Poster Session Abstracts

Oceanário de Lisboa, opened in 1998, is a public aquarium with a total volume of 7 million liters. Fish collection includes c. 350 saltwater teleosts species (2668 specimens in 2010) and 45 elasmobranchs species (180 specimens in 2010). Oceanário de Lisboa’s Animal Health Monitoring Program includes bacterial cultures (BC) and antimicrobial susceptibility testing (AST). A total of 1239 clinical cases were recorded in 11 years (1998-2008) including 891 BC and AST. From the total BC, 57.7% were positive, with an average number of 49 cases per year. A peak of 125 cases occurred in the opening year due to normal public aquarium start-up conditions. Positive correlation (r = 0.918) was found between clinical cases and positive BC. Concerning bacteria families the following was observed: Vibrionaceae – 50%; Pasteurellaceae – 23.5%; Pseudomonaceae – 16%; Flavobactereaceae – 5%; Mycobactereaceae – 1.8%; others – 3.7%. In the first 3 families, the most frequent bacteria included: Vibrio vulnificus (59.7% of total Vibrionaceae) and V. alginolyticus (28.2%); Brevundimonas vesicularis (29.8%); Photobacterium damselae (46.9%) and Mannheimia haemolytica (17%). A total of 55 bacteria species was isolated. A significant association was found between Vibrionaceae at high temperature waters and Pasteurellaceae in low (p = 0.003). The relationship between V. vulnificus (high temperature), V. alginoliticus (low temperature), and water temperature was significant (p = 0.043). Although no apparent antimicrobial resistance trend was observed, therapy is continuously adjusted according to bacteria incidence and antimicrobial resistance assessment. Historical data analysis allows an efficient approach to bacterial disease, being an essential input to prediction of future problems and to correction of husbandry procedures.

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.

Development of Antimicrobial Resistance in Bacteria Associated to Reared Chilean Scallop Larvae under Florfenicol Treatment

In Chilean scallop cultures, the use of florfenicol to prevent larval mortalities is a frequent practice but no studies of its effect on scallop culture have been performed. Samples of scallop larvae from untreated and florfenicol–treated rearing tanks were collected along the period of larval culture from a Chilean hatchery and culturable counts of total and oxytetracycline-, flumequine- and florfenicol-resistant bacteria were determined using VNSS media. Seventy representative florfenicol-resistant isolates were identified by using the BIOLOG system or 16S rRNA gene sequence analysis, and tested for susceptibility to 12 antimicrobials. In addition, the conjugation potential transfer of florfenicol resistance of 10 strains was assayed by filter mating. Significant differences in florfenicol and oxytetracycline resistance were observed between the control (8.81 and 0.61% at day 18) and treated (33.72 and 8.43% at day 18) scallop larvae microbiota along all rearing periods, but no significant differences in flumequine resistance were detected (0.059 and 0.076%, respectively). Florfenicol-resistant strains mainly belonged to the Pseudomonas genus (61.43%). The majority of isolates showed simultaneous resistance to 5–7 antibacterials and were mainly resistant to streptomycin, florfenicol, chloramphenicol and co-trimoxazole and sensitive to cefotaxime, gentamicin, kanamycin, oxolinic acid, flumequine and enrofloxacin. Only a Pseudomonas putida strain displayed simultaneously resistance to florfenicol and oxytetracycline. The results show an important occurrence of selection for florfenicol-resistant bacteria in reared scallop larvae and prompt the need to maintain strict control of florfenicol use in Chilean scallop farming. This study was supported by FONDECYT (grant 1090793).

Microbiological Conditions of a Sole (Solea senegalensis) Production Facility in Portugal

As part of a project that aims to intensify production of sole, a thorough microbiological survey has been conducted to identify the critical points for pathogen development. The survey was conducted in Portuguese mariculture facility, where turbot and European seabass are also reared. The objectives of the survey were to: i) study the distribution of the bacterium Tenacibaculum maritimum, the only pathogen known to be present, through isolation trials and PCR detection from fish, fish mucus, water, sediment and biofilms from representative places in the facility; ii) using the same methodology, search for the presence of other important bacterial fish pathogens; and iii) determine the microbial load of the water in representative places in the facility, in order to evaluate the efficiency of different filters in disinfection. We isolated T. maritimum from several fish and water samples. The DNA of this pathogen was detected by PCR in samples of fish mucus, water, sediment and biofilms, often without its isolation, indicating that this bacterium is widespread in the facility, except for the nursery and hatchery, where it was not detected. None of the important fish pathogenic bacteria such as Vibrio anguillarum, Photobacterium damselae subsp. piscicida, Edwardsiella tarda or Streptococcus parauberis were isolated, or detected by PCR. Bacterial counts in the water showed that the filters were not effective in reducing microbial load. Consequently, major changes have been made to the water circulation in the facility, and the filters are now efficient.

