astrid.holzer@uv.es
3 Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK cs3@stir.ac.uk rw3@stir.ac.uk







Different myxozoan parasite species frequently co-occur in the same fish host, however, nothing is known of the infection dynamics of mixed infections in relation to the age and life stage of fish. The current study investigated the spatial and temporal patterns of invasion, migration and expansion of mixed infections of myxozoans in Atlantic cod, Gadus morhua, and in the brown trout, Salmo trutta, using PCR assays and single- or double-label in situ hybridisation protocols, which were developed specifically for each myxozoan. These techniques allowed for the detection, differentiation and localisation of different myxozoan developmental stages in fish during host development. In G. morhua,  despite concurrent initial infection and continuing exposure of fish to infective stages of the 2 renal myxozoans Gadimyxa atlantica and Zschokkella hildae, proliferation and sporogony of the two species was clearly separated in both time and space. A similar condition was found in S. trutta, where microhabitat separation in the kidney was less obvious but where the development of Chloromyxum schurovi seems to be strongly influenced by the invasion of Tetracapsuloides bryosalmonae. The results strongly indicate that within-host competition, not previously observed in this parasite group, takes place between myxozoan species. The blood stream was identified as the first and possibly most important site of encounter and interaction, especially in species where this site is essential for parasite proliferation prior to host organ invasion. The potential mechanisms of interaction are discussed in relation to the literature on other parasite groups occurring in the blood and to ongoing in vitro studies.





Histopathological Features of the Infection by the Myxosporean Zschokkella hildae Auerbach, 1910 in the Kidney of Atlantic cod (Gadus morhua Linnaeus, 1758).


B Gorgoglione* and C Sommerville

Institute of Aquaculture, University of Stirling, Stirling, Scotland, UK b.gorgoglione@abdn.ac.uk



Zschokkella hildae is a common myxozoan parasite of Atlantic cod, which acts as the vertebrate host in the two-host myxosporean life cycle. Samples were obtained from 200 cultured Atlantic cod aged 1+ from a fish farm on the west coast of Scotland. Z. hildae was identified from spore morphology in fresh squashes and confirmed by PCR using species-specific 18S rDNA. Localisation and distribution were confirmed using ISH. Kidney tissues were examined using standard histological methods. Sporogonic plasmodia of Z. hildae formed large clusters attached to the epithelial lining of the collecting ducts and ureters and commonly occluded the lumen. Rarely, sporogonic plasmodia were seen in interstitial tissue. Pre-sporogonic stages were seen in blood, in interstitial tissue and in the tubular epithelium, suggesting the parasite route into the ureters. Pathological features included granulocyte infiltrations, hyperplasia of supporting-connective tissue, proliferation of interstitial tissue and occasional glomerular oedema. A marked feature of the infection was the rodlet cell invasion of the parasitised tissues, which was always associated with plasmodia. Discharging rodlet cells attached directly to large plasmodia. Our observations provide evidence for the potential pathogenicity of Z. hildae in Atlantic cod.






Chinook Salmon Host Response to Ceratomyxa shasta (Myxozoa)


S J Bjork*1, Y Zhang2, JO Sunyer2 and J L Bartholomew1

1 Department of Microbiology, Oregon State University, Corvallis, OR, USA bjorksa@onid.orst.edu, bartholj@science.oregonstate.edu
2 Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA yonganzh@vet.upenn.edu, sunyer@vet.upenn.edu






Stocks of Chinook salmon (Oncorhynchus tshawytscha) have varying levels of resistance to the myxozoan parasite, Ceratomyxa shasta. In this study, the progress of C. shasta infection and host responses of highly susceptible and resistant Chinook salmon stocks were compared. There were no differences in the invasion of the gills indicating that resistance does not occur at the site of entry. At a sub-lethal parasite dose, resistant fish eliminated the parasite from the blood 2 weeks after infection, limiting the numbers that reach the intestine. On day 12 p.i. infection intensity scores in the intestine were higher in susceptible than resistant fish but inflammation in resistant fish was more severe. The expression of IL-10 and IFNγ in the blood and IL-6, IL-8, IL-10 and IFNγ in the intestine was up-regulated in both strains 12 days after infection.  IL-10 and IFNγ increased in the blood of resistant fish and IFNγ expression increased in the intestine on day 25, whereas all of the susceptible fish died by this time. On day 90, resistant fish had resolved the inflammation and cytokine expression was similar to the control group. Thus, it appears that the inflammatory response of the susceptible strain was delayed and incapable of containing or eliminating C. shasta but resistant fish: 1) limited the number of parasites that reached the intestine through clearance from the blood; 2) elicited a rapid and effective inflammatory response in the intestine that contained and eliminated C. shasta and 3) resolved the inflammation and can recover from infection.









Virulence Properties of Moritella viscosa Extracellular Products


B Bjornsdottir*, T Gudmundsdottir and BK Gudmundsdottir


Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, Iceland
bryndisb@hi.is, tota@hi.is, bjarngud@hi.is




The pathogenic bacterium Moritella viscosa causes winter ulcer disease in fish reared in seawater at low temperatures.  The disease causes significant financial losses in aquaculture, mainly in salmon farming, and thus vaccines are being developed.  It has been shown that extracellular products (ECP) from M. viscosa cause disease symptoms, even leading to death in Atlantic salmon (Salmo salar L.); however, little is known about the virulence of M. viscosa.  The aim of the present study was to evaluate the virulence properties of M. viscosa ECPs.  We obtained ECPs of 22 M. viscosa strains isolated from different geographical regions and host species, and evaluated their lethality to salmon, and their cytotoxic and hemolytic activities, enzymatic activities, and siderophore levels.  The ECPs of strains isolated from salmon showed lower cytotoxic and hemolytic activities than did ECPs of strains isolated from other species of fish.  No correlation was found between lethality to salmon and cytotoxic or hemolytic activities.  Two strains isolated from salmon in Canada were found to be non-virulent, and their respective ECPs were non-lethal.  Variable levels of ECP protein concentration, different enzymatic activities, and siderophore levels could not explain the differences of in vivo and in vitro virulence (that is to say, the lethality to salmon and cytotoxic/hemolytic activities). The results indicate that cytotoxicity assaying of cell-culture models may not be an optimal method for determining the virulence of M. viscosa strains, and that the major extracellular virulence factors of M. viscosa are still unidentified.





Detection of Yersinia ruckeri in Juvenile Cultured Atlantic cod (Gadus morhua) in a Marine Nursery Located in Atlantic Canada.


NY O’Brien1, D Groman*2, J. Giles2 and DS Whelan1


1 Newfoundland and Labrador Department of Fisheries and Aquaculture, Aquatic Animal Health Division, St. John’s Canada. nicoleobrien@gov.nl.ca, darylswhelan@gov.nl.ca
2 Aquatic Diagnostic Services, Atlantic Veterinary College , University of Prince Edward Island, Charlottetown Canada. groman@upei.ca, jgiles@upei.ca






This case study describes a Yersinia ruckeri infection of juvenile Atlantic cod (Gadus morhua), averaging 10 g in weight. In December of 2009, 30,000 Atlantic cod maintained in a marine nursery in Atlantic Canada were reported as experiencing elevated mortality and morbidity (~5% cumulative mortality). Clinical signs observed during an on-site veterinary visit included erythema at the base of the pectoral fins and spiraling within the tanks. Samples were obtained for diagnostic analysis, which included histopathology, bacteriology  and virology.  Samples were negative for Betanodavirus by both tissue culture using E-11 cells and RT-PCR analyses. The bacterial pathogen, Yersinia ruckeri, (Type-01) was isolated from the population.  The confirmatory identification methods for the isolate included, biochemical reactions, serum agglutination, API-20E system and by MALDI-TOF Mass Spectrometry.  The isolate was found to be sensitive by the Kirby Bauer Method to the following antibiotics: Enrofloxacin, Aquaflor, Trimeth/sulfa and Oxytetracycline.  Histopathology examinations revealed that affected fish were suffering from a severe septicemia, with granulomatous/ histiocytic infiltrates present in many of the affected tissue lesions.  The fish were treated with an in-feed antibiotic.  After treatment, mortality and morbidity remained unchanged. The population was subsequently culled and a random sample of the population was obtained to screen for concurrent pathogens/diseases and the prevalence of Yersinia ruckeri was determined.




Characterization of Isolates of Group B Streptococci from Diseased Farmed and Wild Marine Fish from the United States Gulf of Mexico Coast


E Soto, R Wang, JE Wiles, W Baumgartner, and JP Hawke*


Department of Pathobiological Sciences, LSU School of Veterinary Medicine, Baton Rouge, LA USA. esoto1@lsu.edu, rwang@tigers.lsu.edu, wbaumg1@tigers.lsu.edu, jwiles@vetmed.lsu.edu, jhawke1@lsu.edu





Group B streptococci have been described as disease causing agents from various species of marine fish over a wide geographic and host range. Group B Streptococcus sp. have been described as causative agents of fish kills of wild marine and euryhaline species in coastal estuaries of the northern United States Gulf of Mexico coast since the early 1970’s. Isolates of group B streptococci have been obtained from cultured hybrid striped bass (Morone saxatilis x M. chrysops) and cocahoe minnow (Fundulus grandis) from coastal Louisiana fish farms since the late 1980’s. In this study, we identify these United States Gulf of Mexico coast isolates as Streptococcus agalactiae group B type Ib and describe their biochemical phenotype, antibiotic susceptibility, and phylogenetic relationship to other Group B streptococci from fish cultured in Thailand, Ecuador, and Honduras. Genetic analysis was performed using partial sequence comparison of the 16S rRNA gene, sipA which encodes a surface immunogenic protein, cspA which encodes a cell surface-associated protein, and secY which encodes components of a general protein-secretion pathway. Phylogenies inferred from secY and cspA gene sequence comparisons were more discriminative than those inferred from 16S rRNA and sipA gene sequences. In agreement with the molecular findings, biochemical and antimicrobial resistance analysis demonstrated similar profiles for the isolates recovered from the Gulf of Mexico coast when compared to those from other locations. Laboratory challenge methods for inducing streptococcosis in cocahoe minnows were performed using strain LADL-97-151. The lethal dose 50 value, 14 days post-challenge, was 10-9.43 (2 CFU/fish) by intraperitoneal injection. 





A Wide Analysis of Fish Bacterial Pathogen Streptococcus parauberis: Approach to Complementary Genomics and Proteomics

SW Nho*1, SB Park1, HB Jang1, IS Cha1, MA Ha1, YR Kim1, J Hikima1, M Othani1, T Aoki1, 2 and TS Jung1

1 Aquatic Biotechnology Center of WCU project, College of Veterinary medicine, Gyoungsang National University, Jinju, Korea. shyguy0126@gmail.com
2 Laboratory of Genome Science Course of Applied Marine Biosciences Graduate School of Marine Science and Technology, Tokyo, Japan.






Streptococcus parauberis is a causative agent of streptococcosis in different farmed fishes, and is also known to cause cellulitis of soft tissues in humans. Streptococcosis caused by S. parauberis has a drastic impact on Olive flounder (Paralichthys olivaceus) farming, the major aquaculture industry in Korea, causing severe economic losses. This study was performed to identify the antigenic proteins of an S. parauberis isolate by employing immunoproteomic techniques using a specific flounder anti-S. parauberis sera generated by experimental S. parauberis challenge. Sixty-two antigenic proteins from approximately 200 spots, observed on 2-DE profiles of S. parauberis were reacted with the flounder anti-sera. A part of antigenic proteins were identified by mass spectrometry (MS) and peptide mass fingerprinting analyses (http://www.matrixscience.com/) as follows: molecular chaperone, GAPDH, enolase, IMPDH, OCT, FBA, and arginine deiminase. These proteins are associated with invasion, adhesion and escape from defense mechanisms to establish infection within the host environment. Simultaneously, to investigate the phylogeny, metabolism, drug-resistance and virulence of S. parauberis, whole genome shotgun sequencing was conducted using GS-FLX. The S. parauberis genome possesses a single chromosome of approx. 2.1 Mbp containing 2,074 predicted ORFs. Using blast search analyses (http://www.ncbi.nlm.nih.gov/BLAST/), 1,469 of these genes were identified with S. uberis 0140J strain with high query coverage. To identify additional antigenic proteins, the MS data were re-analyzed with database of amino acid sequences deduced from the annotated gene sequences. The proteomics and genomics alliance produces almost complete and accurate gene catalogues for microbial genomes, a comprehensiveness which is essential for efficient systems biology.



Ecological and Epidemiological Aspects of Bacterial Kidney Disease in Marine Phase Chinook Salmon


LD Rhodes*1, CA Rice1, CM Greene1, DJ Teel2, M Trudel3, SL Nance4, T Zubkowski3, E Smith1 and P Moran1


1 NOAA Fisheries, Northwest Fisheries Science Center, Seattle, WA 98112 USA
2 NOAA Fisheries, Northwest Fisheries Science Center, Manchester Research, Laboratory, Port Orchard, Washington, 98366 USA
3 Department of Fisheries and Oceans, Pacific Biological Station, Nanaimo, BC, Canada
4 Aquatic Farms, Ltd., Kaneohe, HI USA







Bacterial kidney disease (BKD) is an endemic disease among Pacific Northwest populations of salmonids.  Infections can occur in freshwater or seawater, but there are few investigations of infection during the marine phase.  A study of neritic (nearshore) habitat utilization by juvenile Chinook salmon (Oncorhynchus tshawytscha) throughout Puget Sound has identified factors associated with infection likelihood as well as factors that do not appear to contribute to infection.  For example, the density of Chinook salmon measured in each tow positively correlated with infection prevalence across a range of spatial scales, suggesting a potential role for density in infection transmission.  Surprisingly, the density of river lamprey (Lampetra ayresis) per tow was a significant risk factor for infection.  When we examined the stomach contents and kidneys of river lamprey, intact cells of the etiologic agent, Renibacterium salmoninarum, were identified, indicating river lamprey could be a transmission vector during emergence to seawater.  Studies of the offshore marine phase have also yielded unanticipated observations.  Fish with levels of bacterial DNA and protein typically associated with morbidity were collected in offshore sampling, demonstrating that substantial infection burdens can be sustained in free-ranging fish.  These types of studies complement freshwater studies by providing information about the epidemiology and ecology of BKD during a poorly characterized part of the salmon life history.






Possible Common Etiological Agent for Strawberry Disease and Red Mark Syndrome in Rainbow Trout.


M Metselaar*1, KD Thompson1, RML Gratacap1, MJL Kik2, SE LaPatra3, SJ Lloyd4, DR Call4, PD Smith5 and A Adams1


1 Aquatic Vaccine Unit, Institute of Aquaculture, University of Stirling, UK  Matthijs.metselaar@stir.ac.uk
2 Department of Pathobiology, Pathology, University of Utrecht, Utrecht, Netherlands
3 Clear Springs Foods, Inc., Buhl, Idaho 83316, USA.
4 Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA
5 Intervet Schering-Plough Animal Health, Aquaculture Centre, Essex, UK








Strawberry Disease (SD), in the USA appears to affect mostly rainbow trout (Oncorhynchus mykiss) and has recently been associated a Rickettsia-like organism (RLO) with SD, based on analysis of 16S rDNA sequences. A disease with similar clinical signs to SD, referred to as Red Mark Syndrome (RMS), has been reported in the UK. The research presented here indicates the presence of a common intracellular bacterium in both SD- and RMS-affected fish. Using two different polyclonal antisera raised against Piscirickettsia salmonis and a number of different anti-P. salmonis monoclonal antibodies (MAbs), a positive reaction was observed in skin lesions, liver, kidney and spleen of RMS-affected fish sampled from several locations within the UK, using immunohistochemistry (IHC). The same panel of anti-P. salmonis antibodies showed similar staining in the tissue of SD-affected fish by IHC. In addition, polymerase chain reaction (PCR) performed (using RLO-specific primers) on RMS-affected fish from the UK, indicated that these samples were positive for the RLO 16S rRNA sequence. These findings suggest that the same aetiological agent may in fact be responsible for RMS in the UK and SD in the USA. Manageable










Isotonic Egg-washing Reduces Bacteria and Viruses on Salmon Eggs in Aquaculture


M Kohara*1, S Ogawa2, H Kasai3 and M Yoshimizu3

1 Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan
2 Nagano Prefectural Fisheries Experimental Station, Akashina, Azumino, Nagano, 399-7102, Japan
3 Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan hisae@fish.hokudai.ac.jp, yosimizu@fish.hokudai.ac.jp







Egg-washing with isotonic solution has been used to improve the artificial fertilization in fresh-water salmon in aquaculture. Usually, unfertilized eggs are rinsed two or three times with isotonic solution before fertilization. We investigated the effect of isotonic egg washing using rainbow trout eggs to reduce colonization with Flavobacterium psychrophilum, Aeromonas. salmonicida,  infectious hematopoietic necrosis virus (IHNV) and Oncorhynchus masou virus (OMV) quantitatively. Newly spawned eggs were divided into three treatment groups that were challenged with varied concentrations of F. psychrophilum: (1) immersion-challenged unfertilized eggs followed by water hardening; (2) immersion-challenged fertilized eggs followed by water hardening; and (3) immersion-challenged eggs that were rinsed with isotonic solution.  Eggs were cultured on modified Cytophaga Agar.  F. psychrophilum was isolated from the contents of eggs at a high prevalence; however, no growth was observed at lower challenge concentrations of F. psychrophilum.  Interestingly, A. salmonicida and Renibacterium. salmoninarum were also isolated from egg contents with a CBB medium when bacterial numbers were high. Although, F. psychrophilum and R. salmoninarum were cultured from the egg contents, A. salmonicida was not cultured at seven days after immersion-challenge. The number of live bacterial cel1s or virus infectivity titers which were eliminated by egg-washing, was 103~5 CFU /ml or 104 TCID50/ml. The ratio of intra-ovum infection with F. psychrophilum was clearly less in rinsed eggs than in non-treated eggs, and result of detection from showered eggs was negative. Results suggest that egg-washing with isotonic solution is effective in reducing certain bacteria and viruses on the egg surface, and may help to decrease the risk of egg infection.





Bioexclusion in Site-Level Biosecurity Plans  

GA Karreman*

Syndel Laboratories Ltd., 958 Chatsworth Road, Qualicum Beach, BC, V9K 1V5 Canada, gracek@wchemical.com




Biosecurity can be defined as a system of processes (i.e., inputs, movements and other activities), each with a set of procedures that taken together minimize the risk of introduction and spread of infectious organisms within or between aquatic animal populations. Biosecurity measures at the site level include bioexclusion (prevention of pathogens from entry), within-site infectious disease control (management of pathogens within a facility) and biocontainment (prevention of pathogens from release). This talk will focus on bioexclusion. When designing a biosecurity plan, an on-site analysis provides the scenario for pathogen introduction into the site. The analysis requires a working understanding of the production biology of the species, the physical layout of the facility and the process flow for operations. It also requires a working knowledge of the epidemiology of the pathogen(s) to be excluded. A risk assessment estimates the risks of introducing pathogenic organisms or disease into an aquatic animal site and provides recommendations for mitigation of those risks. Specific points of pathogen introduction can then be identified by systematically evaluating every step in the process flow for potential introduction of the pathogen into the site.





Effect of Common Aquaculture Chemicals Against Selected Fish Pathogenic Bacteria


SA Smith*


Aquatic Medicine Laboratory, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA USA stsmith7@vt.edu





Bacterial pathogens are one of the most important disease-causing agents in the aquaculture industry.  As most pathogenic bacterial species are water-borne, disinfection of aquaculture systems and equipment is essential for both disease control and for enhanced biosecurity. This study examined a variety of commercially available compounds for their ability to reduce or eliminate specific bacterial species from the water. The compounds evaluated included Clorox®, ethanol, Roccal®, Lysol®, iodine, formalin, Chloramine-T®, glutaraldehyde and Virkon S®.  Each compound was tested at various concentrations over a 60-minute time interval against Mycobacterium marinum, Vibrio anguillarum, V. ordalii, Edwardsiella ictaluri, E. tarda, Aeromonas hydrophila, A. salmonicida subsp. salmonicida, A. salmonicida subsp. achromogenes, Flavobacterium columnare, and F. psychrophilum. In addition, the three Aeromonas species were tested at four different temperatures (22°, 15°, 10°, and 5° Celsius). For M. marinum, ethanol and Lysol® were the most effective disinfectants, each reducing or eliminating the number of detectable bacteria within one minute of contact time. Sodium hypochlorite was moderately effective, but required a minimum contact time of 10 minutes to reduce bacterial counts. For the Vibrio spp., Chloramine-T®, Clorox®, ethanol, iodine, Lysol®, Roccal®, and Virkon S® eliminated all growth of both species of bacteria within one minute of contact time.  All species of Edwardsiella and Aeromonas were killed within one minute of contact time by all compounds except Chloramine-T® and formalin. In addition, all three Aeromonas species were killed within one minute of exposure by the effective compounds regardless of temperature.