Klamath Basin Restoration and Endangered Species: Molecular Profiling of Mucosal Bacterial Communities

DD Iwanowicz*¹, CA Ottinger¹, KR Echols², T Wood³, SP VanderKooi⁴, BH Rosen⁵ and SM Burdick⁴

In 2007, we began a multidisciplinary integrated assessment of factors with potential influence on the survival of the shortnose and endangered Lost River suckers in Upper Klamath Lake (UKL), Oregon. Here, we will focus on mucosal bacterial community profiles identified in these species. From July – September 2008 and 2009, age-0 Lost River and shortnose suckers obtained from Upper Klamath Lake (UKL) or the newly flooded region of the Nature Conservancy Delta Restoration Project were evaluated for skin bacterial flora. Fish mucous samples were examined for bacterial community composition by targeting 16S ribosomal DNA using terminal restriction fragment length polymorphism (tRFLP) analysis for genus-level identification. Data were compared based on fish capture locality. The bacterial flora on the 2008 age-0 suckers exhibited significant differences along a north to south gradient in Upper Klamath Lake. Fish sampled in 2008 from the Tulana Farm region exhibited skin bacterial floras similar to those obtained from near shore areas in either the northern or central regions of UKL. Although there were similarities, fish from localities in both Tulana Farm and UKL exhibited unique bacterial community profiles that permitted accurate assignment of individual fish to a specific locality. Such assignment was achieved via discriminant analysis. We are currently analyzing 2009 data. In addition to data on locality-specific age-0 sucker skin bacterial community composition, we will discuss the impacts these bacteria may have on juvenile sucker health and on delta restoration.

Isolation and Characterization of Pathogenic Aeromonas Hydrophila in Fishes in Northeast China

Fifty-nine Aeromonas hydrophila isolates were obtained and identified from cultured fresh water fishes in northeast China through physiological and biochemical methods. Based on the published 16S rDNA gene sequence and aerolysin gene sequence of Aeromonas hydrophila, synthetic oligonucleotide primers were designed and used to perform PCR amplification of the two conserved gene fragments. The results showed that the detection rate was 90.48% with the PCR method compared to traditional physiological and biochemical methods. Following the physiological and molecular detection, the typing of the Aeromonas hydrophila isolates was done by ERIC-PCR method. The results showed that 59 isolates from the three northeast provinces were divided into 3 genotypes (Ⅰ, Ⅱ and Ⅲ) of which typeⅢ was dominant (54.84% of total isolates). Isolates from Heilongjiang province and Jilin province had a similar trend in the percentage of the sub-types which showed type Ⅲ> typeⅠ> typeⅡ. Among the three sub-types no obvious geographic or specific clustering was shown although the percentage of isolates from the three provinces was different. In summary, typeⅠ, Ⅱ and Ⅲ Aeromonas hydrophila isolates were identified in the main freshwater fish culture areas in the three northeast provinces of China.

Edwardsiella tarda, an enteric Gram-negative bacterium, infects a wide range of fish and causes a systemic fish disease called edwardsiellosis. E. tarda CK41, isolated from Japanese flounder diagnosed with edwardsiellosis, has exhibited a high degree of resistance to ampicillin, kanamycin, streptomycin, and tetracycline. The massive treatment of commercial fish farms with antimicrobial agents has resulted in multiple antibiotic resistant bacteria. Multidrug resistance is a major problem in the cure of pathogenic bacteria. The antibiotic-resistance genes are usually harbored in a plasmid and plasmid-mediated transfer of genes among bacterial strains was considered one of the most important mechanisms for the spread of multidrug resistance. An antibiotic resistant plasmid, pCK41, was purified and transferred into E. coli DH5α for the analysis of antibiotics contained in the plasmid. We have determined the entire DNA sequence of pCK41, which is a 72.8 kb virulence plasmid harbored in E. tarda CK41. A total of 88 open reading frames were annotated, of which 60% have homologous genes of known function, 22% match the putatuve genes in the GenBank database, and the remaining 18% are unknown. E. tarda CK41 were also subjected to a plasmid curing procedure by treatment with novobiocin, resulting in CK108. A virulence test was performed to investigate whether pCK108 had altered virulence properties in vivo, E. tarda CK108 was attenuated in Japanese flounder, zebrafish, and goldfish. In future study, we will investigate roles of pCK41 in E. tarda pathogenesis.

Edwardsiella tarda, a member of the Enterobacteriaceae, causes a systemic disease called edwardsiellosis in marine and freshwater fish. Although the control of edwardsiellosis relies on the use of antibiotics, because of antibiotic resistance problems the use of antibiotics has become more limited in aquaculture farms. Vaccination can be an alternative way to prevent this infectious disease and vaccines prepared using heat- or formalin-inactivated E. tarda are now commercially available. However, the killed vaccine process causes modification of physiochemical and structural properties of surface antigens and may elicit poor and short immune responses due to surface modification. Live attenuated vaccines can overcome those problems. A Crp (cAMP Regulatory Protein), one of global regulators, is a well known virulence factor in many bacterial pathogens. With the application of various genetic and biochemical approaches, we identified and cloned E. tarda crp gene. Analyses of the nucleotide sequences revealed 99% similarity with Salmonella crp gene. Based on the nucleotide sequences of the crp, and its 5' and 3' flanking region we constructed a recombinant suicide plasmid, pBP809, for the crp deletion mutant. Using the allelic exchange method with pBP809, an E. tarda mutant lacking Crp has been generated and named E. tarda CK219. The mutant grew slowly relative to the parent strain, as we seen in other bacteria. The mutant motility also appeared to be defective. We also could not detect changes in sugar metabolism in the mutant. Other research results regarding pathogenesis of the mutant in fish will be discussed further.