The Importance of Hygiene for Live Feed Management: Lessons Learned from New Zealand Groper (Polyprion oxygeneios) Juveniles


S. Anderson*1, I. Salinas1, L. Tubbs2, J.S. Lumsden2, B. Diggles3, V. Webb1, Y. Gublin4, S. Pether4, G. Irvine4 and S. Walker4


1 National Institute of Water and Atmospheric Research (NIWA) Ltd. Private Bag 14901, Wellington 6241, New Zealand. sa.anderson@niwa.co.nz, i.salinas@niwa.co.nz, v.webb@niwa.co.nz
2 Fish Pathology Laboratory, Department of Pathobiology, Ontario Veterinary College, Guelph, Ontario, Canada N1G 2W1. ltubbs@uoguelph.ca, jsl@uoguelph.ca
  DigsFish Services Pty Ltd. 32 Bowsprit Cres. Banksia Beach, Bribie Island, Queensland 4507, Australia. ben@digsfish.com
3 National Institute of Water and Atmospheric Research (NIWA) Ltd, PO Box 147, Ruakaka 0151, New Zealand. y.gublin@niwa.co.nz, s.pether@niwa.co.nz, g.irvine@niwa.co.nz, s.walker@niwa.co.nz










New Zealand hāpuku or groper (Polyprion oxygeneios) is currently undergoing development for aquaculture. This involves the production of groper eggs, larvae, and juveniles. A critical period in groper larvae and juvenile rearing is when the yolk sac is re-adsorbed and the larvae are transitioned onto live feed. For groper, this begins with live rotifers at 9-11dph, followed by transition over a 3-8 day period to live brine shrimp (Artemia salina) at 18-22dph. The present study investigates the possible contribution of Artemia to groper mortality events over 2 production seasons, and describes histopathological changes. In 2008, the introduction of Artemia resulted in inappetance and a period of elevated mortality; dead and moribund fish presented with distended abdomens. Histology revealed a necrotising enteritis in these fish, with empty Artemia cysts present in the intestine. In 2009, a similar increase in mortality occurred when groper juveniles were fed an Artemia diet. In addition to a similar enteritis, a focally extensive hepatitis containing intra-lesional bacteria was observed indicating progression to a systemic bacteremia. In this instance, mortality rates increased rapidly with 100% mortality 6 days after the initial symptoms appeared. Microbiological analysis indicated the presence of Vibrio-like bacteria, later identified as Vibrio ichthyoenteri. This bacterium was also isolated from live Artemia samples, and it is likely to be the pathogen source. It appears that poor Artemia feed quality contributed to juvenile groper mortalities during 2008 and 2009, underlining the critical importance of maintaining hygienic practices for live feed production.




SESSION 10. PARASITOLOGY III: Myxozoan Workshop 3  [TOP]





























Molecular Immunology of Kuruma Shrimp, Marsupenaeus japonicus


I Hirono*1, T Koyama1, A Kaizu1, A Shitara1, MBB Maningas2, FF Fagutao1, H Yamada1, K Komiya1, T Aoki1 and H Kondo1


1 Laboratory of Genome Science, Tokyo University of Marine Science and Technology Tokyo, Japan hirono@kaiyodai.ac.jp
2 Department of Biological Sciences, University of Santo Tomas, Espana, Philippines marybethmaningas@yahoo.com






Because of the importance of penaeid shrimp in world aquaculture, there is much interest in understanding their immune systems to improve their resistance to pathogenic microorganisms. Unequivocal evidence points to the efficiency of RNA interference (RNAi) in studying gene function and their involvement in mounting antiviral responses in eukaryotes. The potency of double stranded RNA (dsRNA) to knock-down gene expression (gene silencing) has been successfully demonstrated in studying gene function in shrimp. From these studies, a shrimp pathogen has been found to be engaged in the innate (non-sequence specific) and RNAi-related (sequence specific) antiviral responses. Recent studies have shown that it is indeed possible to block viral disease progression by injecting shrimp with dsRNA specific to viral genes. Most of the studies conducted that used RNAi technology, however, focused on the shrimp pathogens and few have been done on the host itself. Here, some of our recent research results of shrimp immune-responses against microbial pathogens after in vivo silencing of defense-related genes including clotting protein, TGase, penaeidin-like protein lysozyme and prophenoloxidase is presented. Genomic information of penaeid shrimp is essential for understanding further their immune defense mechanisms. We found several different homologue genes of white spot syndrome virus (WSSV), a virulent pathogen known to significantly affect the health of shrimp. The expression of some of these genes were upregulated in shrimp after WSSV infection.




Characterization of Nitric Oxide Synthase Gene, MjNOS, in Kuruma Shrimp Marsupenaeus japonicus and its mRNA Variants


M Inada*1, S Okugawa1, J Nishi1, M Yoshimine1, R Sudhakaran2, T Kono3, T Mekata4, T Yoshida2, M Sakai2 and T Itami2


1 Graduate School of Agriculture, University of Miyazaki, Miyazaki, Japan agg902u@student.miyazaki-u.ac.jp
2 Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan itamit@cc.miyazaki-u.ac.jp
3 Interdisciplinary Research Organization, University of Miyazaki, Miyazaki, Japan
4 National Research Institute of Aquaculture, Fisheries Research Agency, Tsuiura, Kamiura, Saiki, Oita Pref., Japan mekata@affrc.go.jp








Nitric oxide (NO) signaling is involved in many physiological processes in vertebrates and invertebrates. In mammals, three types of NO synthase (NOS) were reported, namely neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). In crustaceans, NOS plays a significant role in the regulation of nervous system and innate immunity. Here, we describe the entire cDNA sequence (4,616 bp) and the open reading frame (1,187 amino acids) of the kuruma shrimp Marsupenaeus japonicus NOS (MjNOS). Highly conserved amino acid sequences in heme and tetrahydrobiopterin were observed in the oxygenase domain. FMN, FAD and NADPH were found in the reductase domain. When heat-killed Vibrio penaeicida cells were injected into the kuruma shrimp, MjNOS was expressed in the brain, gill, heart, lymphoid organ, intestine and thoracic ganglion. MjNOS expression in the gill reached its peak at 12 hours and decreased to its normal level 24 hours after V. penaeicida injection. In MjNOS mRNA, three variants were detected. Partial deletion of oxygenase domain sequence was found in variant 1 , whereas complete deletion of reductase domain with or without novel 104 bp insertion were found in variant 2 and variant 3. The total NO concentration in hemocytes was determined using the Griess reaction and high concentration was found at 12 hours after injection of heat-killed V. penaeicida cells.






Molecular Cloning and Characterization of the Dicer2 Gene in Kuruma Shrimp Marsupenaeus japonicus

J Nishi*1, S Okugawa1, M Inada1, M Yoshimine1, T Yoshida2, Y Suzuki3, T Kono4, M Sakai2, T Mekata5 and T Itami2  

1 Graduate School of Agriculture, University of Miyazaki, Miyazaki Japan agg905u@student.miyazaki-u.ac.jp
2 Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan itamit@cc.miyazaki-u.ac.jp
3 Faculty of Engineering, University of Miyazaki, Miyazaki Japan suzuki@civil.miyazaki-u.ac.jp
4 Interdisciplinary Research Organization, University of Miyazaki, Miyazaki, Japan
5 National Research Institute of Aquaculture, Fisheries Research Agency, Saiki, Japan mekata@affrc.go.jp









RNA silencing is a common antiviral defense mechanism in many eukaryotes from plants to animals. The generation of microRNA or small interfering RNA is dependent on the RNase ? enzyme Dicer gene. The Dicer gene plays an important role in the RNA silencing, and it has been reported that there are two types of Dicer genes in insects. However, only one type of Dicer gene was reported in kuruma shrimp (Marsupenaeus japonicus). We report here on the characterization of a second Dicer gene (MjDicer2) in kuruma shrimp. This is the first report of the presence of two types of Dicer genes in a crustacean. The full length of MjDicer2 cDNA was 4967 bp, including a 5' untranslated region (UTR) of 119 bp, a 3' UTR of 321 bp, and an open reading frame of 4,527 bp encoding a polypeptide of 1,509 amino acids. Analysis of the deduced amino acid sequence indicated that the mature peptide contained all six recognized functional domains, and showed the highest similarity to the black cottonwood (Populus trichocarpa) Dicer-like protein sequence with a similarity of 28.7%. Quantitative RT–PCR analyses showed a high expression level of MjDicer2 in the gill, lymphoid organ, and heart. MjDicer2 is most highly expressed in hemocytes 2 h after stimulation with white spot syndrome virus (WSSV). This study was supported by the Research and Development Program for New Bio-industry Initiatives and the Japan Society for the Promotion of Science.






Characterization of Tumor Necrosis Factor Ligand Superfamily Gene in Kuruma Shrimp, Marsupenaeus japonicus

S Okugawa*1, R Sudhakaran2, M Inada1, T Mekata3, J Nishi 1, M Yoshimine1, T Yoshida2, Y Suzuki4, T Kono5, M Sakai2 and T Itami2


1 Graduate School of Agriculture, University of Miyazaki, Miyazaki Japan agg905u@student.miyazaki-u.ac.jp
2 Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan itamit@cc.miyazaki-u.ac.jp
3 National Research Institute of Aquaculture, Fisheries Research Agency, Oita Japan mekata@affrc.go.jp
4 Faculty of Engineering, University of Miyazaki, Miyazaki Japan suzuki@civil.miyazaki-u.ac.jp
5 Interdisciplinary Research Organization, University of Miyazaki, Miyazaki, Japan tkono@cc.miyazaki-u.ac.jp










A tumor necrosis factor (TNF) gene has been isolated and characterized in kuruma shrimp, Marsupenaeus japonicus, providing the first conclusive evidence for the existence of the TNF ligand in shrimp. The kuruma shrimp TNF (MjTNF) cDNA was composed of 1868 bp with a 262 bp 50-untranslated region (UTR) and a 220 bp 30-UTR, which was translated into a protein of 462 amino acid residues that included a predicted transmembrane domain of 23 amino acid residues (Trp20-Val42) and the TNF family signature (Pro321-Leu448). Homology analysis of MjTNF showed 30.7% and 26.7% identities with fruit fly (Drosophila melanogaster) Eiger and human (Homo sapiens) ectodysplasin A, respectively. The MjTNF gene was constitutively expressed in unstimulated organs of shrimp such as the muscle, stomach, brain and gill. In lymphoid organ cells, an enhanced expression of the MjTNF gene was observed following stimulation with peptidoglycan and polycytidylic acid. A high expression level of MjTNF was observed in vivo 2 h and 4 h after stimulation with lipopolysaccharide and Vibrio penaeicida, respectively. These observations suggest that MjTNF plays a role in the innate immune defense in kuruma shrimp. The discovery of shrimp TNF will allow a more complete and concrete understanding of shrimp inflammatory responses. This study was supported by Research and Development Program for New Bio-industry Initiatives and Japan society for the promotion of science.





Reciprocal Effects between the Lethal Spiny Lobster Virus PaV1 and the Florida Lobster Fishery


DC Behringer*1, JA Moss2, JD Shields2 and MJ Butler IV3


1 University of Florida, School of Forest Resources and Conservation, Fisheries and Aquatic Sciences, Gainesville, Florida USA behringer@ufl.edu
2 Virginia Institute of Marine Science, Gloucester Point, VA USA jeff@vims.edu, jamoss@vims.edu
3 Old Dominion University, Department of Biological Sciences, Norfolk, Virginia USA mbutler@odu.edu






The Caribbean spiny lobster (Panulirus argus) is one of the most economically valuable fisheries in the Florida Keys and tops this list for most of the Caribbean.  In 2001, the fishery in Florida experienced a major decline in landings of ~30% from which it has not recovered.  This decline was coincident with discovery of a lethal viral pathogen, PaV1, found infecting juvenile lobsters in the Florida Keys.  P. argus has a complex life history and is exploited throughout its range – two factors that have made it difficult to determine the cause of the decline.  Here we describe the first assessment of PaV1 within the fished segment of the population.  We used PCR analysis to measure PaV1 prevalence from lobsters caught in commercial traps throughout the Florida Keys.  We also tested the effect of diseased lobsters within traps on trap attractiveness, and on the transmission of PaV1 to other trapped, but healthy, lobsters. We found a mean prevalence of 11% in the fished population with PCR+ lobsters as large as 95 mm carapace length (76 mm is legal).  We also found that traps harboring an infected lobster caught significantly fewer lobsters than traps containing healthy lobsters.  Furthermore, healthy lobsters confined in traps with diseased lobsters became infected with PaV1 more frequently than those confined with other healthy lobsters. This study demonstrates the indirect and subtle effects that pathogens can have on fishery function through altered animal behavior and the unintended consequences of fishery practices on pathogen epidemiology.





Population Effects of Bitter Crab Disease on Snow and Tanner Crabs From Alaska and Newfoundland.


JF Morado*1, MSM Siddeek2, DR Mullowney3, EG Dawe3, CA Shavey1, PC Jensen1, VC Lowe1 and RJ Cawthorn4


1 NOAA, National Marine Fisheries Service, Alaska Fisheries Science Center, Seattle, WA USA  frank.morado@noaa.gov, christie.sahvey@noaa.gov, pam.jensen@noaa.gov, vanessa.lowe@noaa.gov
2 Alaska Department of Fish and Game, Division of Commercial Fisheries, Juneau, AK shareef.siddeek@alaska.gov
3 Science Branch, Fisheries and Oceans Canada, Northwest Atlantic Fisheries Centre, St John's, NL, Canada darrell.mullowney@dfo-mpo.gc.ca, earl.dawe@dfo-mpo.gc.ca
4 University of Prince Edward Island, Atlantic Veterinary College, AVC Lobster Science Centre, Charlottetown, PEI, Canada C1A 4P3  cawthorn@upei.ca










Bitter Crab Disease (BCD) is a fatal disease of North Pacific snow (Chionoecetes opilio) and Tanner (C. bairdi) crabs, and North Atlantic snow crabs. Both crab species support major commercial fisheries and the disease is caused by a parasitic dinoflagellate of the genus Hematodinium. The method employed in disease monitoring varies and is often dictated by logistical considerations. Regardless of the monitoring method, data from snow and Tanner crab populations are now being sufficiently accumulated for various types of analyses and inclusion into population models. As the Bering Sea Tanner crab stock approaches an overfished level and a rebuilding plan is considered, it was of interest to determine what role, if any, BCD may have on the rebuilding plan. Simulation modeling indicated that BCD would have a delayed, but minimal rebuilding effect on the western stock. However, to compare these potential results with an area of known high prevalence, a similar simulation was performed on the Tanner crab population from Stephens Passage, southeast Alaska. In that instance, under a conservative exploitation rate, rebuilding of the Stephens Passage stock was not possible under short or medium terms. In coastal Newfoundland, BCD is highly prevalent across all pot-sampled size ranges, but most prevalent in small or juvenile crabs. The data indicate that two epizootics have occurred in the study area, but more interestingly, BCD appears to be a good predictor of recruitment strength for subsequent years. The significance of these findings on the respective crab stocks will be discussed further.






SESSION 12. ANTIMICROBIAL USE IN AQUACULTURE: Resistance, Residues and Research  [TOP]





Current Veterinary Approaches to Judicious Antimicrobial Use


AD Scarfe*


American Veterinary Medical Association, Schaumburg, IL, USA  dscarfe@avma.org



Since 1863, the American Veterinary Medical Association (AVMA) has fulfilled its mission and objective to improve animal and human health by seeking input on emerging issues from its members (today numbering ~83,000). Input is channeled through numerous councils, committees, task forces and other entities and utilized to formulate AVMA Executive Board approved opinion (positions and policies) or guidance on numerous issues, including the appropriate use of veterinary drugs. In 1998, to address concern for emerging resistance of pathogens to drugs (particularly antibiotics) during the 1990s, with the assistance of a Steering Committee on Antimicrobial Resistance, the AVMA approved 15 “Principles for the Judicious Use of Antimicrobials.”  These have served as the foundation of numerous subsequent guidance documents and policies involving antimicrobial use in all species – including aquatic animals. Many guidance documents have been developed and distributed in collaboration with other veterinary organizations and government agencies. AVMA policies are also used to guide the profession’s response to legislation and regulations, and in many cases they are also utilized by others. Concerns for resistance to antimicrobials approved for veterinary use increased during the last decade, particularly with respect to the potential implications in human medicine and the use of FDA approved non-therapeutic (growth promotion) antibiotics in livestock. The debates that incurred have, and will continue to influence legislation, regulations, policies, public opinion and guidance (including those at international levels, e.g. Codex Alimentarius, WHO, FAO and OIE) that affect the judicious use of antimicrobials in all animals – including aquatic species.






Aquaculture: Its Contribution to the Aquatic Resistome?


DW Verner –Jeffreys* and C Baker-Austin


Cefas Weymouth laboratory, Weymouth, Dorset, UK david.verner-jeffreys@cefas.ac.uk



The continued and accelerating development of antimicrobial resistance (AMR) is one of the most serious threats to animal, plant and human health we are presently facing.  The lack of development of new antibiotics, the persistence, even in the absence of selection, of antimicrobial resistance in several bacterial pathogens means that our ability to treat bacterial infections in farmed animals and man is increasingly threatened.  There is strong scientific evidence that much of the resistance in bacterial pathogens is encoded by ‘mobile’ genetic elements capable of efficient cross genus transfer of resistance genes sometimes in large genetically linked clusters.  Together with the resistance genes, come a whole host of genes of unknown function which may confer an entirely different selectable phenotype.  The reservoir of genes which transfer under the selective pressure of antibiotic usage is largely unknown. A large body of scientific evidence shows that bacteria harboring these mobile AMR genes are widely disseminated, and often at high prevalence, in all environments studied to date (e.g. soil, mining waste, livestock production sites, water treatment effluent, marine and freshwater water column and sediment samples). The potential threat that these environmental  ‘reservoirs’ of mobile AMR genes pose to human and animal (including fish)  health has not yet been ascertained,  but is likely to be considerable. For this presentation we will particularly focus on the aquatic environment and discuss the extent to which aquaculture practices are affected by, and may contribute to, the aquatic ‘resistome’.






Efforts by the U.S. Food and Drug Administration’s Center for Veterinary Medicine to Address Antimicrobial Resistance in Aquaculture


J Matysczak*


US Food and Drug Administration, Rockville, MD, USA.  Jennifer.matysczak@fda.hhs.gov



Antimicrobial drugs have been widely used in human and veterinary medicine for more than 50 years with tremendous benefits to both human and animal health.  However, a number of scientific reports have raised concerns over the use of medically important antimicrobial drugs in food-producing animals. The FDA has recommended phasing in measures that would limit the use of medically important antimicrobial drugs in food-producing animals to uses that are considered necessary for assuring animal health and that include veterinary oversight or consultation.  This presentation will provide a brief introduction to how the FDA considers antimicrobial resistance in its review of a new animal drug application for food-producing animals, its efforts to encourage judicious use of antimicrobials in aquaculture domestically and in collaboration with international governments and organizations, and the recent draft guidance published for comment.






Resistant or Susceptible May Not Always be Your Answer


RA Miller*


Center for Veterinary Medicine, U.S. Food and Drug Administration, Rockville, MD USA




In recent years, new terminology has emerged to more accurately describe and interpret in vitro antimicrobial susceptibility test results [minimal inhibitory concentrations (MICs) and zone diameters (ZDs)].  Epidemiologic cut-off values [also called wild-type cutoffs] separate populations on the basis of MIC and/or ZD distributions, and are used to predict the presence of resistance mechanisms in a given bacterial isolate.  These are not to be confused with the more robust clinical breakpoint categories, susceptible, intermediate, and resistant, which have been validated in the animal and are defined by MIC and/or ZD values.  Technically, it is incorrect for clinicians to describe any aquatic isolate as susceptible or resistant, with one exception; if oxytetracycline and/or oxolinic acid is tested against an isolate of Aeromonas salmonicida (see CLSI Supplement M42/M49-S1 for details).  To define an isolate as susceptible would mean the clinician has validated the isolate’s in vitro test result and correlated it previously with a positive clinical outcome in the patient (population).  Instead, this isolate should be categorized as wild-type (i.e., there are not likely to be resistance mechanisms present) or nonwild-type (the isolate likely possesses acquired and/or mutational resistance mechanisms).  Recent survey data has shown fish diagnostic laboratories use a wide range of ZDs to define their lab-specific clinical breakpoints.  However, a more harmonized approach to how in vitro susceptibility data is generated, reported, and interpreted could lessen some of the confusion encountered when interpreting data from within a country, much less across countries, and simultaneously maintain prudent antimicrobial use principles.






Development of Antimicrobial Susceptibility Testing Methods for Flavobacterium columnare and F. psychrophilum.


CM Gieseker*1and TD Mayer1


US Food and Drug Administration, Center for Veterinary Medicine, Office of Research, 8401 Muirkirk Road, Laurel, MD 20708 USA  charles.gieseker@fda.hhs.gov




The goal of this study was to develop reliable broth microdilution methods to test the drug susceptibility of the fish pathogens Flavobacterium columnare and F. psychrophilum.  Broth microdilution methods that use dilute 1:5 Mueller-Hinton broth (4g/L) were developed and/or confirmed, then used to test F. columnare (n=23, 28°C) and F. psychrophilum (n=23, 18°C) to determine the appropriate drug concentration ranges to use for standardized testing.  The quality control bacteria Escherichia coli, ATCC25922 and Aeromonas salmonicida, ATCC 33658 were also tested along with the Flavobacteria at both temperatures.  Custom frozen broth microdilution panels were used to test the susceptibility of these isolates to ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, oxolinic acid, oxytetracycline, sulfamethoxazole/trimethoprim, and sulfadimethoxine/ormetoprim.  To validate these broth microdilution methods, F. columnare (n=24, 28°C) and F. psychrophilum (n=24, 18°C) isolates were tested simultaneously by agar dilution and broth microdilution for their susceptibility to florfenicol and oxytetracycline.  The minimum inhibitory concentration (MIC) results from broth microdilution were similar to agar dilution.  An intra-laboratory trial was conducted with 4 technicians separately testing the susceptibility of F. columnare (n=6), F. psychrophilum (n=6) and both QC bacteria to ampillicin, florfenicol, oxytetracycline, and sulfadimethoxine/ormetoprim using our broth microdilution methods.   The technicians agreed >90% within one MIC dilution for both QC bacteria at both test temperatures and for most of the Flavobacteria isolates.  These results demonstrate that these broth microdilution methods are robust and can be used to standardize antimicrobial susceptibility testing for F. columnare and F. psychrophilum.



Analysis of Bacterial Antibiotic Resistant Genes in Aquaculture


JY Wang*, YJ Geng, RX Wang and J  Feng


South China Sea Fisheries Institute, Chinese Academy of Fishery Sciences, Guangzhou, China wjy104@163.com




Fifty-four bacterial strains derived from aquaculture sources in different regions were tested for the drug sensitivity of 10 common antibiotics determined by Kirby-Bauer disk diffusion according to NCCLS. With specific primers designed according to the reported sequences in GenBank, polymerase chain reaction (PCR) assay were used to amplify the antibiotic resistant genes and study the mechanisms of drug resistance. Forty-two of the 54 gram-negative bacteria were identified as resistant bacteria (77.8%), and 37 strains (68.5%) had multi-resistances for resistance to more than 3 antibiotics.  Resistant genes on plasmids were detected using PCR methods. Nine strains were tested including trimethoprim-sulfamethoxazole resistance gene sul2, 3 strains possessed chloramphenicol resistant gene cat2, 4 strains had chloramphenicol resistant gene cat3, only 1 strain was amplified to have chloramphenicol resistant gene cat4, and 3 strains were detected with kanamycin resistant gene aadB. Detection ratios of resistant genes in plasmid were 50%, 27%, 36%, 9%, 60% respectively.











Apicomplexan Parasites in the Brain of an Immature Indo-Pacific Green Sea Turtle


M Flint*1,2, JC Patterson-Kane3, CJ Limpus4 and PC Mills2

1 Aquatic Animal Health, College of Veterinary Medicine, University of Florida, Gainesville FL USA. Mark.Flint@MyFWC.com  
2 Veterinary-Marine Animal Research, Teaching and Investigation (Vet-MARTI) unit, School of Veterinary Science, The University of Queensland, St. Lucia, Queensland Australia. p.mills@uq.edu.au
3 School of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, United Kingdom. j.patterson-kane@vet.gla.ac.uk
4 Queensland Department of Environment, Resources and Management, Brisbane, Queensland Australia. col.limpus@derm.qld.gov.au 










Apicomplexan protozoan parasites of interest to aquatic animal caregivers and indigenous people include Eimeria, Neospora, Isospora and Toxoplasma spp. Toxoplasma gondii has been responsible for isolated to mass mortality events in several marine mammal species. However, it has not been shown to cause a true infectious state in wild reptiles. In October 2007, a 79.5 cm curved carapace length, large immature male green turtle (Chelonia mydas) stranded in Moreton Bay, Australia. Initial clinical examination revealed it to have a buoyancy disorder (“floater syndrome”), be emaciated and lethargic, and display neurologic deficits (right-sided head tilt).  After 12 days of palliative care at SeaWorld, Australia the turtle died. At necropsy the turtle was emaciated, a fishing hook was embedded in the gastric mucosa, and 10-15  1-mm diameter cream, nodular lesions, were noted in the small intestinal serosa. Tissue specimens were fixed in 10% neutral buffered formalin, processed routinely for histologic examination and stained with hematoxylin and eosin. The brain contained multiple protozoal cysts intermingled with inflammatory changes; the latter associated with spirorchiid trematode eggs.  There was meningeal fibroplasia with evidence of infiltration by mixed leukocytes. Diagnosis based on histopathology was cerebral toxoplasmosis, however immunolabelling for Toxplasma gondii was inconclusive and negative for Neospora spp. Molecular analysis of the formalinized sample was unsuccessful. Ultrastructurally, the protozoal elements showed features consistent with being Apicomplexans. Very little is known about such parasites in marine turtles and any possible clinical effects. Detailed investigation including conducting complete gross and histological necropsy examinations inclusive of the brain is warranted.






The Importance of Emerging Exotic Viral Pathogens of Ornamental Fish in Australia


AE Rimmer*, JA Becker, A Tweedie and RJ Whittington


Farm Animal and Veterinary Public Heath, Faculty of Veterinary Science, University of Sydney, Camden, Australia anneke.rimmer@sydney.edu.au




The movement of ornamental fish through exporting and importing practices provides a transmission pathway for the introduction and establishment of exotic viral pathogens.  The viral agents cyprinid herpesvirus-2 (CyHV-2) and megalocytivirus (gourami iridovirus) represent significant exotic pathogens in Australia, having been implicated as the cause of severe disease and mortality in intensively raised fish and ornamental fish.  The disease threat to the aquaculture industry, and to the native fish of Australia, has driven the development of molecular diagnostic tests capable of detecting these ornamental fish viruses. Optimized PCR assays for these pathogenic viruses were employed in a survey of ornamental fish from Australian pet shops.  Twelve of 37 goldfish (Carassius auratus) tested positive for CyHV-2, while 8 of 23 dwarf gouramis (Colisa lalia) and 2 of 7 blue/gold gouramis (Trichogaster trichopterus) tested positive for megalocytivirus.  The murray cod (Maccullochella peelii peelii) is also susceptible to megalocytivirus.  An iconic species of high conservation value in Australia, the murray cod is a member of the Family Pericichthydae, several members of which are listed as vulnerable or endangered in Australia, including trout cod (Maccullochella macquariensis), golden perch (Macquaria ambigua ambigua), and Macquarie perch (Macquaria australasica).  To further study the impact of megalocytivirus on Australian fish, an experimental challenge model was developed in murray cod.  The outcomes from this research have important implications for both quarantine policy and management of ornamental fish in Australia. 





Correlations of Swimming Patterns with Vertebral Deformities in Captive Sandtiger Sharks, Carcharias taurus


EE Tate* 1, 2, PA Anderson1 and IK Berzins3

1 The Florida Aquarium Center for Conservation, Tampa, Florida USA panderson@flaquarium.org
2 The University of Tampa, Tampa, FL USA  etate07@gmail.com
3 John G. Shedd Aquarium, Chicago, IL USA  Iberzins@sheddaquarium.org






Swimming behavior is hypothesized to contribute to the development of vertebral deformities in captive sandtiger sharks (Carcharias taurus). Public aquaria across the United States were surveyed and asked to record video of individual C. taurus swimming from the top and from the side of the exhibits for ten minutes per view. Fifty-three individuals from 11 institutions were recorded, and the records sent for ethological observation and analysis. Thirteen sharks had vertebral deformities (via gross appearance, radiography, and/or veterinary diagnosis), the remaining 40 were healthy. Data was recorded and analyzed using JWatcher software. Behaviors measured included duration and time percent swimming in a direction, gliding percentage, and tail beat duration. Combined, C. taurus spent a median of 98.9% of time swimming and 0.62% of time gliding; furthermore, healthy sharks spent a median of 0.67%  of time gliding versus a median 0% for afflicted sharks, suggesting an increased swim-to-glide ratio. All sharks swam asymmetrically (in a circular pattern, clockwise or counter-clockwise) at a median of 99.7% of the time. Specimens with vertebral deformities had tail beat durations of 3.36 ± 0.22 s vs 2.72 ± 0.10 s for healthy sharks.  Stereotyped swimming patterns in accordance with an increase in swim-to-glide ratios support the hypothesis that swimming patterns, especially in correlation with smaller tanks and exhibits, may be causative of, or at least may exacerbate, vertebral deformity in C. taurus. Behavioral enrichment programs and implementation of larger and non-circular tank designs are recommended to decrease the likelihood of the deformity occurring in captive specimens.





Prospecting for Biomarkers of Health in Whale Sharks, Rhincodon typus, Using Metabolomic Methods


ADM Dove*1, J Leisen2, M Zhou2, K Lim-Hing2, DH Webb1, L Gelbaum2, J Byrne3, M Viant3, F Fernandez2 and J Kubanek2


1 Georgia Aquarium Research Center, Georgia Aquarium, Atlanta, GA USA adove@georgiaaquarium.org
2 Georgia Institute of Technology, Atlanta GA Julia.kubanek@biology.gatech.edu
3 School of Biosciences, University of Birmingham, Birmingham UK m.viant@bham.ac.uk





Whale sharks are plankton-eating sharks that live in tropical oceans throughout the world; they are the largest of all fishes.  Despite their size, or perhaps because of it, relatively little is known about their physiology or biochemistry.  The long-term management of a population of whale sharks in an aquarium in Atlanta has provided opportunities to redress this situation.  Terminal illness in two of six animals allowed for comparison of serum chemistry between healthy and moribund animals, with the goal of identifying promising biomarker compounds to serve as indicators of health in this species.  Serum was analyzed by complimentary metabolomic methods that could identify and sometimes quantify unknown metabolites in serum samples.  Several 1D and 2D 1H nuclear magnetic resonance experiments were followed by analyses using DART mass spectrometry that confirmed putative identifications and also provided extensive lists of candidate compounds based on mass/charge ratios of individual metabolites.  Serum of whale sharks included a diverse array of metabolites, among which osmolytes (particularly TMAO), amino acids and hydroxy acids were the most frequently encountered.  Multivariate analyses were used to show metabolic trajectories of the moribund animals.  Homarine, or n-methyl picolinic acid, was a prominent driver of these trajectories; direct quantification by NMR confirmed that it had a significantly lower concentration in moribund whale sharks compared to healthy animals and is therefore a promising biomarker.  This is the first application of metabolomics to sharks and the first report of homarine in an elasmobranch, but its function remains unknown. 






Megalocytiviruses of Ornamental Fish and Implications for Aquaculture Species


J Go*1, M Lancaster2, K Deece1, O Dhungyel1 and RJ Whittington1


1 Faculty of Veterinary Science, University of Sydney Australia jeffey.go@sydney.edu.au
2 Victorian Institute of Animal Science, Attwood, Australia




Research was undertaken to determine whether viruses present in ornamental fish are linked to emerging viral diseases in aquaculture. The present study was prompted by a disease outbreak in February 2003 in Australian native Murray cod (Maccullochella peeli peeli) resulting in losses of up to 90% in two groups of fingerlings at a Victorian aquaculture facility. Histology revealed pathology consistent with Megalocytivirus infection, including those found in ornamental fish, and distinct from Epizootic Haematopoietic Necrosis Virus – the only systemic piscine iridovirid known to occur naturally in Australia. Using a molecular epidemiological approach, DNA was extracted from samples collected during the disease outbreak, and PCR primers were designed to amplify DNA from the major capsid protein (MCP), ATPase and RNA polymerase genes as well as CY15 and IRB6 amplicons. Sequencing results from PCR products indicated that there was extremely close homology between the Murray cod iridovirus (MCIV) isolate and viruses in the genus Megalocytivirus, particularly infectious spleen and kidney necrosis virus (ISKNV) and dwarf gourami iridovirus (DGIV) sequences in Genbank. Screening of routine gourami mortalities from pet shops in Sydney, using PCR primers designed against the MCP gene, revealed 22% were positive for Megalocytivirus DNA. When organ filtrates of PCR positive samples were injected intraperitoneally into Murray cod juveniles, 29 of 30 died within 21 days, and cohabitation of juvenile Murray cod with live gouramis resulted in transmission of Megalocytivirus through water and death of 11 of 30 Murray cod. Sequencing of this Megalocytivirus isolate derived from dwarf gouramis revealed 99.95% similarity, over 4527 nucleotides, between this isolate and MCIV, suggesting a likely origin of MCIV through the trade in ornamental fish.






Correlations of Capture, Transport, and Nutrition with Vertebral Deformities in Captive Sandtiger Sharks (Carcharias taurus)


PA Anderson*1, DR Huber2, DE Noaker2 and IK Berzins3


1 The Florida Aquarium Center for Conservation, Tampa, Florida 33602 USA  panderson@flaquarium.org
2 The University of Tampa, Tampa, FL  USA  dhuber@ut.edu  dnoaker@ut.edu
3 John G. Shedd Aquarium, Chicago, IL USA  Iberzins@sheddaquarium.org





Captive sandtiger sharks (Carcharias taurus) commonly develop vertebral deformities.  U.S. public aquaria participated in a three-stage epidemiological study of resident sharks:  1) a history and husbandry survey, 2) hematology, clinical chemistry, and radiography requested during health exams, and 3) a necropsy protocol upon expiration or euthanasia.  Eighteen aquaria submitted data and/or samples from 73 specimens, including 19 affected sharks (35.2%).  Smaller sharks caught off the Rhode Island coast and/or by pound net demonstrated a higher prevalence of deformity than larger sharks caught from other areas or with hook and line.   Affected sharks had lower leukocrits, disputing an infectious etiology, but suggesting a chronic stress response to the condition.  Affected sharks were also deficient in zinc, potassium, and vitamins C and E relative to healthy sharks.  Zinc and vitamin C play critical roles in proper cartilage/collagen development and maintenance. Flexural stiffness of intact vertebral columns and compressive stiffness, yield strength, and yield strain of individual vertebrae from healthy animals were greater than those from deformed animals; attributable to deficits in both the quantity and distribution of mineral within the vertebrae of deformed specimens. Additionally, compressive stiffness and ultimate strength of vertebrae from healthy specimens were still lower than those of other species tested, suggesting either a species-specific predisposition for vertebral deformity, or abnormalities in tissue modeling due to nutrient deficiencies from captive diets.  Hence, aquarists should consider choice of collector and size at capture, and should supplement diets to provide nutrients required for proper development and maintenance of cartilaginous tissue.







Ulcerative Mycoses Caused by Aphanomyces invadans and Ochroconis sp. in Wild and Cultured Marine Fish Species.


JP Hawke*, W Baumgartner, and E Soto


Department of Pathobiological Sciences, LSU School of Veterinary Medicine, Baton Rouge, LA, USA. jhawke1@lsu.edu, wbaumg1@tigers.lsu.edu, esoto1@lsu.edu.




Ulcerative mycotic diseases are described from wild populations of red drum Sciaenops ocellatus and black drum Pogonius cromis and cultured cobia Rachycentron canadum. This is the first report of an invasive fungal infection in cobia. The diseases were diagnosed from specimens voluntarily submitted to the Louisiana Aquatic Diagnostic Laboratory at the School of Veterinary Medicine, Louisiana State University. Adult red drum and black drum were collected from the inshore marsh habitat in Louisiana and the juvenile cobia (10-15 cm) were from a recirculating culture system.  Specimens presented with sharply demarcated, circular to irregular, ulcerative lesions in the skin and penetrating into the underlying muscle tissue.  Oomycete and dematiaceous hyphomycete fungi were isolated by aseptic necropsy on peptone yeast glucose (PYG) agar from infected muscle tissue. The Aphanomyces colonies grew slowly on the agar medium at 25°C and produced white velvety colonies that grew above and below the agar surface. The hyphomycete fungus Ochroconis sp. also grew slowly at 25°C and produced brown pigmented colonies on the surface of PYG agar. Histopathology of Aphanomyces infected fish revealed non-septate hyphae penetrating deep into the muscle surrounded by granulomatous inflammation and multinucleated giant cells. Histopathology of Ochroconis infections revealed melanized, septate, fungal hyphae surrounded by epithelioid granulomatous inflammation in skin, muscle, intestine, and kidney tissues.  Identification of the pathogens was accomplished using a combination of morphological characters and molecular analysis by amplification of the internal transcribed spacer (ITS) region located between the 18S and 28S rRNA genes and sequencing.






Exophialiosis in Bonito (Sarda chiliensis) from a Public Aquarium


K Hirokawa*1, M Murray2, A Reed1 and ES Weber III1


1 Aquatic Animal Health Laboratory, University of California at Davis, Davis, CA, USA khirokawa@gmail.com, aigialos@yahoo.com, epweber@undavis.edu
2 Monterey Bay Aquarium, Monterey, CA USA MMurray@mbayaq.org





Two wild-caught pacific bonito (Sarda chiliensis) from Monterey bay died during the quarantine without prior clinical signs. The gross necropsy findings included skin ulceration, enlarged spleen, swollen kidney with black nodules, granulomas in the spleen, and black fungal hyphae were identified in renal impression smears using light microscopy. Tissue samples were submitted for histopathologic examination to the University of California, Davis. The results of histopathologic examination revealed black septate fungus in the kidney and skin. The kidney and skin cultures grew exophiala species, and Vibrio alginolyticus and Photobacteruim damsela from kidney, skin, and blood. Exophiala samples were sent to a laboratory in Texas for speciation. Previously, exophiala species have been cultured from marine and fresh water fish including striped jack, Atlantic salmon, sandlance, smooth dogfish, and leafy sea dragon in captive and cultured animals. In the current case, while the origin of infection is not yet established for the bonito, the previously reported cases all involve captive or cultured fish. Thus far, no wild animal cases of exophialiosis have been reported, and nosocomial infection is suspected in the current case. As far as we are aware, this is the first case of exophialiosis reported in bonito. Exophiala are ubiquitous in soil and water, and is a significant pathogen which can cause systemic disease, resulting in significant mortality and morbidity in some fish. It has also caused severe disease in valuable aquarium fish such as sea dragons. The prevention and control of systemic mycotic disease is critical in maintaining healthy aquarium animal collections.







Effects of Environmental Temperature on the Kinetics of Ichthyophoniasis in Juvenile Pacific Herring (Clupea pallasii)


JL Gregg*1, JJ Vollenweider2, CA Grady1, RA Heintz2, and PK Hershberger1


1 U.S. Geological Survey, Western Fisheries Research Center, Marrowstone Marine Field Station, Nordland, WA USA jgregg@usgs.gov
2 NOAA National Marine Fisheries Service, Alaska Fisheries Science Center, Auke Bay Laboratories, Juneau, AK USA





Ichthyophonus, a widely distributed parasite of the Atlantic and Pacific Oceans, significantly impacts commercially important fish stocks through acute epizootics (fish kills), chronic persistent mortality, reduced fitness, and reduced product quality.  Primarily considered a disease of older adult cohorts, Ichthyophonus may also play a role in depressing survival of juvenile fish and thus limiting their recruitment to fisheries.  Here we present the results of experiments conducted to examine the effect that Ichthyophonus has on juvenile Pacific herring (Clupea pallasii) undergoing a simulated over-winter fast.  These experiments were carried out at three temperatures and have given us new insights into the effects that environmental temperature has on disease processes and mortality.  Temperature had no effect in herring with established disease; however, temperature did affect mortality in newly infected fish.  Both lowering and raising the temperature tended to reduce mortality.  Warmer temperatures likely improve host immune function, while cooler temperatures slow parasitic growth.  When the exposure dose was reduced, we detected an inverse relationship between infection prevalence and temperature.   These data indicate that the environmental temperature at the time of parasite exposure may determine the prevalence of disease in wild fish populations over ensuing months and years.




Molecular Detection of Ichthyophonus in Walleye Pollock and the Search for Planktonic Reservoirs Using Quantitative PCR


VL Lowe*1,2, CS Friedman1 and JF Morado2


1 School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA USA  carolynf@u.washington.edu
2 NOAA Fisheries, National Oceanic and Atmospheric Administration, Alaska Fisheries Science Center, Seattle, WA USA Vanessa.Lowe@noaa.gov, Frank.Morado@noaa.gov





Ichthyophoniasis is a disease of economic significance in both wild and cultured fisheries and is caused by the protist, Ichthyophonus. The parasite has low host specificity and is acquired through feeding on infected prey. Zooplankton may act as potential reservoirs for Ichthyophonus and aid in transmission to fish hosts.  An Ichthyophonus-like parasite was identified in walleye pollock, Theragra chalcogramma, an ecologically and commercially important North Pacific fish. Intense parasite infections can adversely affect product quality by altering cooked fillet texture, causing increased fillet screening by the industry. To estimate Ichthyophonus prevalence in walleye pollock and identify infected prey items, a probe-based quantitative PCR (qPCR) assay has been developed based on the 18S rDNA gene of Ichthyophonus. Paired samples for qPCR and histology were collected from the Bering Sea in 2006 and 2007 to compare detection methods. Age-0 pollock (<11cm) were examined by histology and plankton samples collected in 2007 were screened using qPCR. Parasite prevalence in age-0 fish was estimated at 2%, and increased with fish size to 28% in adult pollock.  Suspect Ichthyophonus has been detected in mixed plankton samples, but these results should be approached with caution without confirmation by other methods.  On-going studies are focused on parasite detection in sorted plankton taxa found in the diet of age-0 pollock. Validation of this assay delivers an additional tool for Ichthyophonus research and may aid in the characterization of alternative infection pathways.







A New Microsporidian from Common Stingray Dasyatis pastinaca in the Mediterranean Sea


A Diamant*1,, M Goren2, MB Yokeş3, BS Galil4, Y Klopman2, D Huchon2, A Szitenberg2 and SÜ Karhan5


1 National Center for Mariculture, Israel Oceanographic and Limnological Research, Eilat, Israel diamant@agri.huji.ac.il
2 Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
3 Haliç University, Faculty of Arts & Sciences, Department of Molecular Biology & Genetics, Okmeydani, 34384 Istanbul, Turkey.
4 National Institute of Oceanography, Israel Oceanographic and Limnological Research Haifa, Israel
5 Division of Hydrobiology, Department of Biology, Faculty of Science, Istanbul University, Istanbul, Turkey.










A new microsporidian infecting the Mediterranean common stingray Dasyatis pastinaca was found in Iskenderun Bay, Turkey. Fish samples were taken by bottom trawl during summer 2009. The parasite invaded the stingray disc muscles, producing spindle-shaped subcutaneous swellings and tumor-like masses measuring up to 11 cm in length. The masses contained a yellowish-white substance which contained degraded host tissue and parasites developmental stages. The ripe spore contained a single nucleated sporoplasm and had a large posterior vacuole. Prevalence was 21% in a sample of 143 host individuals examined. While normal appearing, uninfected individuals reached 3.8 kg, all infected stingrays were within the weight class of 300 - 800 g. There is only a single previous report of a microsporidian in a cartilaginous fish: and infection in captive sharks in a public aquarium. The present study reports on the first documentation from a wild elasmobranch population. Analyses of the 16S rRNA gene indicated that this microsporidian belongs to the Pleistophoridae. It clustered with species of the genera Heterosporis Schubert, 1969 and Ovipleistophora Pekkarinen, Lom & Nilsen, 2002. However, the morphology, development and host differed distinctly from all currently known species, and thus the new genus Dasyatispora is erected, with the new species named D. levantinae gen. et sp. nov.




Diversity of Microsporidia Among Marine Parasitic Copepods (Caligidae) in the Northeast Pacific Ocean


SRM Jones*, G Prosperi-Porta and E Kim


Department of Fisheries and Oceans, Pacific Biological Station, Nanaimo, British Columbia Canada. simon.jones@dfo-mpo.gc.ca




Desmozoon lepeophtherii is a microsporidian parasite of the salmon louse (Lepeophtheirus salmonis) infesting farmed Atlantic salmon in Scotland and Norway.  Microsporidia with similarities to D. lepeophtheirii were observed in salmon lice from Puget Sound, Washington.  The prevalence of microsporidia in salmon lice was further documented by examining specimens from Puget Sound (PS) and British Columbia (BC), Canada, and those belonging to five other Lepeophtheirus species from BC.  Analyses of the small subunit ribosomal gene amplified from 157 copepods revealed three distinct microsporidian sequences that differed in host association, prevalence and geographic distribution.  Type A was found only in L. salmonis with a prevalence of 15% in PS and 4% in BC. The genetic distance of Type A from that of D. lepeophtherii was 0.004.  Type B occurred in L. salmonis (43% prevalence in PS, 30% in BC), L. cuneifer (50% prevalence) and L. parviventris (30% prevalence).  The genetic distance between Types A and B was 0.525.  Type C was obtained from a single specimen of L. hospitalis and differed from Types A and B with genetic distances of 0.507 and 0.287, respectively.  Type A clustered with strong support among the enterocytozoonids whereas Types B and C were weakly supported among microsporidia of marine origin.  Microsporidia associated with sequence types A and B displayed distinct patterns of development and tissue distribution.  Thus a geographic variant of D. lepeophtherii and two novel microsporidia occur among caligid copepods in the northeast Pacific Ocean.







Effect of Essential Oils on the Viability of Caligus rogercresseyi Using a Novel in vitro Assay Based on the Inhibition of Lice Frontal Filament Formation.


J Troncoso* and J González


EWOS Innovation, Colaco Research Center, Av. Benavente 550 piso 10A, Puerto Montt, Chile jose.troncoso@ewos.com




Three in vitro assays were performed to assess a product based on a mixture of essential oils obtained from cinnamon, eucalyptus and oregano, against the sea lice Caligus rogercresseyi. Two assays were conducted to determine the LD50 of the oils against copepodids and adult lice, respectively. The first trial was performed using 10 different doses of the essential oil mix ranging from 0.175 to 175 μL/L. In the second assay, seven different doses of the essential oil mix were tested, ranging from 0.175 to 52.5 μL/L. A third assay was performed using a novel method to evaluate the degree of inhibition of the formation of the frontal filament of copepodids. In this last assay, four different concentrations of the essential oil mix were tested: 0, 0.175, 6.869 and 13.563 μL/L. The essential oils induced mortality of copepodids and adults of Caligus rogercresseyi. The calculated LD50 against copepodids of C. rogercresseyi was 21.6 ppm (18.5-25.8, 95% credible interval) at 48 h of exposure. Similarly, the calculated LD50 against adults of C. rogercresseyi was 22.1 ppm (18.4-27.4, 95% credible interval) at 48 h of exposure. The method to test the inhibition of the formation of the frontal filament was used as a measure of copepodid viability. The dose of essential oils needed to inhibit the formation of the frontal filament in 80% was significantly lower than the LD50 required to kill copepodids (0.175 ppm vs 21.6 ppm). The method to determine the capacity of essential oils to inhibit the formation of the frontal filament of copepodids of C. rogercresseyi proved to be a sensitive test to evaluate potential anti-sea lice compounds




Conceptual Framework for the Use of Parasites of Fishes as Bioindicators of Acute and Chronic Environmental Perturbation after the 2010 Deepwater Horizon Oil Spill (DHOS) in the Gulf of Mexico


SA Bullard*1 and GW Benz2


1 Department of Fisheries and Allied Aquacultures, Aquatic Parasitology Laboratory, Auburn University, Auburn, AL 36849, USA ash.bullard@auburn.edu
2 Department of Biology, Middle Tennessee State University, Murfreesboro, TN, USA gbenz@mtsu.edu





Fish parasites comprise a large portion of marine biodiversity but have been virtually ignored as Gulf of Mexico (GoMex) bioindicators. Shifts in parasite diversity, prevalence, and intensity resulting from the DHOS could indicate spill-related changes to water quality, abundances and immunological health of free-living organisms, or the GoMex food web. Ectoparasites with direct life cycles (no intermediate host or food-web mediated transmission) may be sentinels for acute spill effects, as they are typically small, have high surface area to volume ratios, and remain immersed in seawater. Endoparasites with indirect life cycles (intermediate host[s] required) involving food-web mediated transmission may be sentinels for detecting chronic spill effects, as they reside in a host where they are less vulnerable to toxins and have larvae requiring predator/prey transmission. Environmental perturbations affecting food-web structure (chronic effect) typically impact endoparasites more than ectoparasites. A ratio of the numbers of ectoparasite species to endoparasite species per fish species per geographic locality has been informative regarding oil pollution events: a relatively smaller ratio signals acute environmental effects (i.e., ectoparasites die; endoparasites survive), a relatively larger ratio signals chronic environmental effects (i.e., ectoparasites recolonize hosts as water normalizes; endoparasites are extirpated as food-web links are broken by loss of intermediate hosts). Our recently funded RAPID Response study (National Science Foundation grant DEB-1048523) focuses on these matters in hope of gaining critical new insights about parasites as bioindicators on a regional, basin scale while providing insights on the environmental “ripple effects” of the DHOS. Supported by NSF grant DEB-1048523.






Molecular Identification, Ultrastructure and Histopathological Studies of Microsporidian Parasite of Transparent and Albino Puntius tetrazona


O Chang, K Li, F Wang, C Liu and S Wu


Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, Guangdong, China changouqin@163.com




Specimens of the transparent and albino Puntius tetrazona, obtained by intraspecies hybridization at the Pearl River Fisheries Research Institute, were discovered to have numerous cystic masses in their muscle tissue.  After pressure was applied to the muscle tissue, many sporocysts and spores were observed.  We set out to identify these spores using ultrastructural, histopathological, and molecular methods.  Under a transmission electron microscope, the mature spores were uninucleate, ovoid or pyriform, and approximately 7-8 μm in length and 3-4 μm in width.  They possessed a polar tube surrounding the posterior vacuole, which occupied about half of the spore’s total volume.  Under a scanning electron microscope, sporocysts with a thick, dense wall were observed in the muscle tissue, and many mature ovoid spores were present within the sporocysts. To further characterize the parasite, we prepared histopathological sections from the muscle of infected fish, and stained them with hematoxylin and eosin.  The spores and sporocysts were in different developmental stages, all of which developed within a wall-covered structure called the sporophorocyst. Together, these ultrastructural and histopathological features suggested that the spores represent a microsporidian Pleistophora parasite, which was confirmed by molecular methods.  Furthermore, when healthy transparent and albino Puntius tetrazona were fed and injected with spores isolated from infected muscle, the ultrastructural and histopathological features described above were observed.  Our results suggest that the microsporidian parasite was Pleistophora, and that the transparent and albino Puntius tetrazona would be a good model organism for Pleistophora infection.






Isolation and Identification of a Novel Reovirus by Using a Newly Established Cell Line Derived from Kidney of Channel Catfish, Ictalurus punctatus, Rafinnesque


LB Zeng*, J Xu, XL Li, XS Luo, Y Xiao, and Y Zhou

Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Jingzhou City, Hubei China zenglingbing@gmail.com




A cell line, designated CCK, derived from the kidney of Channel catfish, Ictalurus punctatus, Rafinnesque, has been newly established and characterized. A novel reovirus has been isolated in CCK cells from channel catfish fingerlings suffered with severe hemorrhage, and has been identified as channel catfish reovirus (CCRV). In an artificial infection test in fish, the typical naturally occurring symptoms of channel catfish hemorrhage could be reproduced. The isolated virus could cause typical cytopathic effect in CCK cells. Electron microscopy observation of ultra-thin section samples of CCRV infected CCK cells revealed that the virus replicated in the cytoplasm, was present in crystalline arrays, and had a non-enveloped double capsid with a diameter of 60-70nm. Frozen-thawed, 56oC 1h, chloroform and ether had no significant effects on CCRV titre, 65oC 1h could significantly inactivate the viral infectivity. The CCRV genome SDS-PAGE analysis and nuclease sensitivity test showed that the virus genome was the same as that of viruses in Aquareovirudae, and consisted of 11 segments of dsRNA assigned into three classes L1, L2, L3; M1, M2, M3 and S1, S2, S3, S4, S5 with a range of size from 0.9 to 4.4bp. The cloning and sequencing of the CCRV S4 segment indicated the nucleic acid number of CCRV S4 was 909bp, which was exactly the same in length as that of GCRV S4 (AF403396) and GSRV S4 (AF403407) segments. The BLAST of CCRV S4 sequence in NCBI GenBank showed that it had a 99% and 90% similarity in sequence to the GCRV S4 and GSRV S4 segments, respectively.







Re-activation of Koi Herpesvirus (KHV) in Persistent Infected Fish Used for Virus Detection After Non-lethal Sampling


SM Bergmann*


Friedrich-Loeffler-Institut (FLI), German Reference Laboratory for KHVD, Institute of Infectology, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany sven.bergmann@fli.bund.de




KHV disease (KHVD) has spread world-wide by transfer of infected but non-diseased carp (Cyprinus carpio). Due to limited sensitivity of CCB or KF-1 cells, molecular tools represent the “gold standard” for KHV detection. As a herpesviral characteristic, fish become “healthy” persistent carriers after infection. In persistent phase, virus load can decrease often to 5 genomic particles measured by real-time PCR. The reaction in fish after KHV infection depends on water temperatures, virus concentration and virulence as well as on immune status of the fish. While concentrations such as 105 TCID50 / ml at 23°C may lead to a severe KHVD outbreak, concentrations of 102 TCID50 / ml at 12°C cause often a persistent asymptomatic infection (Meyer 2007). Virus eclipse represents a severe diagnostical problem. Routinely used detection methods are in an unfavourable position because of false-negative results. To overcome this and to provide a loophole, KHV infected carp which had shown KHVD for 2 - 3 weeks p.i., were kept under the same conditions (20°C, re-circulating system) further on. These carp, without any sign of KHVD, were controlled weekly for KHV presence by non-lethal sampling. In a basin, fish were netted to simulate transportation. Daily, gill swabs and dropping samples were taken and investigated by molecular methods (Bergmann et al. 2010). In daily taken swabs an individually different increase of the virus load was detected between days 1 and 3 after netting. In droppings, KHV was only detected at day 3 after netting. Generally, virus concentration decreased dramatically in swabs from day 4 after netting. Even this stress did not provoke KHVD. In conclusion, it was shown that non-lethal sampling methods after stress induction are a useful tool for diagnosis of KHV in persistent infected fish without killing those carp.




Detection of KHV in Healthy Non-carp Carrier Fish in Polish Inland Waters by PCR.


J Kempter, M Kielpinski, R Panicz* and J Sadowski 


Department of Aquaculture, West Pomeranian University of Technology, Szczecin, Poland remigiusz.panicz@zut.edu.pl



Wild- as well as cultured fishes were examined for the presence of the Koi-Herpes-Virus (KHV) genome. The epizootic map of the Odra River drainage was compiled based on the results covering 17 fish-rearing facilities and 49 open-water sites.  In total, 525 fish specimens were studied. A potential for transmitting the virus to common carp by: vimba, grass carp, and silver carp was also tested. The transmission experiment was based on co-habitation of specific pathogen free (SPF) carp with vimba, tench,  and grass carp.  The studies were carried out in three experimental treatments, differing in temperature regimes, each in two repetitions. Obtained results clearly showed that virus transmission in tench and vimba aquariums were present in all temperatures (18, 23, 27 °C).  The second set of experiments focused on testing inter-specific hybrids (Cyprinus carpio × Carasius auratus, Cyprinus Carpio × Carasius carasius) for their resistance to KHV infection and for their chances to develop clinical signs of the disease. The fish farms, diagnosed herewith as KHV positive, should not rear carp for a minimum of two seasons.  In the meantime, they may culture other fish species after performing disinfection procedures. It should be emphasized that a full rearing potential of an aquaculture enterprise, following a disease outbreak, is very costly and it cannot be successful without public money. Leaving such fish farm on its own, without public support and with a KHV-positive label would result in the farm bankruptcy.  The data gathered within the present project were compiled to create the epizootic map of the Odra River drainage, which is intended to provide the relevant information to fish farmers and users of open waters about the current KHV-infection state of this drainage.






Analysis of Attenuation in a Gene 50 Deleted Channel Catfish Virus (Ictalurid herpesvirus 1) Construct Suggests That More is Involved than Lack of Gene 50 Expression.


Larry A. Hanson*1, Nathalie Vanderheijden2, Joseph A. Martial2, Dusan Kunec1 and Lorelei Ford1


1 Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USA hanson@cvm.msstate.edu
2 GIGA-Research - Unité de Biologie Moléculaire et Génie Génétique, Université de Liège, Sart Tilman, Belgium






We had previously established a recombinant channel catfish virus (CCVdl50) that was highly attenuated and has potential for use as a CCV vaccine (Vanderheijden et al. US patent 6,322,793).  This virus was established by replacing the gene 50, which encodes an excreted glycoprotein, with Escherichia coli lac Z gene.  In this study we evaluated the cause of the attenuation observed in CCVdl50. Because gene 50 is close upstream to the dUTPase gene, we generated CCV recombinants deleted in just the 5’ region of gene 50 using infectious BAC technology. These recombinants did not express the excreted glycoprotein but were only moderately attenuated, suggesting that combined effects may have attenuated CCVdl50. To further evaluate this phenomenon we generated a series of CCVdl50 revertants by co-transfecting the gene 50 encoding region with CCVdl50 genomic DNA into CCO cells.  The resulting revertants expressed the gene 50 glycoprotein and regained some virulence.  However they were still highly to moderately attenuated compared to the wild type.  The revertants demonstrated some variation in the size of the gene 50 and we hypothesize that the repetitive nature of gene 50 sequence caused sequence variability in the revertants, confounding this analysis.  The combined data suggest that the attenuated nature of CCVdl50 is caused by interrupting gene 50 expression and another uncharacterized factor.







A Fatal Reovirus is Highly Prevalent in Blue Crab, Callinectes sapidus, in Aquaculture and Soft Shell Crab Production Facilities


HA Bowers1, L Carrion2, GA Messick3, O Zmora4, A Hanif1, R Jagus1 and EJ Schott*1


1 Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science, Baltimore, MD USA  schott@umces.edu
2 Coveside Crabs, Inc., Dundalk, Maryland  USA  ldcupuc@verizon.net
3 Center for Coastal Environmental Health & Biomolecular Research at Charleston, USDOC/NOAA/NOS/NCCOS, Oxford, Maryland USA  gretchen.messick@noaa.gov
4 Department of Marine Biology, University of Maryland Baltimore County, Baltimore, MD USA  zmorao@umbc.edu









The blue crab, Callinectes sapidus, supports a $150 million/year fishery in Atlantic and Gulf states of the USA.  Fishing pressure has necessitated harvest restrictions in many states. Fishermen can increase the value their catch by setting aside pre-molt animals (“peelers”) for production of high value soft-shell crabs.  However, crabs in soft-shell production facilities typically suffer mortalities of 25-50%.  We suspected that virus infections were responsible for some mortalities. By enriching double stranded RNA from crabs, we uncovered a reo-like viral RNA genome pattern in over 50% of the dead and dying animals but in fewer than 5% of healthy peelers.  Virus passage was accomplished by injection of crab homogenate, and was always fatal.  Electron microscopic analysis of virus-injected crabs shows reo-like virions within hemocytes.  Analyses of dying peelers from other soft-shell systems in MD, GA and FL revealed a >60% prevalence of reovirus.  We cloned part of the reovirus genome, and designed and validated a sensitive Rt-PCR to detect it.  Using these molecular tools, we have screened wild blue crabs collected by a NOAA/NCCOS project to assess Chesapeake Bay health.  The prevalence of reovirus was <4%, though a single sample showed a prevalence of 66%. The effects of this virus on blue crab populations in the wild may be underappreciated. We are continuing to monitor and conduct transmission experiments to better understand the impact of this virus on wild crabs and to understand why the prevalence in captive crabs is so high. 






Metagenomic Studies Reveal Novel Viruses in Shrimp


TFF Ng*1, S Alavandi2 and M Breitbart1 


1 University of South Florida, Saint Petersburg, FL, USA terryfeifan@gmail.com
2 Central Institute of Brackishwater Aquaculture, Chennai, India




The emergence of viral pathogens is a serious threat to aquaculture and fisheries worldwide. However, our response to viral pathogens has been largely reactive – a new pathogen is usually not discovered until it has already reached epidemic proportions. Current diagnostic methods such as PCR, immunological assays, and pan-viral microarrays are limited in their ability to identify novel viruses. Viral metagenomics, which involves viral particle purification and shotgun sequencing, has proven to be useful for describing novel viruses. Using viral metagenomics, this study investigated the virus diversity in hepatopancreas, heart and hemolymph from asymptomatic wild shrimp (Farfantepenaeus duorarum) collected from Tarpon Springs, Florida. Metagenomic sequencing of DNA and RNA viruses purified from these shrimp led to the discovery of a novel circovirus and two novel nodaviruses. The novel shrimp circovirus had a single-stranded DNA genome of 1955 nucleotides, and shared less than 50% amino acid identity to any known viruses in the Genbank database. All previously described circoviruses infect birds or pigs, making this shrimp circovirus the first circovirus described in an invertebrate. The shrimp circovirus represented a phylogenetic branch distinct from the known avian and porcine circoviruses. Two novel nodaviruses, which shared less than 60% amino acid identity to known shrimp nodaviruses and likely represent a novel virus genus, were also identified in the shrimp. Future research needs to examine the prevalence and ecological impact of these viruses; however, this study has demonstrated the potential of viral metagenomics to characterize novel viruses in aquatic species, and its application for viral disease prevention and monitoring.












Special Session













Construction and Screening of a Library of Attenuated Mutants in Edwardsiella ictaluri, Identification of Genes Involved in Bacterial Invasion and Potential as Vaccines


S Menanteau-Ledouble* and ML Lawrence


Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, MS, USA smenanteau@cvm.msstate.edu, lawrence@cvm.msstate.edu 




Attachment and invasion of host cells are very important factors in the virulence of a number of pathogenic bacteria. Previously, we investigated these mechanisms in the enterobacterial catfish pathogen Edwardsiella ictaluri by constructing a random MarTxT7 library of E. ictaluri mutants harboring the bioluminescence plasmid pAKLux. This library was then screened in a series of three nested challenges to identify mutants that showed decreased ability to colonize catfish epithelium. We identified 18 colonization-deficient mutants that were then confirmed to be attenuated in another challenge.  Gene mutations were identified by single primer PCR and sequencing and included a dipeptide transport system, an outer-membrane lipoprotein, as well as a putative adhesin.  Four of these mutants were used in a vaccination trial using 14 day old catfish fry followed 4 weeks later by experimental exposure to wild-type E. ictaluri.  Results suggested that these mutants are attenuated and provide a limited level of protection against ESC. This study allowed not only for the identification of several genes involved in skin colonization but also yielded several promising candidates for the development of vaccines against this challenging pathogen



Efficacy of a Parenteral and Oral DNA Vaccine in Fish for Aquatic Mycobacteriosis


SA Smith *


Aquatic Medicine Laboratory, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia USA stsmith7@vt.edu





Aquatic mycobacteriosis is a chronic progressive disease that leads to systemic infections and death.  It is an economically important bacterial disease of virtually all species of freshwater, brackish and marine fish, and is most commonly caused by the intracellular pathogens, Mycobacterium marinum, M. fortuitum, or M. chelonae.  While the immune response against aquatic Mycobacterium spp. has been characterized, an effective vaccine against mycobacteriosis has not been developed.  Because of the potential to induce a protective immune response against intracellular pathogens, a DNA vaccine was chosen as an appropriate construct for aquatic mycobacteriosis. The vaccine was assembled by cloning the M. marinum 85A gene in the pcDNA 3.1 eukaryotic expression vector. Hybrid striped bass (Morone saxatilis x M. chrysops), a documented susceptible species, were used as a model to test the vaccine.  Intramuscular or intraperitoneal vaccinations of the pCMV-85A construct were administered on days 0 and 14, along with appropriate positive and negative control vaccinations. Blood samples were obtained every two weeks from the fish to evaluate the humoral immune response by ELISA. On day 70, the fish were challenged with live M. marinum.  Morbidity and mortality observations, along with routine histological examination and splenic bacterial counts were used to determine the relative degree of protection among the different vaccination groups. Experimental groups administered the DNA vaccine demonstrated a reduced severity of pathology and a slower progression of disease. Based on these results, the vaccine was compounded with chitosan or polyactide-co-glycolide (PLG) nanoparticles and orally administered to separate groups of hybrid striped bass. Blood samples were again obtained every two weeks from the fish to evaluate the humoral immune response by ELISA. Only the fish administered the PLG-DNA vaccine orally had an increased humoral immune response similar to fish administered the DNA vaccine intramuscularly.





The Identification and Characterization of Interleukin-17 Family Genes and Receptors in Fish

H Korenaga*1, T  Kono2, H Kuse1, H Takayama1 and M  Sakai1


1 Faculty of Agriculture, University of Miyazaki, 1-1 Gakuenkibanadai-nishi, Miyazaki, Japan hiro102wes@gmail.com
2 Interdisciplinary Research Organization, University of Miyazaki, 1-1 Gakuenkibanadai-nishi, Miyazaki, Japan tkono@cc.miyazaki-u.ac.jp






Interleukin-17 (IL-17) is a new cytokine family with six members (A-F) identified in human. The IL-17 family has been classified as pro-inflammatory cytokines because of its ability to induce the expression of many mediators of inflammation properties. The uniqueness of IL-17 family as a separate cytokine family was proved by the isolation of a receptor, IL-17R, which binds IL-17 family and is required for its signaling. Five additional receptors (IL-17RA-E) have been identified in human. In fish, there are few reports on the identification for IL-17 family ligands (L) / receptors (R). In this study, we describe a detailed identification, cloning and genomic characterization of five IL-17 family ligands from medaka (Oryzias latipes) and genomic characterization of IL-17 family receptors from Fugu, zebrafish, stickleback, and medaka genome database. The screening of medaka genome database revealed the existence of five scaffolds containing six IL-17 ligand genes (AF-1, 2, 3, C, D and N). Among these genes, IL-17N identified the particular gene in teleost from analyzing the available genome database. Phylogenetic analysis using neighbor-joining method showed that medaka IL-17 family ligands clustered with other teleost IL-17 counterparts. Expression analysis by RT-PCR revealed expression of IL-17 family ligand genes in normal tissues and LPS-stimulated intestine. The expression patterns of IL-17 ligand family genes were different with other teleosts. In the LPS-stimulated intestine, the expression of IL-17 family genes was significantly increased. Moreover, the existence of IL-17 family receptors (RA, RC, RD and RE) was isolated from fish genome database.






Pathogen Recognition Proteins in Rainbow Trout Plasma (Onchorhynchus mykiss)


S Russell*, K Young, M Edwards, A Peterson, A Reid and JS Lumsden. 


Fish Pathology Laboratory, Department of Pathobiology, University of Guelph, Guelph, ON, Canada sprussel@uoguelph.ca




Soluble innate immune proteins are essential components of defense and play a critical role recognizing and eliminating infectious agents.  Using binding assays, 2D PAGE and mass spectrometry we identified two rainbow trout plasma proteins, ladderlectin (RTLL) and intelectin (RTInt), which bound chitin and various Gram-negative bacteria that cause disease in trout. Similar studies found RTLL, but not RTInt, bound to purified viral hemorrhagic septecimia virus.  RTLL is a C-type plasma lectin while RTInt is an X-type lectin with homology to carp, human, and murine intelectins. Both lectins show mannose specific, calcium dependent binding to target surfaces. To quantify both proteins, rabbit polyclonal antibodies were produced for use in enzyme immunoassays.  Neither lectin was induced in trout experimentally infected with Aeromonas salmonicida or injected with either A. salmonicida lipopolysaccharide (LPS) or a commercial A. salmonicida vaccine.  However, in tissues of clinically diseased rainbow trout, both proteins were localized by immunohistochemistry in intimate association with bacteria, fungi and protozoa and within inflammatory leukocytes. These studies demonstrate that both proteins are not detectably induced during an acute phase response but they provide further evidence that RTLL and RTInt have microbial binding abilities similar to other soluble pattern recognition receptors in higher vertebrates.





Antibody Response in Ranched Southern Bluefin Tuna (Thunnus maccoyii) Naturally Infected with the Blood Fluke Cardicola forsteri


NT Kirchhoff*, MJ Leef, V Valdenegro, C Hayward, and BF Nowak


National Centre for Marine Conservation and Resource Sustainability, University of Tasmania, Launceston, Australia nicolek1@amc.edu.au, mleef@amc.edu.au vav@amc.edu.au, Hayward.Craig@saugov.sa.gov.au, bnowak@amc.edu.au





The acquired immunity to a natural infection of blood fluke, Cardicola forsteri, was investigated in ranched southern bluefin tuna (Thunnus maccoyii) (SBT) in Port Lincoln, South Australia.  Cardicola forsteri is a aporocotylid blood fluke, which infects southern bluefin tuna.  Prevalence of infection increases from 5-10% when SBT are introduced into the ranching pontoons, to 100% after three months of ranching.  This presentation will characterize the speed of antibody generation, antibody activity at various stages of blood fluke infection, and the specificity of antibody response.   Approximately 400 ranched SBT were used for the investigation, representing ranching durations spanning from 0 (wild) to 21 weeks post transfer into ranching pontoons.  Antibody activity was quantified in serum through an ELISA using adult C. forsteri as an antigen source.  The specificity of the antibody response was examined against the other common parasites of ranched SBT: Caligus chiastos, Hexostoma thynni, Pseudocycnus appendiculatus, and Euryphorus brachypterus.  Histology of the heart and gills was used to characterize the presence of Cardicola forsteri eggs, number of eggs per square centimeter within the heart, and presence or absence within the gills.  Antibody activity was then correlated against the different stages of infection.  Speed of antibody response to infection was also calculated in relation to culture duration and stage of infection. This study is the first to detail the egg stage of Cardicola forsteri infection within ranched SBT.  Results indicate 100% prevalence of eggs within the heart after 3 months ranching and an increase in specific antibody activity with culture duration.






The Zebrafish Model – A Flow Cytometry-based Investigation of Mechanisms of Endocytosis


C Hohn*, S-R Lee, LM Pinchuk, and L Petrie-Hanson


Department of Basic Sciences, College of Veterinary Medicine, Mississippi State, MS USA. hohn@cvm.msstate.edu




Zebrafish are utilized as models for human and fish diseases. Mutant zebrafish in particular are being utilized to investigate mechanisms of host-pathogen interaction. We established a zebrafish model for the infectious disease Enteric Septicemia of Catfish caused by the intracellular pathogen Edwardsiella ictaluri. Rag1-/- mutant zebrafish, known to lack an acquired immune system, are useful to investigate the innate immune functions like endocytosis during infection. With an average length of 3 – 5 cm and blood yield of 10 μl per zebrafish, commercially available endocytosis test kits are non-practical for use in these fish. We describe a new procedure utilizing flow-cytometry to overcome this limitation. Pooled zebrafish kidney hematopoietic tissues were used as a source of phagocytic cells for flow cytometry based endocytic assays. FITC-Dextran, Lucifer Yellow and FITC-Edwardsiella ictaluri were used to evaluate selective and non-selective mechanisms of uptake in zebrafish phagocytes. We show for the first time that zebrafish phagocytes utilize macropinocytosis and Ca2+ dependant endocytosis mechanisms. Furthermore, 2 hour in situ exposure of kidney cells to live E. ictaluri altered subsequent fluid phase uptake mechanisms in phagocytic cells. We conclude that flow cytometry is a quick, reproducible, and objective method to evaluate the endocytic capacity of zebrafish renal phagocytes. The proposed method is easy to implement and will enable researchers to study the effects of pathogens, environmental toxins, and stress on fish immune health. It also provides an immunological method that makes zebrafish a more attractive model system for addressing immunology related questions.







Detection and Quantification of Renibacterium salmoninarum in Fluid Samples by Solid Phase Cytometry


CL McKibben1, DG Elliott*1, and J-L Drocourt2


1 U.S. Geological Survey, Western Fisheries Research Center, Street, Seattle, USA cmckibben@usgs.gov, dgelliott@usgs.gov
2 Chemunex SA, 3, Allée de la Seine, Ivry sur Seine, France  jl.drocourt@aeschemunex.com





Many methods commonly used for detection of waterborne pathogens or indicator organisms (e.g., bacteria, protozoa, zooplankton and phytoplankton) have limitations in speed, sensitivity, and accuracy. A new technique, solid phase laser scanning cytometry (SPC), has been developed for rapid detection, identification and quantification of low numbers of organisms in filterable samples. Organisms are first captured by membrane filtration and labeled with fluorescent dye. Fluorescent probes for organism identification (via immunofluorescence or fluorescence in situ hybridization) or assessing viability are often used. The SPC instrument scans the entire surface of the filter in less than 5 minutes, records and analyzes each fluorescent event, and discriminates target cells from background and auto-fluorescence. The position of each event is graphically displayed on a computer screen, and a motorized microscope stage allows the operator to visually validate each result. We are testing SPC for detection of the salmonid pathogen Renibacterium salmoninarum in water samples and in ovarian fluid of spawning female fish. For optimization of R. salmoninarum detection by SPC, we tested several commercial polyclonal and monoclonal primary antibodies (unlabeled and fluorochrome-labeled) and polyclonal secondary antibodies (labeled), as well as procedures to reduce background fluorescence and to increase and prolong specific fluorescence signals. We optimized SPC pre-scan instrument settings and post-scan discriminant settings for R. salmoninarum detection, and are testing its specificity, sensitivity, and repeatability according to standard procedures.






Aeromonas salmonicida subsp. achromogenes and the Effect of the Autoinducer Synthase AsaI on Bacterial Virulence


J Schwenteit, T Gudmundsdottir, B Bjornsdottir and BK Gudmundsdottir*


Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, 112 Reykjavik, Iceland IW650@gmx.de; tota@hi.is, bryndisb@hi.is, bjarngud@hi.is




Quorum sensing (QS) is a cell–cell communication system that enables bacteria to synchronize gene expression with population density.  LuxIR-type QS via N-acyl-homoserinelactones (AHLs) autoinducers are used by many Gram-negative proteobacteria for intraspecies QS.  LuxI is the autoinducer synthase and LuxR is an AHL-dependent transcriptional regulator.  The Gram-negative fish pathogenic bacterium Aeromonas salmonicida possesses the LuxIR-type quorum sensing (QS) system, termed AsaIR, and secretes only one type of N-acyl-homoserinelactone (AHL), N-butanoyl-L-homoserine lactone (C4-HSL).  This study was performed to investigate the role of QS in Aer. salmonicida subsp. achromogenes virulence and pigment production.  A knock out mutant of AsaI, constructed by allelic exchange, did not produce a detectable QS signal and its virulence in fish was significantly impaired, as LD50 of the AsaI-deficient mutant was 20 fold higher than that of the isogenic wt strain and the mean day to death of the mutant was significantly prolonged.  Furthermore, the expression of two virulence factors, a toxic peptidase, AsaP1, and a repeat in toxin, RTX, and a brown pigment were reduced in the mutant. AsaP1 production was inhibited by synthetic QS inhibitors (N-(propylsulfanylacetyl)- L-homoserine lactone; N-(pentylsulfanylacetyl)- L-homoserine lactone; and N-(heptylsulfanylacetyl)- L-homoserine lactone) at concentrations that did not affect bacterial growth.  The observation that the Aer. salmonicida subsp. achromogenes LuxIR-type QS system regulates virulence and the ability of synthetic QSI to inhibit an important virulence factor without affecting bacterial growth, makes Aer. salmonicida subsp. achromogenes an interesting target organism for the development of novel antibacterial therapeutics.






Weissella sp. Outbreaks in Brazilian Rainbow Trout (Onchorynchus mykiss) Farms:  Aetiologic Agent, Genotyping, Transmission Routes and Antibiotic Resistance Profile


HCP Figueiredo*1,2, CAG Leal2 , FAA Costa2 and GA Carvalho- Castro2


1 General Coordinator of Aquatic Animal Health, Ministry of Fisheries and Aquaculture, Brasília, DF, Brazil.
2 AQUAVET, Laboratory of Aquatic Animal Diseases, Department of Preventive Veterinary Medicine, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.






Since 2006, rainbow trout (Onchorynchus mykiss) farms in Brazil have been suffering with outbreaks of a hemorrhagic septicemic disease. A similar illness was described in China as the first case of Weissella sp. infection in fish in 2009. In the summers of 2009 and 2010, seven outbreaks in Brazilian rainbow trout farms were studied to address important aspects of disease. The clinical signs observed were anorexia, lethargy, exophthalmia, ascites, and hemorrhages in the mouth, tongue and eyes. A total of 107 bacterial isolates from 44 fish were obtained. Biochemical tests and 16S rRNA sequences of Brazilian strains showed similarity (100%) with sequences of Weissella sp. isolated in China. The genetics of the strains were characterized by pulsed-field gel electrophoresis (PFGE), using the restriction enzyme smaI, and results indicated that the strains were clone related. The bacteria were isolated from the intestinal tract of diseased rainbow trout but not from healthy ones. In order to fulfill the Koch´s postulate and address the potential infectious routes of bacteria, rainbow trout were experimentally infected by intraperitoneal injection, immersion, and cohabitation between diseased and healthy fish with no direct contact. The disease was successfully reproduced and bacteria could be transmitted through the water. The antibiotic resistance profile for five antibiotics were determined by disc diffusion test (CLSI,2006). Based on normalized resistance interpretation (NRI) analysis, all isolates were resistant to sulfonamide and susceptible to florfenicol, and a few strains had reduced susceptibility to erythromycin, oxytetracycline and norfloxacyn. This is the first description of multiple cases of Weissella sp. infection in rainbow trout farms including PFGE genotyping, antibiotic resistance profile, and transmission routes for disease.






Analysis of the Serological Variability of Vibrio tapetis, Causative Agent of BRD in clams


S Balboa, A Bastardo, A Doce, JL Barja, AE Toranzo, JL Romalde*

Departamento de Microbiología y Parasitología. CIBUS-Facultad de Biología. Universidad de Santiago de Compostela. Spain




The etiological agent of brown ring disease (BRD) was identified more than ten years ago as a new member of the genus Vibrio designated as Vibrio tapetis. First studies indicated that strains of this pathogen constituted a homogenous group on the basis of their phenotypic and genotypic characteristics. However, as new strains were isolated from different hosts, including fish, some variability within the bacterial species was demonstrated, allowing for the recognition of three main genetic subgroups, correlated to host type.  A total of 28 strain of V. tapetis with different geographical and host origin were employed to this study, including isolates from Manila clam (Ruditapes philippinarum), carpet-shell clam (R. decussatus), Pullet carpet-shell clam (Venerupis pullastra), cockle (Cerastoderma edule), halibut (Hippoglossus hippoglossus), corking wrasse (Symphodus melops), corb (Umbrina cirrosa) and wedge sole (Dicologoglossa cuneata). The study of these isolates was done using the following serological techniques; slide agglutination and dot-blot assays, as well as analysis of LPS, total and outer membrane proteins and extracellular proteins. Three antisera, obtained against the O-antigen of representative strains of the three groups previously described for this bacterial species, were used for the analysis.  Most strains could be divided into three major groups on the basis of both slide agglutination and dot-blot assays, which coincided with the genetic groups previously described. In addition, the wedge sole, corb and corking wrasse isolates showed some cross-reactivity, being arranged in two new groups. LPS and protein analyses confirmed these results.





Mycobacterial Infections in Resident Chesapeake Bay Striped Bass Morone saxatilis


CB Stine*1,2, AM Baya1,3, L Hungerford3, MA Matsche 4, L Pieper 4, K Rosemary 4, C Driscoll 4 and AS Kane1,5,6


1 University of Maryland, Department of Veterinary Medicine, College Park, MD USA
2 United States Food and Drug Administration, Center for Veterinary Medicine, Laurel, MD USA cynthia.stine@fda.hhs.gov (current address)
3 Maryland Department of Agriculture, College Park, MD USA ambaya@umd.edu, Laura.Hungerford@fda.hhs.gov
4 Maryland Department of Natural Resources, Sarbanes Cooperative Oxford Laboratory Oxford, MD USA MMatsche@dnr.state.md.us, LPieper@dnr.state.md.us, KRosemary@dnr.state.md.us, CDriscoll@dnr.state.md.us
5 University of Maryland, Department of Epidemiology and Preventative Medicine, Baltimore, MD USA
6 Emerging Pathogens Institute, University of Florida, Gainesville, FL USA (current address) kane@ufl.edu













Striped bass, aged 0+ to 3+, were collected from the Upper Bay and the Choptank, Nanticoke, Pocomoke and Potomac rivers in 2003-2005 to investigate the risk factors associated with mycobacterial colonization.  Three multivariate models of mycobacterial colonization were fit in order to account for putative variations in migratory behavior at different ages.  As 0+ striped bass remain within their natal river, location of capture other than the Pocomoke River, presence of grossly observable parasites and presence of bacteria other than mycobacteria were associated with prevalence of mycobacterial infections.  As 1+ fish remain close to their natal river, only location of capture other than the Pocomoke River was associated with prevalence of colonization.  2+ and 3+ striped bass generally migrate within the Chesapeake Bay and presence of acanthacephalans in the intestine and presence of moderate to severe trichodinid infections on the skin and gills were associated with prevalence of mycobacterial colonization.  The existence of multiple risk factors, and the variability introduced into the models by age-dependant life history factors, indicate mycobacterial colonizations in Chesapeake Bay striped bass are a result of a series of complex processes.




Genetic Diversity of Mycobacterium chelonae in Aquatic Environments as Determined by PFGE


S Sanchez*1, C Still1, J Lane1, J Saliki1, A Camus2, B Bossart3, T Clauss3, T Mullican3 and A Dove3


1 Department of Infectious Diseases and Athens Veterinary Diagnostic Laboratory, College of Veterinary Medicine, The University of Georgia, Athens, GA USA ssanchez@uga.edu
2 Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, GA USA.
3 The Georgia Aquarium, Atlanta GA USA.







Mycobacteria are aerobic, mycolic acid containing bacteria that are characteristically acid-fast. All members of this group fall within one genus Mycobacterium, which includes important pathogens for both fish and mammals. The most common of these opportunistic pathogens is M. chelonae. In spite of the importance of this bacterium, very little is known about its genetic diversity in animal and aquatic environments. The purpose of the present study was to study the genetic relatedness of isolates obtained from enclosed aquatic environments, fish, mammals and biofilms. Fifty-five M. chelonae isolates were grown in Muller-Hinton broth media to a 4.0 MacFarland standard. Pulsed-field gel electrophoresis was performed using restriction enzyme XbaI and DraI. The generated Tiff images were uploaded to Bionumerics (version 4.6, Applied Maths, Kortrijk, Belgium), using the Dice coefficient to analyze the similarity of banding patterns, and the unweighted pair group method using arithmetic averages (UPGMA) for cluster analysis. The data generated by both enzymes showed that M. chelonae isolates cluster by source, such as water, fish or mammal species they were isolated from, and differed from each other in 1 to 2 bands. This is expected as they are genetically very homogenous. Nevertheless, some isolates identified as M. chelonae phenotypically, and by DNA sequencing, had PFGE patterns that differed in 5-10 bands. This seems to indicate that some M. chelonae may be adapted to a particular host or niche. Furthermore, there may be some isolates that current diagnostic methodology fails to properly identify as rapidly growing Mycobacterium at the species level.










The Frequency and Distribution of Genetic Strains of Hematodinium, a Parasitic Dinoflagellate of the Blue Crab (Callinectes sapidus) along the Delmarva Peninsula, VA


KM Pagenkopp*1, JD Shields1, J Xiao1, TL Miller2, HJ Small1, AR Place3, and KS Reece1


1 The Virginia Institute of Marine Science, The College of William and Mary, 1208 Greate Road, Gloucester Point, VA, USA pagenkopp@vims.edu, jeff@vims.edu, jxiao@vims.edu, hamish@vims.edu, kreece@vims.edu
2 Biodiversity Program, Queensland Museum, PO Box 3300, South Brisbane, QLD 4101. Australia terrence.miller@qm.qld.gov.au
3 Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Research, 701 East Pratt Street, Baltimore, MD, USA place@umbi.umd.edu









The dinoflagellate Hematodinium sp. is a significant pathogen of blue crabs on the Eastern seaboard of the USA. We designed 15 microsatellite markers (9 polymorphic and 6 monomorphic) from an enriched microsatellite library constructed from a non-clonal culture of Hematodinium from a blue crab (Callinectes sapidus). Using the microsatellites, we genotyped Hematodinium strains in blue crabs, and alternate hosts, from six sites along the Delmarva Peninsula.  Multiple genetic strains of Hematodinium were found, some of which were seen throughout the entire region, while others were only found in the southern portion of the Delmarva Peninsula. The reasons for this distribution are still being investigated, but possibilities include a source of Hematodinium from other geographic regions, or varying environmental tolerances among different strains. Additionally, simultaneous infections were identified, indicating that multiple genetic strains occur in blue crabs; up to four different strains of Hematodinium were found in an individual crab. Strains of the parasite were compared to those in alternate hosts, as well as to those from blue crabs from Georgia and Florida.





Discovery and Characterization of New Perkinsus Species


KS Reece*1, GP Scott1, CF Dungan2, PM da Silva3, C Adams1,4 and EM Burreson1


1 Virginia Institute of Marine Science, College of William & Mary, Gloucester Point, VA  23062, USA kreece@vims.edu
2 Maryland Department of Natural Resources, Cooperative Oxford Laboratory, Oxford, MD  21654, USA
3 Universidade Federal de Sergipe, Centro de Ciências Biológicas e da Saúde, Departamento de Engenharia Agronômica.  Av. Marechal Rondon, s/n Jardim Rosa Elze, 49100-000 Sao Cristovao, SE, Brasil
4 Swarthmore College, 500 College Avenue, Swarthmore, PA 19081, USA









For decades since the first description of Perkinsus marinus in 1950, there were only a few recognized species in the genus Perkinsus.  However, modern molecular detection and characterization techniques have facilitated the identification and description of five new species within the past 10 years.   In addition, P. atlanticus was synonymized with P. olseni, and P. andrewsi with P. chesapeaki. We have used PCR, in situ hybridization (ISH), DNA sequence-based phylogenetic analyses, and field and laboratory studies to examine the host and geographic distributions of Perkinsus species worldwide.  Potential molluscan host tissues from six continents were screened for parasite DNAs using PCR primers designed initially to target members of the genus Perkinsus based on approximately 50 different internal transcribed spacer (ITS) region sequences from three Perkinsus species.  These primers successfully amplify the DNA of all currently described Perkinsus species. The PCR primers along with an ISH probe targeting the large subunit ribosomal RNA gene of Perkinsus species have been key tools for identifying and characterizing many of these species.  We found a potential new Perkinsus species in Australian abalone, whose ITS-region sequences group with those from clams being imported into Florida from Vietnam for the aquarium trade.  We are currently screening Vietnamese molluscs for this parasite. Most recently we have discovered a new strain or species of Perkinsus in Saccostrea culcullata oysters from India, which based on genetic data is closely related to P. beihaiensis described from southern Chinese oysters and a Perkinsus sp. recently found in Brazilian Crassostrea gasar oysters.






Haplosporidiosis in Bivalve Molluscs: New Perspectives on Haplosporidium costale, Agent of SSO Disease in Crassostrea virginica


NA Stokes* and RB Carnegie


Department of Environmental and Aquatic Animal Health, Virginia Institute of Marine Science, P.O. Box 1346, Gloucester Point, VA 23062, USA stokes@vims.edu, carnegie@vims.edu




The protistan parasite Haplosporidium costale infects the eastern oyster, Crassostrea virginica, and is the agent of SSO (seaside organism) disease.  The parasite has been reported from higher salinity waters (>25psu) on the Atlantic coast of the USA from Virginia to Maine, causing peak mortalities in May-June. In the absence of sporulation, this parasite is indistinguishable from the notorious pathogen Haplosporidium nelsoni, and differentiating plasmodial infections by H. costale from the more common infections by H. nelsoni has long been problematic. Recent analyses by our laboratory and others have shown that the distribution, and in some cases also the impact, of H. costale is greater than previously appreciated. Detection of H. costale by morphological, and/or DNA approaches, has revealed its presence in oysters from a broad geographical range, including Atlantic Canada, California, China and Australia; furthermore, infections have been detected in three host species, C. virginica, Crassostrea gigas and in Saccostrea glomerata. Infections have been detected in nearly every month of the year, not only in spring as previously known, and unexpectedly high prevalences (reaching 44%) have been observed in Virginia embayments that previously have not been considered H. costale-enzootic. There have been enough moderate to heavy H. costale infections to indicate that up to 10% of oysters are experiencing serious SSO disease and mortality. Species-specific in situ hybridizations have confirmed that the observed plasmodia are H. costale and not H. nelsoni. A contrasting size-specific infection profile has emerged, with more H. costale infections in larger oysters and more H. nelsoni infections in smaller oysters. We hypothesize that this may reflect the difference between acute (H. nelsoni) and chronic (H. costale) infection and disease.






Oyster Parasites Common to Lake Worth Lagoon, Florida


SE Laramore*1 and J Scarpa2


1 Harbor Branch Oceanographic Institute at Florida Atlantic University, 5600 US 1 North, Fort Pierce, FL 34946  USA  slaramo1@hboi.fau.edu
2 Harbor Branch Oceanographic Institute at Florida Atlantic University, 5600 US 1 North, Fort Pierce, FL 34946  USA  jscarpa1@hboi.fau.edu






Three eastern oyster (Crassostrea virginica) reefs in the Lake Worth Lagoon, a 21-mile long estuary in Palm Beach County, Florida, were monitored for two years (March 2008-February 2010). Sites were natural reefs located at John D. MacArthur Beach State Recreation Park at the northern end of the lagoon and at Ibis Isle in the central lagoon.  The third site was a created reef at the Snook Islands Natural Area in the central lagoon. Oysters (n=5-6) were collected monthly from each site to determine prevalence and intensity of Dermo (Perkinsus marinus) using Ray’s fluid thioglycollate medium and other parasites using standard H&E histological techniques. Water temperature was similar between sites, but salinity was on average 5 ppt higher and more stable at the northern site (MacArthur). Dermo prevalence was similar at all three sites (50-100% spring/summer/fall) and lowest at all sites during the winter, as well as during low-salinity events at the central sites. Dermo intensity ranged from 0-4.5 Mackin scale with an average annual intensity range of 0.85-1.2; MacArthur had the highest and Snook Island the lowest. MSX (Haplosporidium nelsoni) was not found at any site. Other parasites detected histologically included the gregarine protozoan Nematopsis sp., turbellarians, the trematode Bucephalus, the cestode Tylocephalum, and ciliates. Symbiotic parasites (cnidarian Eutima and pea crab Pinnotheres) were also present. Annual, seasonal, and site variation prevalence was noted for different parasites. The diversity of parasites found in oysters is indicative of a healthy ecosystem that supports a diverse assortment of other species.






Potential Bivalve Reservoirs of Bonamia sp. in Florida’s Indian River Lagoon  


SE Laramore*1 and AL Lave2


1 Harbor Branch Oceanographic Institute at Florida Atlantic University, 5600 US 1 North, Fort Pierce, FL 34946   slaramo1@hboi.fau.edu
2 Jacksonville University, 2800 University Blvd. North, Jacksonville, FL 32211  alave@jacksonville.edu





In 2007, suminoe oysters (Crassostrea ariakensis) and eastern oysters (Crassostrea virginica) exposed to Indian River Lagoon, FL waters were found to be PCR positive for Bonamia sp., although infection was histologically confirmed only in C. ariakensis. Both species were also infected with Dermo (Perkinsus marinus). In the summer of 2010 we began to look for bivalve reservoirs of Bonamia sp. in the Indian River Lagoon. Ten to twenty representatives of various bivalve species were collected from eight locations within the southern portion of the Indian River Lagoon (St. Lucie Co. and Martin Co.) and tested for the presence of the Bonamia sp., P. marinus, and Haplosporidium nelsoni (causative agent of MSX) using polymerase chain reaction (PCR). Bivalves collected and tested varied by location, but included various oyster species (C. virginica, Ostreola equestris, Isognomon alatus, I. bicolor), mussels (Brachidontes exustus, B. modiolus, Geukensia granossima, Ischadium recurvum), arks (Arca imbricata, A. zebra), and clams (Mercenaria mercenaria, M. campechiensis, Chione elevata). PCR positive results have been obtained for Bonamia sp. in C. virginica, I. alatus, I. bicolor, and G. granossima and for P. marinus in C. virginica, I. alatus, and G. granossima.  Analysis (PCR and histology) is ongoing; however the presence of Bonamia may indicate a wider geographic range for Bonamia than previously reported in the US.






Evolutionary Ecology of Crassostrea virginica and Its Parasites


RB Carnegie*1 and EM Burreson2


Department of Environmental and Aquatic Animal Health, Virginia Institute of Marine Science, P.O. Box 1346, Gloucester Point, VA 23062 USA carnegie@vims.edu, gene@vims.edu




The oyster Crassostrea virginica has long been plagued by the parasites Perkinsus marinus and Haplosporidium nelsoni.  In the 1950s in the Chesapeake Bay, USA, H. nelsoni joined an established C. virginica-P. marinus system. The goal of our study was to determine how the arrival of H. nelsoni perturbed this system. We proceeded along two tracks: First, we analyzed data from contemporary samples of wild and sentinel oysters and placed this data in the context of a sixty-year span; second, we re-evaluated archival histological materials to assess changes in parasite presentation and disease.  Data analyses have revealed decreasing H. nelsoni infection in wild oysters despite increasing infection pressure on naïve sentinels, suggesting that wild oysters have become more resistant to H. nelsoni.  The oysters have also become more resistant to disease caused by P. marinus, though this parasite continues to result in substantial mortality.  Since the mid-1980s, disease caused by P. marinus has seen a dramatic intensification, which has been attributed to a greater abundance of P. marinus since the drought years of that decade.  Retrospective histopathology, however, revealed a fundamental change in the parasite presentation since 1986:  relatively large and scarce P. marinus cells that divide by schizogony have been replaced by relatively small and abundant cells that divide in binary fashion.  We hypothesize that this change relates to the observed disease intensification and an increase in parasite virulence and, furthermore, that it is a response by P. marinus to the introduction of H. nelsoni.










Comparison of in-vitro and in-vivo Efficacy of Florfenicol for Treatment of Francisella asiatica Infection in Tilapia


E Soto*1, RG Endris2 and JP Hawke1

1 Louisiana State University, Department of Pathobiological Sciences, School of Veterinary Medicine, Baton Rouge, LA USA1 esoto1@lsu.edu,  jhawke1@lsu.edu
2 Intervet/Schering-Plough Animal Health, Roseland, New Jersey USA richard.endris@sp.intervet.com






Francisellosis caused by the emergent fish pathogen Francisella asiatica is an important bacterial disease in warm water aquaculture. The lack of effective treatments led us to investigate the efficacy of florfenicol to F. asiatica in vitro and to potentially treat piscine francisellosis in vivo. The minimal inhibitory concentration (MIC) of florfenicol for F. asiatica, as determined by the broth-dilution method, was 2 µg/ml. In tilapia head kidney derived macrophages (THKDM) assays, addition of florfenicol to the medium at 10 µg/ml resulted in uptake of the drug and significantly reduced bacterial loads. Moreover, cytopathogenicity assays done in infected THKDM also demonstrated drug efficacy as concentrations of lactate dehydrogenase (LDH) released from infected THKDM were significantly lower in macrophages treated with florfenicol (p<0.001) than controls.  In medicated feed trials, treated fish were fed 15mg/kg of florfenicol for 10 days, either at 1, 3, or 6 days post-challenge. Immersion challenges resulted in 70% mortality in non-treated fish.  Fish fed medicated feed 1 and 3 days post infection showed significantly higher survival rates (p<0.001). Bacterial concentrations in the spleen of surviving fish were evaluated by enumerating colony forming units (CFU) on cystine heart agar (CHA) plates 30 days post-challenge. Significantly higher numbers (p<0.001) of bacteria were recovered from non-treated fish than from fish fed medicated feed 1 and 3 days post-infection. There were no significant differences in bacterial burden between fish treated 6 days post-infection and untreated controls. If administered during early infection, florfenicol may effectively treat piscine francisellosis, including via intracellular penetration and bacterial clearance.  





The Administration of a Bronopol-Based Chemical Treatment to Control Ichthyophthirius multifiliis (Ciliophora) Infections


SM Picón-Camacho*, JE Bron and AP Shinn


Aquatic Parasitology Research Laboratory, Institute of Aquaculture, University of Stirling, UK sara.picon@stir.ac.uk, jeb1@stir.ac.uk, aps1@stir.ac.uk




Whitespot infections caused by the ciliate protozoan Ichthyophthirius multifiliis cause serious economic losses, annually and globally, for commercial and ornamental freshwater aquaculture. Few effective chemotherapeutants are available for its control in commercial-scale aquaculture. The present study explores the usage of bronopol, commercialized by Novartis Ltd. as PycezeTM, as a biocide for the treatment of I. multifiliis through a series of in vitro and in vivo trials. In vitro exposure of tomonts to bronopol (0, 20, 50 and 100 mg L-1) killed 0%, 76.2%, 97.2% and 100% after 30 min. Long exposures to 1 mg L-1 bronopol (up to 48 h) were successful in reducing the numbers of theronts surviving after 12 h (29.16%) and 24 h (0% survival). In the in vivo trial, the efficacy of continuous low doses of bronopol was explored in reducing the number of trophonts establishing on juvenile O. mykiss. The effect of bronopol on the time of infection was investigated by starting the administration of 2 mg L-1 24 h prior to the infection, and for 72 h afterwards. The effect of bronopol on maturing trophonts and tomonts was assessed through the delivery of 1, 2 and 5 mg L-1 at 10-36 d post-infection. The trial showed that 2 mg L-1 administered prior to infection reduced the number of theronts surviving by 35-48%. The 2 and 5 mg L-1 bronopol in the second cycle of infection reduced the number of trophonts by 46-83%, while doses during the third cycle gave further reductions of 83-97%.






Efficacy of Florfenicol, Copper Sulfate and Potassium Permanganate in Controlling a Natural Infection of Aeromonas hydrophila and Flavobacterium columnare in Sunshine Bass, Morone chrysops Female ´ Morone saxatilis Male


AM Darwish*


Harry K. Dupree-Stuttgart National Aquaculture Research Center, Agricultural Research Service, United States Department of Agriculture, Stuttgart, AR, USA Ahmed.Darwish@ars.usda.gov




Sunshine bass (Morone chrysops female ´ Morone saxatilis male) naturally infected with Aeromonas hydrophila and Flavobacterium columnare were randomly assigned to six treatments: 1) two treatments of waterborne exposures to copper sulfate (CuSO4), at 2.1 and at 4.2 mg/L (approximately one and two percent the water alkalinity) for 24 h, 2) two treatments of waterborne exposures to potassium permanganate (KMnO4) at 2 mg/L above the KMnO4 demand (PPD) for 24 h and at 10 mg/L above the PPD for 1 h, 3) a treatment of florfenicol-medicated feed at 15 mg/kg of fish body weight (BW) for 10 d, and 4) non-treated fish as control.  The fish were observed for 32 d.  The control fish had 71% survival compared to, 88 and 79% survival for the CuSO4 treatments at 2.1 and at 4.2 mg/L, respectively, 72 and 77% survival for the KMnO4 treatments at 2 mg/L for 24 h and at 10mg/L for 1h, respectively, and 90% survival for the fish treated with florfenicol-medicated feed at 15 mg/kg of fish BW for 10 d.  The final survival of fish fed florfenicol-medicated feed (90%) was significantly higher (P<0.1) than the control fish (71%).  According to logrank comparison the survival curves of the florfenicol-medicated treatment and the CuSO4 treatment at 2.1 mg/L for 24 h were significantly different from the control fish; the rate of death was significantly higher in the control fish.  This is the first controlled study to demonstrate the beneficial effect of florfenicol and CuSO4 against a mixed infection of A. hydrophila and F. columnare.  




Evaluation of Novel Oral Therapeutics Against Monogenean Parasites


LA Tubbs*1,2, B Wang3 and M Hamann3


1 National Institute of Water and Atmospheric Research Ltd, Viaduct Precinct, Auckland Central, New Zealand
2 Fish Pathology Laboratory, Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada (Present address) ltubbs@uoguelph.ca
3 Department of Pharmacognosy, School of Pharmacy, University of Mississippi, MS 38677, USA mthamann@olemiss.edu








A fundamental aspect of drug discovery strategies is the ability to characterise the relationship between the pharmacodynamics (PD), pharmacokinetics (PK), efficacy and toxicity of potential therapeutics. The present study evaluated several marine compounds as potential oral therapeutics against a monogenean parasite of kingfish (Seriola lalandi) using in vitro techniques and experimental dosing. A panel of 35 compounds were initially screened by immersing detached parasites in equimolar concentrations (1μM, 10μM and 100μM) of each compound; efficacy was compared to an established anthelmintic (praziquantel (PZQ)). The most effective compounds were advanced to a secondary screen using a modified Ussing chamber, where parasites were attached to a piece of excised kingfish skin then exposed to each compound via a supportive media, providing an in vitro assay which highly effectively models the oral dosing of an infected fish. Four compounds with efficacy comparable to PZQ were selected in this manner for in vivo assessment of toxicity and PK. Toxicity was determined using a dose-escalation approach to determine the maximum tolerated oral dose within the range 1 to 500 mg.kg-1 body weight. The relative PK of the least toxic, most effective, compound was determined in the skin, muscle and plasma of kingfish using LC-MS using two empirically derived, repetitive dosing regimens: 3 days with a 12h dosing interval and 5 days with a 24 h interval. The plasma concentration of this compound was between 10 and 100 fold lower than the concentration in muscle and skin, indicating rapid clearance. These results demonstrate useful mechanisms for predicting the outcome of potential therapeutics.






Using Vaccines in Aquaculture


H Mitchell*


Aqua Life Veterinary Services, Aquatic Life Sciences (Western & Syndel), 12015 115th Ave NE, Kirkland, WA 98034 USA  hughm@aqualifesci.com




Vaccines are relatively old medical technologies pre-dating germ theory and have been credited with no less than revolutionizing human and animal medicine.  Their commercial use in aquaculture is less than 40 years old, however their application in many fish species still disproportionately languishes behind that of terrestrial animals. Not unlike terrestrial production medicine, the current use and understanding of fish vaccines can be controversial. As recognized in agriculture medicine: “The veterinary practitioner is often asked for advice about the use of vaccines for the control of infectious diseases of food producing animals. There is often more controversy and uncertainty about the efficacy of these vaccines than about almost any other topic in livestock production”. How can the terrestrial experience be used to foster more development, use and effectiveness in fish production?  Part of the answer lies in understanding exactly what a vaccine is: “…when veterinarians recommend use of vaccine, the unspoken, implied expectation is that the product will end disease losses.  Avoidance of this misunderstanding requires precise and accurate communication of reasonable expectations.”  The history of fish vaccinology with current status in aquaculture will be briefly reviewed.  The various reasons for vaccinating will be discussed, including variables involved in defining and assessing vaccine efficacy.  The approach to using vaccines within a fish production environment will be outlined in the context of an integrated fish health management program where the other essential interacting components include:  genetics; risk factor reduction; biosecurity and pathogen load reduction; and early diagnosis and treatment.








Health and Reproductive Consequences for Sciaenops ocellatus (Perciformes: Sciaenidae) Infected with the Ovarian Parasite Philometra floridensis (Nematoda: Philometridae)


MD Bakenhaster*1, S Lowerre-Barbierrie1, Y Kiryu1, S Walters1, and EJ Fajer-Avila2


1 Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, St. Petersburg, FL USA Micah.Bakenhaster@myfwc.com, Susan.Lowerre-Barbierrie@myfwc.com, Yasu.Kiryu@myfwc.com, Sara.Walters@myfwc.com     
2 Centro de Investigación en Alimentación y Desarrollo, A.C., Unidad Mazatlán en Acuicultura y Manejo Ambiental, C.P. Mazatlán Sinaloa, México efajer@ciad.mx







The parasitic nematode Philometra floridensis is known to infect the ovary of the economically important fish Sciaenops ocellatus, but its life history, the factors influencing host susceptibility, and potential pathogenic effects are unknown.  Here we report new information on these topics based on evaluations of reproductively mature wild hosts, both infected and uninfected, collected from the Gulf of Mexico off Tampa Bay, Florida.  To check for ontogenetic differences in host susceptibility to the nematode, we evaluated the length and age of fish vis-à-vis nematode prevalence.  About 46% of female fish, and no male fish, were infected with P. floridensis, with prevalence and intensity tending to be higher in larger specimens.  To evaluate health and reproductive consequences of infection, we looked for parasite effects on histological conditions, condition factor (K), batch fecundity estimates (BF), and gonadosomatic index (GSI).  We observed substantial pathological changes in ovarian tissue associated with female nematodes but only in regions close to worms.  Both K and BF were higher for infected fish at a given size.  Together, these results suggest that P. floridensis has a negative impact on its host’s reproduction at the cellular level but not at the organismal level.  Several factors, including the higher prevalence of infection in larger and older hosts, may have prevented our detection of some health impacts; however, at the intensities observed in this study, infection did not appreciably reduce reproductive fitness.






Unknown Clinostomum sp. Associated with Pathology in Channel Catfish (Ictalurus punctatus) in Mississippi (USA)


CM Doffitt*1, L Khoo2, LMPote1 and DT King3.


1 Department of Basic Sciences, College of Veterinary Medicine (CVM), Mississippi State University, Mississippi State, MS USA lpote@cvm.msstate.edu, doffitt@cvm.msstate.edu
2 Department of Population and Preventative Medicine, Thad Cochran Warmwater Aquaculture Center, (CVM/MSU), Stoneville, MS USA lhk2@msstate.edu
3 USDA/Wildlife Services/National Wildlife Research Center, Mississippi State, MS USA tommy.king@aphis.usda.gov








As the commercial catfish industry has expanded in the southern United States, there has been an increase in piscivorous bird populations in this region.  Many of these birds are infected with digeneans that utilize fish as intermediate hosts. Clinostomum marginatum and C. complanatum have been reported in channel catfish; however, these infections do not usually cause morbidity or mortality.  Recently a clinostomoid-like trematode was identified that was associated with morbidity and high parasite loads.  A challenge study was done to determine the pathology of this parasite and confirm its genomic identification.  Cercariae were isolated from snails, Planorbella trivolvis, collected from commercial catfish ponds.  Parasite-free channel catfish fingerlings were exposed to 200 cercariae/fish for 2 hours.  The fish were sampled at 7, 14, 21, 28, 40, 80, and 120 days post-infection (dpi). Metacercariae were observed at 14 dpi in the musculature, ocular cavity, and oral cavity.  By 120 dpi, the metacercariae were well-developed with most internal structures evident.  At all sampling points, pathology was limited to the metacercarial site; however, the infected catfish showed reduced growth rates and a relatively high parasite load (up to 24 metacercariae/fish). Histological analysis of the metacercariae showed little host immunological response or histopathology.  Sequence analysis of cercariae and metacercariae revealed a 99% homology with a Clinostomum sp.  The high parasite infection combined with a lack of mortality and severe pathology associated with this trematode is unusual.  More research is needed to identify this species and its avian host. Research supported by The Berryman Institute.






DNA Barcoding Platyhelminths that Parasitize Aquatic Animals


SA Locke*1, JD McLaughlin2 and DJ Marcogliese1


1 St. Lawrence Centre, Environment Canada, Montreal Canada sean.locke@ec.gc.ca
2 Department of Biology, Concordia University, Montreal Canada




The ability to distinguish species of pathogens can play an important role in monitoring and controlling disease in aquaculture and wildlife management. DNA barcoding is a method of distinguishing species using sequence from a standardized region of the cytochrome oxidase I gene in the mitochondrion. In a recent survey of 20 fish species in the St. Lawrence River near Montreal, DNA barcodes from ~1000 parasites collected revealed much higher diversity and, in most cases, higher host specificity than expected in metacercariae in the Diplostomoidea (Platyhelminthes: Digenea) (Locke et al, 2010, Int J Parasitol 40, 333-343; Locke et al, 2010, Mol Ecol 19, 2813-2827). Here we present additional results from ongoing sampling that is expanded in terms of both geographic coverage and host taxa, including amphibians, mammals, birds and additional fish species from across North America. The two main patterns observed in the St. Lawrence River – widespread host specificity and high diversity among diplostomoids – are not contradicted by the results of the expanded sampling program. DNA barcodes have also been obtained from other trematode taxa and from cestodes. The performance, applications and limitations of DNA barcoding are discussed in the context of wildlife parasitology.






Life Histories of the Trematodes Found in Double-crested Cormorant Populations in the Mississippi Delta and the Potential Impact of these Parasites on Commercial and Wild Fish Species Found in this Region


MM O’Hear*1, LM Pote1, DT King2, CM Doffitt1, C Panuska1 and LE Miranda3


1 Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762 mohear@cvm.msstate.edu, lpote@cvm.msstate.edu
2 United States Department of Agriculture/Wildlife Services/ National Wildlife Research Center, Mississippi State, MS 39762 tommy.king@aphis.usda.gov
3 Department of Wildlife, Fisheries and Aquaculture, Mississippi State University, Mississippi State, MS 39762 smiranda@cfr.msstate.edu








The population of double-crested cormorants (Phalacrocorax auritus) has steadily increased over the last few decades in the Mississippi Delta.  This piscivorous bird serves as a definitive host for numerous trematodes, many of which have immature stages that infect numerous fish species.  The goal of this study was to identify and determine the impact of these trematodes on both commercial and wild fish populations in the Mississippi Delta.  All adult trematodes were enumerated and identified from cormorants (n=35) collected in close proximity to commercial catfish ponds in the Mississippi Delta from January 2003 to March 2004. Trematodes found in the cormorants were Pseudopsilostoma varium, Drepanocephalus spathans, Austrodiplostomum ostrowskiae, Hysteromorpha triloba and Ascotyle longus.  Three of these A. ostrowskiae, H. triloba and A. longus are reported to have fish as an intermediate host while the life cycles of the other two are unknown. In addition, fish were collected from an ox-bow lake located near commercial catfish operations that had a large resident cormorant population. Fish were examined externally and internally for the presence of parasites. A large percentage of these fish were infected with Posthodiplostomum minimum, which was found in multiple organs in 7 fish species. The only larval trematode stage recovered from this fish population was P. minimum. Research is underway to identify the hosts for P. minimum in this lake and identify the life stages of P. varium and D. spathans using molecular sequence comparisons.  Research was partially supported by the Berryman Institute.




Infection Dynamics of Ecto- and Endoparasites of Channel Catfish (Ictalurus punctatus), Blue Catfish (Ictalurus furcatus), and C×B Hybrid Catfish (Female Ictalurus punctatus × Male Ictalurus furcatus) in Earthen Pond Aquaculture


TN Truong*1 and SA Bullard2


1 Department of Fisheries and Allied Aquacultures (FAA), Aquatic Parasitology Laboratory (APL), Auburn University, Auburn, AL USA, tnt0004@auburn.edu
2 FAA, APL, sab0019@auburn.edu





It is reported that C×B hybrid catfish (HC) exhibit better growth, feed conversion ratios, tolerance to low dissolved oxygen, and dress-out percentages, but few controlled, in-pond studies have quantified parasite prevalence and biodiversity in HC compared with channel (CC) and blue (BC) catfishes. This twelve-month study is underway to reveal the ecological and taxonomic diversity of parasites infecting HC. Fingerlings of HC, CC, and BC were communally stocked at equal density (750 individuals/species/pond) in three 0.1 acre earthen ponds at the EW Shell Fisheries Center (Auburn, AL) in January 2010 and sampled monthly (10 specimens per catfish species per pond) for the presence of ecto- and endoparasites using stereomicroscopy and subsequent histology of brain, eye, gill, skin, fins, kidney, swim bladder, heart, liver, spleen, intestine, and stomach. Six major taxa of parasitic metazoans were found: Monogenea (HC– 104 infected of 106 examined [98.1%]; CC– 63 of 63 [100%]; BC– 54 of 56 [96.4%]), Cestoda (HC– 31 of 106 [29.2%]; CC– 24 of 63 [38.1%]; BC– 15 of 56 [26.8%]), Copepoda (HC– 9 of 106 [8.5%]; CC– 15 of 63 [23.8%]; BC– 15 of 56 [26.8%]), Nematoda (HC– 1 of 106 [0.9%]; CC– 1 of 63 [1.6%]; BC– 0%), Unionidae (HC– 5 of 106 [4.7%]; CC– 0%; BC– 0%), and Myxozoa (HC– 22 of 106 [20.8%]; CC– 50 of 63 [79.4%]; BC– 3 of 56 [5.4%]). These preliminary results show no evidence that HC is refractory to initial infection by each major metazoan parasite taxon that infects its parental species.






Observation and Description of Five New Species of Cestode Parasites in Fishes from Ogba River, Benin City, Edo State, Nigeria.


EE Obano*


Department of Fisheries, Faculty of Agriculture, University of Benin, Benin City, Edo State, Nigeria.   efeobano2002@yahoo.com




The entire length of Ogba River is about 200 km, with many settlements and villages, whose inhabitants are mainly farmers and fisher folks that depend on the river for their domestic, farming and fishing needs. The study area was the bridge segment of the Ogba River, which is about 10m in width with steep sandy slopes, bordered by a large multipurpose agricultural farm complex, a forest reserve, a zoological garden and an open drainage channel fed by the Benin City Master drainage system. A survey of the helminth fish parasites was carried out at Ogba River, Benin City, Edo State.  The following helminth parasites were recovered: Caryophyllaeides sp, Crescentovitus sp, Bialovarium sp, Lytocestus sp, Monobothrium sp, Stocksia sp, Cucullanus sp, Procamallanus laeviconchus, and Spinitectus sp.  Cestodes accounted for 78% of the infection, while the nematodes accounted for 22%. The main location of parasite occurrence in fish was the intestine.  Sex did not influence the degree of helminth parasite infection in fishes.  Small sized and younger fishes were mostly infected.  Five of the parasites (Stocksia sp, Monobothrium sp. Bialovarium sp, Crescentovitus sp and Caryophyllaeides sp) were new parasites recorded in the fishes of Ogba River. 








Results of 2009 Surveillance Efforts for Viral Hemorrhagic Septicemia Virus in Lake Ontario and Lake Superior


ER Cornwell*1, GE Eckerlin2, RG Getchell1, GH Groocock1, TH Thompson3, WN Batts3, G Kurath3, JR Winton3, PR Bowser1, MB Bain4, and JW Casey1.

1 Aquatic Animal Health Program, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY USA  erc58@cornell.edu, rgg4@cornell.edu, ghg3@cornell.edu, prb4@cornell.edu, and jwc3@cornell.edu
2 Department of Environmental and Forest Biology, SUNY College of Environmental Science and Forestry, Syracuse, NY USA  geeckerl@syr.edu
3 USGS Western Fisheries Research Center, Seattle, WA 98115 USA  tthompson@usgs.gov, bbatts@usgs.gov, gkurath@usgs.gov, jwinton@usgs.gov
4 Department of Natural Resources, Cornell University, Ithaca, NY USA  Mark.Bain@Cornell.edu










Viral Hemorrhagic Septicemia Virus (VHSV) was first isolated in the Great Lakes from an archived muskellunge (Esox masquinongy) collected from Lake St. Clair in 2003.  In 2005, the first mass mortality event in the Great Lakes attributable to VHSV occurred in freshwater drum (Aplodinotus grunniens) in Lake Ontario.  Throughout 2005, 2006, and 2007, additional mortality events occurred throughout Lake Ontario, Huron, Michigan, and Erie, as well as smaller inland lakes in the Great Lakes basin.  To date, no mortalities attributable to VHSV have been reported in Lake Superior.  We conducted a surveillance effort of asymptomatic fish in Lake Ontario and Lake Superior in 2009 to determine whether VHSV was still present in Lake Ontario, and if there was evidence for its presence in Lake Superior.  All fish were first screened using a qRT-PCR assay, and presumed positive tissues are currently being confirmed using traditional cell culture techniques.  Using qRT-PCR, we detected VHSV in 382 out of 2,010 fish in Lake Ontario, with an apparent prevalence of 27% in round goby (Neogobius malenostomus) and 8% in yellow perch (Perca flavescens).  In Lake Superior, we collected 847 fish at 7 sites and detected VHSV at 4 sites.  Our results were independently confirmed by re-isolation of RNA and nested RT-PCR amplification of a different region from original tissues at a separate laboratory.  This is the earliest evidence for the presence of VHSV in Lake Superior. Our results confirm that VHSV remains in Lake Ontario and suggest that it is present in Lake Superior.



Generation of a Mutant Viral Hemorrhagic Septicemia Virus of the Great Lakes Strain Which is Attenuated in Yellow Perch (Perca flavescens)

A Ammayappan1, TM Thompson2, G Kurath2 and VN Vakharia*1

1 Department of Marine Biotechnology, University of Maryland Baltimore County, Baltimore, MD USA  vakharia@umbc.edu
2 U.S. Geological Survey, Western Fisheries Research Center, Seattle, WA USA  gkurath@usgs.gov



Viral hemorrhagic septicemia virus (VHSV), belonging to the genus Novirhabdovirus in the family of Rhabdoviridae, causes a highly contagious disease of fresh and saltwater fish worldwide. Recently, a novel genotype of VHSV, designated IVb, has invaded the Great Lakes causing large-scale epidemics in wild fish. An efficient reverse genetics system was developed to generate a recombinant VHSV of genotype IVb from cloned cDNA. The recombinant VHSV (rVHSV) was comparable to the parental wild type strain both in vitro and in vivo, causing high mortality in yellow perch (Perca flavescens). A modified recombinant VHSV was generated in which the NV gene was substituted with an enhanced green fluorescent protein gene (rVHSV-?NV-EGFP), and another recombinant was made by inserting the EGFP gene into the full-length viral clone between the P and M genes (rVHSV-EGFP). The in vitro replication kinetics of rVHSV-EGFP was similar to rVHSV; however, the rVHSV-?NV-EGFP grew 2 logs lower. In yellow perch challenges, wtVHSV and rVHSV induced 82% to 100% cumulative percent mortality (CPM), respectively, whereas rVHSV-EGFP produced 62% CPM and rVHSV-?NV-EGFP caused only 15% CPM. No reversion of mutation was detected in the recovered viruses and the recombinant viruses stably maintained the foreign gene after several passages. These results indicate that the NV gene of VHSV is not essential for viral replication in vitro and in vivo, but it plays an important role in viral replication efficiency, pathogenicity, and attenuation. 



Development of Tools to Forecast the Potential for Viral Hemorrhagic Septicemia Epizootics in Wild Fish Populations


PK Hershberger*1, JL Gregg1, MK Purcell2, CA Grady1, JC Woodson2 and JR Winton2


1 U.S. Geological Survey, Western Fisheries Research Center (WFRC), Marrowstone Marine Field Station, Nordland, WA USA  phershberger@usgs.gov
2 U.S. Geological Survey, WFRC, Seattle, WA USA





Traditional disease surveillance efforts in wild fishes are based on periodic infection and disease surveys combined with mobilization of epidemiological investigations in response to observed fish kills.  This approach, although well-suited for identifying pathogens of concern and providing a list of probable causes for the observed epizootics, is likely to overlook acute disease epizootics that occur non-synchronously with scheduled surveillances or those that occur among highly migratory species that move outside the surveyed area.  Further, the traditional approach provides no capacity for mitigating or preventing the impacts of diseases to the affected populations.  We contend that by understanding basic disease mechanisms unique to the host-pathogen relationships, we can develop population-screening tools that are capable of forecasting the potential for future disease epizootics.  These tools can then be employed to design adaptive disease management strategies intended to prevent or mitigate population-level impacts of these diseases.  Using epizootiological and immunological principles unique to viral hemorrhagic septicemia (VHS) in Pacific herring, we describe the development of several tools (an in vitro fin explant assay, a passive immunization assay, and an Enzyme-Linked-Immunosorbent Assay (ELISA) to detect herring antibodies against VHSV) that may be capable of forecasting the potential for future VHS epizootics in wild herring populations.




Experimental Susceptibility of Sea Lamprey and Tiger Muskellunge to VHSV IVb

RG Getchell*1, GH Groocock1, ER Cornwell1, LL Coffee2, GA Wooster1, and PR Bowser1

1 Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY,  USA  rgg4@cornell.edu, ghg3@cornell.edu, erc58@cornell.edu, gaw5@cornell.edu, prb4@cornell.edu
2 Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA  llc77@cornell.edu





During 2009 the experimental susceptibility of sea lamprey Petromyzon marinus and tiger muskellunge Esox masquinongy X Esox lucius to the Great Lakes strain of viral hemorrhagic septicemia virus (VHSV IVb) was tested by intraperitoneal (IP) injection of 106 pfu of the MI03 isolate.  Prior to challenge, 30 wild lamprey from Cayuga Lake were assayed for VHSV using cell culture and a quantitative RT-PCR (qRT-PCR) method.  No clinical signs of VHSV were observed in the fish collected. Five-fish pools of liver, heart, spleen, anterior and posterior kidney were assayed for VHSV by cell culture, and all pools were negative.  Sixty lamprey from Cayuga Lake were subsequently collected for the susceptibility experiment, which was carried out at 10 ºC.  Nine of thirty lamprey challenged were positive by cell culture and 26 of 30 positive by qRT-PCR.  No mortalities occurred during the 28-day trial.  The mean quantity of VHSV measured by qRT-PCR was not significantly different at days 1, 3, 7, 14, or 28.  None of the 30 media-injected controls tested positive by qRT-PCR or cell culture. The tiger muskellunge were obtained from the NYSDEC’s South Otselic Hatchery and held at 15 ºC for at least 2 weeks.  The tiger muskellunge were temperature shocked by dropping the tank water to 10 ºC the day of the viral challenge.  None of the 25 media-injected controls tested positive by qRT-PCR or cell culture.  All 55 fish injected IP with VHSV tested positive by qRT-PCR and cell culture.  There was 85% mortality during the 14-day trial.




Detection of VHSV IVb Within the Gonads of Great Lakes Fish Using In Situ Hybridization


LJ Al-Hussinee*and JS Lumsden


Fish Pathology Laboratory, Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Netherlands lalhussi@uoguelph.ca




Among several species of the Great lakes, freshwater drum (Aplodinotus grunniens) and muskellunge (Esox masquinongy) have been impacted by viral hemorrhagic septicaemia virus genotype IVb (VHSV IVb) in the spring of 2005 and 2006. The viral agent belongs to the genus Novirhabdovirus, and globally this agent causes high economic impact to aquaculture. Fish with VHSV IVb from Ontario waters were characterized by moderate to marked necrosis of the myocardium and other tissues and by a vasculitis. The pattern of lesions varied with the species affected to some degree. Immunohistochemistry (IHC), using a rabbit polyclonal antibody to purified VHSV IVb, identified viral antigen in tissues with lesions but also in the oocytes and testicular tissues of several fish species.  In the current research, we employed in situ hybridization (ISH) to examine the ovaries from naturally infected freshwater drum and muskellunge, as well as experimentally infected rainbow trout (Oncorhynchus mykiss) and fathead minnows (Pimphales promelas). Although the ovaries and testes of fish infected with VHSV IVb had no significant histological changes, viral RNA was present to varying degrees within these tissues. The abundance of viral RNA was moderate to marked within oocytes of muskellunge and freshwater drum with lesser amounts in oocytes of rainbow trout and fathead minnow. Viral RNA was mainly situated inside yolk granule and/or surrounding yolk vacuoles of oocytes, and around primary and secondary spermatocytes. As determined by ISH, VHSV IVb may reside with oocytes and spermatozoa; however, survival of virus within gametes needs to be determined. This method is also another useful technique to study the pathogenicity of VHSV infection.






Humoral Immune Response of Muskellunge, Esox masquinongy, to Viral Hemorrhagic Septicemia Virus


EV Millard*1 and M Faisal1, 2


1 Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, S-112 Plant Biology Building, Michigan State University, East Lansing, MI   USA millarde@cvm.msu.edu
2 Department of Fisheries and Wildlife, S-110 Plant Biology Building, Michigan State University, East Lansing, MI USA faisal@cvm.msu.edu






Viral Hemorrhagic Septicemia Virus (VHSV) genotype IVb emerged in the Laurentian Great Lakes causing widespread fish kills.  There are mounting concerns about the effects this pathogen may have on the health of resident fish populations.  Our previous studies have demonstrated the high susceptibility of the esocid muskellunge, Esox masquinongy, to VHSV-IVb, though additional studies have provided evidence that this species can survive infection if the dose of exposure is relatively low.  Therefore this study was designed to determine if circulating anti-VHSV antibodies are present in muskellunge survivors.  Serum samples were collected from two groups of muskellunge; adults from Lake St. Clair (Lake Erie watershed, Michigan, USA) over four years, and juveniles that were experimentally exposed to VHSV.  Initially, sera were analyzed for the presence of antibodies by the complement-dependent 50% plaque neutralization test.  Our findings indicated that Lake St. Clair muskellunge mount a strong antibody response that ranged from <40 to 40,960, and that high titers were detectable even in years when the virus itself was not isolated from the lake.  In the case of experimentally infected muskellunge, antibodies could be measured as early as 5 weeks post infection and titers ranged from <40 to 20,480.  High titers were still detectable at 16 weeks.  The samples were then assessed using a recently developed competitive enzyme-linked immunosorbent assay (cELISA), using VHSV recombinant proteins as the antigen.  This approach provides a comparison between the two assays and gives an accurate account of the dynamics of anti-VHSV antibodies in a susceptible species.   





Viral Hemorrhagic Septicemia (VHS) Surveillance on Wisconsin Fish Farms


MJ Kebus*


Wisconsin Department of Agriculture, Trade & Consumer Protection, Division of Animal Health, Madison WI USA Myron.kebus@datcp.state.wi.us




This study reflects work conducted between the Wisconsin Department of Agriculture, Trade and Consumer Protection (DATCP) and the United States Department of Agriculture, Animal and Plant Health Inspection Service (APHIS), Veterinary Services under a Cooperative Agreement Award. In Wisconsin the DATCP is responsible for health requirements of imported fish, farm-raised fish and all fish stocked into waters of the state. Wisconsin has an abundance of trained veterinarians who are available to fish farmers for VHS sampling and other disease work including observational surveillance. The DATCP has collaborated with the University of Wisconsin-Madison, School of Veterinary Medicine, to develop an online Fish Health Medicine Certificate Program with accompanying hands-on training that has trained over 230 private practice, state, and federal veterinarians from the U.S. in fish farm veterinary medicine, including VHS sample collection.  All fish being stocked into state waters are required to have a fish health certificate prior to stocking.  This requirement has been in place prior to the diagnosis of VHS in the Great Lakes. Since the APHIS Amended VHS Federal Order was put in place in the fall of 2006, the volume of fish health certificates has increased from 100 to 500 annually.  This increase resulted in part due to the requirements of the Amended Federal Order and in part due to the response by the VHS-affected states to add additional fish health certificate requirements beyond those required by APHIS for import into their states.  As a result of the increased volume of fish health certificates, there is now a desire from Wisconsin fish farmers, natural resource agencies and DATCP to develop electronic fish health certificate and an electronic database to help analyze and manage the fish health certificates in Wisconsin. 











Biology and Genetic Characterization of the Fish and Amphibian Herpesviruses (Family: Alloherpesviridae)


TB Waltzek*1, GO Kelley2, ME Alfaro3, T Kurobe1, AJ Davison4 and RP Hedrick1


1 Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA,  USA tbwaltzek@ucdavis.edu
2 California Department of Fish and Game, Fish Health Laboratory, Rancho Cordova, CA, USA gokelley@dfg.ca.gov
3 Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA, USA michaelalfaro@ucla.edu
4  MRC Virology Unit, Institute of Virology, University of Glasgow, Glasgow UK a.davison@mrcvu.gla.ac.uk










Phylogenetic relationships among herpesviruses (HVs) of mammals, birds, and reptiles have been studied extensively, whereas those among other HVs are relatively unexplored. We have reconstructed the phylogenetic relationships among 13 fish and amphibian HVs using maximum likelihood and Bayesian analyses of amino acid sequences predicted from parts of the DNA polymerase and terminase genes. The relationships among 6 of these viruses were confirmed using the partial DNA polymerase data plus the complete sequences of the terminase, helicase, and triplex protein genes. The position of these viruses among all other sequenced HVs was also investigated using the complete terminase gene. The results established the monophyly of the fish and amphibian HVs (Alloherpesviridae) separate from the HVs of mammals, birds, and reptiles (Herpesviridae) and the single recognized HV of bivalve mollusks (Malacoherpesviridae) in the order Herpesvirales. Two major clades in the family Alloherpesviridae were recognized: one consisting of viruses from cyprinid and anguillid hosts and the other of viruses from ictalurid, salmonid, acipenserid, and ranid hosts. A comparison of virus and host phylogenies suggested that closely related HVs in this family may have coevolved with their hosts, whereas significant codiversification was not apparent for the more distantly related viruses.




Genogroup-Specificity of DNA Vaccines Against Infectious Hematopoietic Necrosis Virus (IHNV) in Rainbow Trout (Oncorhynchus mykiss)


Ma. M D Peñaranda*1, 2, 3, S LaPatra4, and G Kurath1,2


1 Graduate Program in Pathobiology, Department of Global Health, University of Washington, Seattle WA USA mmdpena@uw.edu
2 Western Fisheries Research Center, Biological Resources Discipline, USGS, Seattle, WA USA gkurath@usgs.gov
3 Present affiliation: Southeast Asian Fisheries Development Center – Aquaculture Department, Tigbauan, Iloilo, Philippines
4 Clear Springs Foods, Inc., Research Division, Buhl, Idaho USA scottl@clearsprings.com









Infectious hematopoietic necrosis virus (IHNV) is a fish rhabdovirus that causes significant mortality in salmonid species. IHNV has three major genogroups, designated M, U, and L. Host-specific virulence of M and U genogroup IHNV has been established, both in the field and in experimental challenges, with M isolates being highly virulent in rainbow trout (Oncorhynchus mykiss) and U isolates being more virulent in sockeye salmon (Oncorhynchus nerka). In the present study, the efficacy of DNA vaccines containing either M (pM) or U (pU) virus glycoprotein gene was investigated during intra- and cross-genogroup challenges in rainbow trout. Correlates of protection was also investigated by looking at the expression of the interferon-stimulated gene Mx-1, and the production of neutralizing antibodies (NAbs) following pM or pU DNA vaccination. At 7 days post-vaccination (early antiviral response), both pM and pU were highly protective against either M or U challenge. At 28 days post-vaccination (late antiviral response), the homologous pM vaccine was significantly more protective than pU against M IHNV challenge, while both pM and pU induced comparably high protection against U IHNV challenge. Mx-1 gene expression, measured as an indicator of the host innate immune response, was significantly higher in pM than pU vaccine when injected in rainbow trout at equal vaccine dose. Neutralizing antibodies produced in response to the two vaccines did not show a clear correlation with protection, suggesting that there are other adaptive immune response mechanisms that are important in providing late protection against IHNV in rainbow trout. 






Vector Potential of the Salmon Louse (Lepeophtheirus salmonis) in the Transmission of Infectious Haematopoietic Necrosis Virus (IHNV)


E Jakob*1,2, DE Barker1and KA Garver2


1 Fisheries & Aquaculture Department, Vancouver Island University, Nanaimo, BC Canada Eva.Jakob@viu.ca, Duane.Barker@viu.ca
2 Fisheries & Oceans Canada, Pacific Biological Station, Nanaimo, BC Canada Kyle.Garver@dfo-mpo.gc.ca






Previous studies have isolated viral and bacterial aquatic pathogens from various piscine ectoparasites, including leeches and parasitic crustaceans, implicating them as possible carriers and/or reservoirs of aquatic pathogens. However, the role of ectoparasites in the transmission of aquatic diseases remains unclear. To this end, we investigated the potential of the salmon louse, Lepeophtheirus salmonis, to harbour the aquatic rhabdovirus IHNV and transmit it to naïve Atlantic salmon (Salmo salar) under laboratory conditions. One hour waterborne immersion trials of lice with 105  PFU/ml IHNV revealed that lice can acquire and then harbour infectious virus up to 24 hours post exposure. Furthermore, studies investigating the parasitism of the salmon louse on IHNV infected Atlantic salmon demonstrated that lice are able to acquire high titers of IHNV. Moreover, in a preliminary trial it was investigated whether lice are able to transfer the virus to naïve fish (a) after being exposed to IHNV through waterborne exposure and (b) after feeding on IHNV infected fish. These studies provide the first evidence to support a direct role of L. salmonis in the transmission of IHNV to Atlantic salmon. 




Estimation of Viral Parameters Associated with Waterborne Transmission of Infectious Hematopoietic Necrosis Virus (IHNV) in Atlantic Salmon.  

KA Garver*1, AM Grant1, and J Richard1

Fisheries & Oceans Canada, Pacific Biological Station, Nanaimo, B.C. Canada  Kyle.Garver@dfo-mpo.gc.ca, Amelia.Mahony@dfo-mpo.gc.ca, Jon.Richard@dfo-mpo.gc.ca



Multiple epizootics of Infectious Hematopoietic Necrosis Virus (IHNV) have occurred in farmed Atlantic salmon in the marine waters of British Columbia.  During an outbreak occurring from 2001-2003, IHNV spread to 36 farm sites resulting in the loss of over 12 million fish due to either mortality or culling.  To better understand the transmission of IHNV in marine salmon sites, we empirically determined the minimum infectious dose and shedding rates for IHNV in Atlantic salmon as well as the stability of virus in seawater.  Waterborne challenges ranging in viral dose from 10-104 PFU/ml demonstrated that the minimum dose required to induce mortality in Atlantic salmon post-smolts was 10 PFU/ml.  Ultrafiltration of seawater collected from flow-through virus challenge tanks (104 PFU/ml, 1h, 10oC) revealed detectable levels of shed virus beginning at 7 days post-challenge.  Peak shedding was observed at day 16 and was estimated to be 2 x 105 PFU fish-1 hour-1. Virus stability studies indicate that IHNV decay rates in raw seawater is rapid (~105 PFU/day) yet dependent on temperature and local microbial flora.  This information is being incorporated in a water circulation model to provide more accurate geospatial predictions of risk for IHNV in farmed Atlantic salmon.




Molecular Characterization and Infection Kinetics of Erythrocytic Necrosis Virus (ENV) in Pacific Herring


EJ Emmenegger*1, JA Glenn1, WN Batts1, CG Grady2, JL Gregg2, SE Roon2, JR Winton1, and PK Hershberger2


1 USGS Western Fisheries Research Center, Seattle, WA USA eemmenegger@usgs.gov, jglenn@usgs.gov, bbatts@usgs.gov, jwinton@usgs.gov
2 USGS Marrowstone Marine Field Station, Nordland, WA USA cgrady@usgs.gov, jgregg@usgs.gov, sroon@usgs.gov, phershberger@usgs.gov






Viral erythrocytic necrosis (VEN) periodically causes epizootics in populations of herring and some salmonids in coastal marine waters of the Atlantic and Pacific Oceans.  VEN is classically diagnosed by microscopic examination of Giemsa-stained blood films for the presence of inclusion bodies within the cytoplasm of affected erythrocytes. Little is known about VEN disease progression or the causative agent, erythrocytic necrosis virus (ENV), because it fails to show cytopathic effect in any known cell line. ENV is presumed to be an iridovirus based on electron micrographs, however viral identity has not been confirmed. We are attempting multiple strategies to molecularly characterize ENV and have initiated a study to understand the kinetics of viremia during an ENV infection. We followed the course of the disease after specific pathogen-free Pacific herring (Clupea pallasii) were exposed to the putative iridovirus and we demonstrated that the prevalence of erythrocytic inclusion bodies increased from 0% at 0-4 days post-exposure, to 94% after 28 days. The kinetics of viremia throughout this period will be quantified by enumerating virions in paired blood and spleen/kidney samples using transmission electron microscopy.  A better understanding of the relationship between viral load and cytoplasmic inclusion bodies will facilitate collection of blood samples containing the highest concentration of virus, which will aid in the molecular identification of ENV and ultimately in the development of a diagnostic PCR assay. 










Recombination Activation Gene 1 (rag1) Mutant Zebrafish for Immunology Research


L Petrie-Hanson, C Hohn* and L Hanson


Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS USA hohn@cvm.msstate.edu




We confirmed the presence of the 1 gene point mutation rag1t26683 in our rag1-/- mutant population. Mutants survived caudal fin sampling by continuous bath treatment in 42 mg/L aquarium salt and 0.04 g/L Jungle Fungus eliminator during recovery. Rag1+/-, rag1-/- and rag1+/+ adults were differentiated by visible implant elastomer (VIE) tagging. The dorsal fin base was the optimal site and optimal colors were red, pink, orange and yellow. Peripheral blood differentials indicated that mutants had decreased lymphocyte populations (34.7%) compared to wild types (70.5%), and increased granulocyte populations (52.7%) compared to wild types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. FACS Calibur flow cytometric analyses demonstrated that 7% and 43% of gated cells from mutants were lymphocytes and myeloid cells, respectively, while wild types had 26% and 21% lymphocytes and myeloid cells, respectively. Reverse transcriptase polymerase chain reaction (RT-PCR) assays demonstrated mutant zebrafish kidney hematopoietic tissue expressed mRNA encoding Non-specific Cytotoxic cell receptor protein-1 (NCCRP-1) and Natural Killer cell (NK) lysin but lacked T cell receptor (TCR) and immunoglobulin (Ig) VH1 transcript expression, while wild type zebrafish kidney hematopoietic tissue expressed NCCRP-1, NK lysin, TCR and Ig VH1 transcript expression.





Adhesion Dynamics of Flavobacterium columnare to Channel Catfish (Ictalurus punctatus) and Zebrafish (Danio rerio) after Immersion Challenge


O Olivares-Fuster, SA Bullard, A McElwain, and CR Arias*


Department of Fisheries and Allied Aquacultures, Auburn University, AL USA ariascr@auburn.edu



Adhesion of Flavobacterium columnare to skin and gill of channel catfish (Ictalurus punctactus) and gill of zebrafish (Danio rerio) was evaluated by immersion challenge followed by bacterial plate count and confirmatory observations of gill-adhered bacterial cells using scanning electron microscopy.  Two strains of F. columnare (ARS-1 and BGFS-27 from genomovar I and II, respectively) were compared. At 0.5 h post-challenge, both strains adhered to gill of channel catfish at comparable levels (106 colony forming units (CFU)/g) but significant differences in adhesion were found later in the time course.  Channel catfish was able to effectively reduce ARS-1 cells on gill; whereas, BGFS-27 persisted in gill beyond the first 24 h post-challenge. No significant difference was found between both strains when adhered to skin, but adhered cell numbers were lower (103 CFU/g) than those found in gill and were not detectable at 6 h post-challenge. Adhesion of BGFS-27 cells to gill of zebrafish also occurred at high numbers (>106 CFU/g) while only <102 CFU/g of ARS-1 cells were detected in this fish. The results of the present study show that particular genomovars of F. columnare exhibit different levels of specificity to their fish hosts.




Effects of the Anesthesia Agents Benozocaine and Tricaine Methanesulfonate (MS-222) on Immune Parameters of Tadpoles of the Cuban Tree Frog (Osteopilus septentrionalis)


B F Sears*, P Snyder, and J R Rohr


Department of Integrative Biology, University of South Florida, Tampa, FL USA bsears@usf.edu,  psnyder@usf.edu, jrohr@usf.edu




Benzocaine and tricaine methanesulfonate (MS-222) are widely used in the anesthetization of poikilothermic vertebrates. While the physiological effects of anesthesia via these agents have been characterized in fish and adult amphibians, little is known about the physiological consequences of anesthesia in larval amphibians. Because anesthesia is frequently employed when studying the behavior of larval amphibians, including their avoidance of parasites, we tested whether anesthesia reduces immunocompetence by reducing the number of circulating white blood cells (WBC). Tadpoles of the Cuban tree frog (Osteopilus septentrionalis) were exposed to artificial spring water (ASW; control), MS-222, or benzocaine. Animals were sacrificed at intervals post-anesthesia, and blood smear slides were prepared for microscopical examination of circulating WBC. Benzocaine did not result in significantly different numbers of circulating WBC when compared to the artificial spring water control. In contrast, MS-222 did significantly reduce levels of circulating thrombocytes. We are currently conducting experiments to examine changes in resistance to experimental trematode infections after anesthesia.





Antiviral Activities of an Intracellular Viral RNA Sensor, MDA5 in Japanese Flounder (Paralichthys olivaceus)


M Ohtani*1, J Hikima1, TS Jung1 and T Aoki1, 2

1 Aquatic Biotechnology Center, College of Veterinary Medicine, Gyeongsang National University, Gyeongnam, South Korea makky34@gmail.com, j.hikima@gmail.com, and jungts@gnu.ac.kr
2 Laboratory of Genome Science, Tokyo University of Marine Science and Biotechnology, Minato, Tokyo, Japan aoki@kaiyodai.ac.jp






Infected viral RNAs are sensed with pattern recognition receptors including toll-like receptors (TLRs) and retinoic acid inducible gene-like receptors (RLRs). Melanoma differentiation-associated gene 5 (MDA5), a member of RLRs, recognizes the viral RNA in cytoplasm and enhances the antiviral response in the host cells. MDA5 gene in Japanese flounder (Paralichthys olivaceus) was cloned. The MDA5 transcripts in the kidney infected with viral hemorrhagic septicemia virus (VHSV) showed a significant increase in the expression levels as well as the MDA5 expression in the kidney leukocytes stimulated with poly I:C in vitro. To reveal the antiviral activity of the flounder MDA5, five expression constructs containing pcDNA4-MDA5 (full-length), pcDNA4-MDA5ΔRD (regulatory domain deleted), pcDNA4-MDA5ΔCARD (CARD deleted), pcDNA4-MDA5ΔHeli (RNA helicase region deleted) and pcDNA4-Empty (as control) were transfected into the flounder natural embryo (HINAE) cell line. After 48 hours transfection, the transfected cells were infected with VHSV or infectious pancreatic necrosis virus (IPNV) at 15?C. The cytopathic effect appearance of the virus-infected HINAE cells transfected with pcDNA4-MDA5, pcDNA4-MDA5ΔRD and pcDNA4-MDA5ΔHeli was delayed in contrast to the pcDNA4-Empty or -MDA5ΔCARD. To confirm the antiviral responses, the expression level of interferon inducible genes (Mx and ISG15) in HINAE cells transfected with the expression vectors were quantified by Q-PCR. The expressions of Mx and ISG15 mRNAs were induced by pcDNA4-MDA5, pcDNA4-MDA5ΔRD and pcDNA4-MDA5ΔHeli in the presence of poly I:C. These results suggested that the flounder MDA5 induced the inhibition of viral replication through the induction of interferon inducible gene expressions.




Lymphocyte Deficient Zebrafish Demonstrate Specific Protection Following Bacterial Re-exposure


C Hohn and L Petrie-Hanson*


Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS USA, lora@cvm.msstate.edu




Recombination activation gene 1 deficient (rag1-/-) mutant zebrafish lack functional B and T lymphocytes, and therefore are entirely dependant on their innate immunity for protection against pathogens. The innate immune system is thought to lack adaptive characteristics that provide enhanced protection to a second exposure of the same pathogen. In the present study we evaluated the ability of rag1-/- mutant zebrafish to mount such a protective response to the intracellular bacterium Edwardsiella ictaluri. Following secondary challenge with a lethal dose of homologous bacteria 4 and 8 weeks after a primary vaccination dose, both wild-type and lymphocyte deficient rag1-/- mutant zebrafish demonstrated equivalent levels of protection. Heterologous bacterial exposures did not provide protection in either wild-type or rag1-/- mutant zebrafish. Adoptive leukocyte transfers from previously exposed mutants conferred protective immunity in naïve mutants when exposed to homologous bacteria. Our findings show that a component of the innate immune system mounted a response that provided long-term specific protection. Further, adoptive cell transfers demonstrated that kidney interstitial leukocytes from lymphocyte deficient zebrafish transferred this protective immunity. This is the first report of any lymphocyte-deficient vertebrate mounting a protective secondary immune response to a bacterial pathogen, and suggests that innate immune cells can mediate adaptive immunity.




Generation of a Monoclonal Antibody Specific for ORF68 of Koi Herpesvirus


T Aoki*1, I Hirono1, H Kondo1, T Takano1, S Unajak1, T Taki2, J Hikima3 and TS Jung3


1 Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Tokyo, Japan, aoki@kaiyodai.ac.jp, hirono@kaiyodai.ac.jp, h-kondo@kaiyodai.ac.jp, takano5320@hotmail.com, and fscissmn@ku.ac.th
2 Department of Microbiology, Faculty of Hygienic Technology, School of Allied Health Sciences, Kitasato University, Kanagawa, Japan, tatabox@kitasato-u.ac.jp
3 Aquatic Biotechnology Center, College of Veterinary Medicine, Gyeongsang National University, Jinju, Gyeongnam, South Korea, j.hikima@gmail.com, and jungts@gmail.com









Outbreak of Koi herpesvirus [KHV or Cyprinid herpesvirus 3 (CyHV-3)] disease in carp is still a serious problem worldwide. KHV is related closely to other two cyprinid herpesviruses, including pox herpesvirus [CHV or Cyprinid herpesvirus 1 (CyHV-1)], haematopoietic necrosis herpesvirus of goldfish [Cyprinid herpesvirus 2 (CyHV-2)], and KHV. It is said that these viral species have common antigens, therefore, identifying specific antigens from KHV is important to possibly control the disease caused by this virus. In a previous study, two major KHV antigenic proteins, encoded by open reading frame (ORF) 62 and ORF68 were identified by immunoscreening from the genomic phage library using KHV-specific rabbit polyclonal antibody. To generate the monoclonal antibody (MAb) against ORF62 or ORF68 proteins, BALC/c mice were immunized with the recombinant antigenic proteins of ORF62 or ORF68. The spleen cells from immunized mice were fused with Sp2/o myeloma cells using polyethylene glycol. After the screening by enzyme-linked immunoassay (ELISA) and Western blot analysis, we obtained one MAb against ORF68 (7C6) but no MAb against ORF62. In Western blot analysis, the anti-ORF68- MAb demonstrated specific reaction with KHV-infected KF-1 cells lysate but not with CHV- or CyHV-2-infected KF-1 cells lysate. Similar results were shown in fluorescent antibody (FA) test using infected KF-1 cells and immunohistochemical (IHC) investigation conducted by haematoxylin and eosin (H&E) stain for infected liver sections or FITC detection for infected spleen sections. These suggested that the anti-ORF68-MAb (7C6) is capable of specifically detecting the KHV ORF68 protein.





Histopathologic Effects of 17 Beta-estradiol on the Gonads of Larval Xenopus laevis


JC Wolf *1, I Lutz2, W Kloas2, TA Springer3, HO Krueger3, LR Holden4 and AJ Hosmer5

1 Experimental Pathology Laboratories, Inc., Sterling, VA USA  jwolf@epl-inc.com
2 Inland Fisheries, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany  werner.kloas@igb-berlin.de  ilka.lutz@igb-berlin.de
3 Wildlife International Ltd., 8598 Commerce Drive, Easton, MD, 21601 USA  tspringe@wildlifeinternational.com  hkrueger@wildlifeinternational.com
4 Sielken & Associates Consulting, Inc., Bryan, TX USA  larry@sielkenassociates.com
5 Syngenta Crop Protection, Inc., Greensboro, NC USA. alan.hosmer@syngenta.com










Traditionally, histopathology has been used in a limited fashion to investigate reproductive effects of xenoestrogens in anurans.  The purpose of this study was to fully characterize the histopathologic effects of 17 beta-estradiol (E2) on the gonads of larval Xenopus laevis exposed under tightly controlled laboratory conditions.  This study was performed as three separate experiments at two separate facilities, and involved more than two thousand control and E2-exposed frogs.  Microscopic findings were recorded according to standardized criteria and terminology that were ultimately refined by the process.  Throughout all experiments, the most consistent effects of E2 exposure were increased proportions of phenotypic females, and increased prevalences (as compared to untreated controls) of mixed sex, dilated testicular tubules, dividing gonocytes in the testis, and internal melanophores in the testis.  The diagnosis of dilated testicular tubules was an especially robust E2 indicator.  These experiments demonstrated the benefits of a detailed histopathologic examination for detecting endocrine-active effects.






Mortalities, Skin Lesions and Intersex in Smallmouth Bass: Possible Association with Contaminants of Emerging Concern


VS Blazer*1, LR Iwanowicz1, DD Iwanowicz1 and H Ellery1,2


1 National Fish Health Research Laboratory, U.S. Geological Survey, Kearneysville, WV USA    vblazer@usgs.gov; liwanowicz@usgs.gov; diwanowicz@usgs.gov
2 Cooperative Fish and Wildlife Unit, West Virginia University, Morgantown, WV USA  hellery@usgs.gov





Since 2002, major fish kills of centrarchids have occurred in the Potomac, James and Susquehanna river drainages.  The mortalities have varied in location and severity and are generally accompanied by skin lesions. In the Potomac and James rivers it is primarily the mature, adult smallmouth bass and redbreast sunfish that are affected in the spring. In certain areas population effects have been reported. Surveys over the past six to eight years have indicated that a variety of bacterial and viral pathogens are present, heavy parasite loads are observed and other lesions such as papillomas, gill lamellar fusion, oxidative damage and a high prevalence of intersex or testicular oocytes are noted. No one pathogen is consistently isolated and the variety of lesions suggest the fish are immunosuppressed, and hence susceptible to a number of potential pathogens, including Aeromonas salmonicida, motile Aeromonads, Flavobacterium columnare and largemouth bass virus. Heavy infestations of trematode metacercariae and a number of myxozoan parasites are observed. The coincidental finding of high prevalence and the severity of testicular oocytes at all areas at which skin lesions and mortalities occur suggests that contaminants of emerging concern may be involved, particularly estrogenic chemicals. Many of these have been shown to influence disease resistance and be reproductive endocrine-modulators. Sources of these chemicals (natural and synthetic hormones, pesticides, herbicides, personal care products, and pharmaceuticals) include human wastewater treatment plant effluent, stormwater runoff and agriculture. Ongoing studies including chemical analyses of water and fish tissue, and landscape analyses, will be discussed.




Investigating Fish Kills in Young-of-Year Smallmouth Bass in the Susquehanna River Drainage.


L Iwanowicz*1, V A Blazer1, C Starliper1, H Ellery and K Crawford2


1 US Geological Survey, Leetown Science Center, Fish Health Branch, 11649 Leetown Rd. Kearneysville WV, 25430  USA   Luke_Iwanowicz@usgs.gov
2 US Geological Survey, Columbia Environmental Research Center, 4200 New Haven Road, Columbia,  MO  65201-8709, USA   






Smallmouth bass mortality throughout the Potomac and Susquehanna River drainages has attracted the concern of the public and resource management agencies during recent years. In the Susquehanna River drainage, SMB mortality in the Susquehanna is primarily observed in young-of-year (YOY) bass. Flavobactrerium columnare has been isolated from bass during mortality events, but histological evidence suggests that this pathogen may not be the singular or primary cause of mortality. Chronic granulomatous inflammation in multiple tissues was consistently identified in YOY bass examined in 2007. Interestingly, within these areas of inflammation a putative rickettsia-like organism has been noted. Largemouth bass virus has been isolated from moribund bass and large myxozoan cysts have been observed in the skeletal muscle of these fish. Extensive bacterial and viral culture efforts in addition to culture independent methods are in progress to identify the assortment of microbes associated with these mortality events.  Here results of our findings from 2007-2010 will be reported.






Disappearing Golden Shiner Phenomenon- Is Hydrogen Sulfide the Culprit?


AM Kelly* and S Kumaran

University of Arkansas at Pine Bluff, Fish Health Services, Lonoke, AR 72086, USA
akelly@uaex.edu,  skumaran@uaex.edu




Throughout the year, when water temperatures are between 70 and 750C, golden shiner (Notemigonus crysoleucas) stocks seem to disappear from ponds that have been in production for two or more years.  Investigations into the possible causes for this phenomenon have lead to the conclusion that hydrogen sulfide may play a role. Since the toxic form of hydrogen sulfide is more abundant at lower pH values, a study conducted in ponds with fluctuating pH and temperatures was conducted. Twenty-five ponds on a commercial fish farm were outfitted with a cage containing 12 sentinel fish.  In these 25 ponds, monitors were used to record daily fluctuations in pH and temperature.  Cages were checked twice a week and the number of fish in the cages and their health status were recorded.  At the end of the study, the number of live fish remaining was also recorded.  Hydrogen sulfide concentrations were taken in ponds before and after disturbing the mud bottoms.  Ponds were categorized into low <0.005-0.01 mg/L, medium 0.01-<0.75 mg/L and high >0.075 mg/L hydrogen sulfide producing ponds. Fish in the low hydrogen sulfide ponds had significantly higher survival rates (61.8%) versus those in the medium (17.7%) and high (3.3%) hydrogen sulfide ponds.  Future studies include determining median lethal levels for hydrogen sulfide to golden shiners at various pH values. Studies on the hydrogen sulfate and hydrogen sulfide concentration in ponds as well as the 24 hour hydrogen sulfide cycle will be presented.



Contents - Scientific Sessions - Plenary Abstracts - Poster Abstracts - Special Session Abstracts - Author Index