Flavobacterium columnare, the causative agent of Columnaris disease, infects a wide variety of freshwater and brackish water fishes. The gram-negative bacterium has been linked to significant losses in commercial fish species, particularly farm-raised channel catfish (Ictalurus punctatus) in the southeastern United States. Using modifications to previously established protocols, we designed oligonucleotide primer and probe combinations specifically targeting a 203 bp nucleotide region of the chondroitin AC lyase gene (GenBank AY912281). Following primer optimization, the linear dynamic range was established for two separate isolates representing two different genomovars. There were no significant differences between the two isolates, suggesting limited copy number variation of the target gene between the two genomovars. For both isolates, the assay was found to be highly repeatable and reproducible, with a coefficient of variation between runs of < 3.0%, indicating an acceptable level of precision. The linear dynamic range for both isolates covered 6 orders of magnitude ranging from 50 to 0.0005 ng of genomic DNA. The specificity of the assay was determined against 5 taxonomically or ecologically relevant isolates; Flavobacterium johnsoniae, Pseudomonas aeruginosa, Aeromonas salmonicida, Edwardsiella tarda, Edwardsiella ictaluri. There was no amplification of the target sequence for any of these non-target organisms. The development of this assay lays the foundation for future projects utilizing qPCR for the detection of columnaris in comparative susceptibility experiments and epidemiological studies quantifying the agent in pond water.

Sequence Analysis of the 16S-23S Intergenic Spacer Regions of Flavobacterium columnare

Lorelei Ford*¹, Estaban Soto¹, Brian Scheffler², Mark Lawrence¹and Larry Hanson¹

The 16S, 23S, and 5S ribosomal RNA (rRNA) genes are highly conserved sequences in bacteria and are often used for phylogenetic classification. Less conserved regions between the structural sequences are intergenic spacer regions (ISRs), which can be used to differentiate strains of the same bacterial species. This study evaluated/compared the 16S-23S ITS of 70 isolates of Flavobacterium columnare, an important pathogen of cultured fish. We used PFGE to separate I-CeuI restriction fragments from type strain ATCC 49512 and sequenced and analyzed the resulting ITSs. We found that the genome of this species harbors at least five rRNA operons with ISRs that are very similar and contain the same tRNA encoding sequences. However, there were sequence differences between the ISRs, and each of the five ISRs had a unique sequence. This intragenomic variation in ISR sequences could cause misleading results in phylogenetic studies if PCR primers amplify more than one ISR. We developed a PCR assay that allows amplification, cloning, and sequencing of the variable region of one selected ISR. Because this assay is specific for one ISR, its use will yield more consistent results in phylogenetic studies.

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

Flavobacterium columnare is a rod shaped, Gram-negative member of the Cytophaga-Flavobacterium-Bacteroides group that can cause columnaris disease in a broad spectrum of fish species. Isolation of F. columnare in the lab is relatively fastidious because it is easily outcompeted by other accompanying bacteria present in the fish. Even after primary isolation, F. columnare cultures required regular attention in the lab since they have to be passed onto fresh media every other day. Flavobacterium columnare cells lose viability rapidly when left on media after they reach the stationary phase and also become unculturable under refrigeration conditions. Some bacterial species are capable of developing resistant forms under low nutrient conditions as a survival strategy. The objective of this study was to test the effect of starvation on F. columnare cells. Resuspending F. columnare cells in low nutrient media resulted in viable cultures over a several week period. Expected changes in morphology, based on studies performed on other species, were reduction in cell size and shortening of bacilli towards coccoid forms. However, using scanning electron microscopy we observed unique morphologies of starved cells. The long F. columnare bacilli coiled themselves forming structures that resemble a ‘cinnamon roll’. Two weeks into starvation, more than 98% of the cells have adopted this conformation. Members of the genus Flavobacterium are known to produce spherical degenerative forms usually considered nonviable, often referred to as sperophlasts, when growing in liquid cultures. However, the forms we observed are not sperophlasts but ‘coiled’ structures, some of which seemed to be covered by a matrix. Contrary to the non-viable sperophlasts, the F. columnare coiled cells seem to be perfectly viable when inoculated into fresh media. The role that these starved forms play in F. columnare biology and the potential consequences that having a resistance form could have on columnaris epidemiology are unknown at this point but deserve further investigation.

Hemolytic activity of cells of smooth and rough phenotypes of Flavobacterium psychrophilum was investigated in two different assays, a microplate and an agarose hemolysis assay, using rainbow trout erythrocytes. Smooth cells showed a high, and rough cells a low, concentration dependent, hemolytic activity in the microplate assay. In the agarose assay, both phenotypes showed a rather weak hemolytic activity, with two distinct hemolytic patterns. The hemolytic activity of the cells was not regulated by iron availability and cell-free extracellular products did not show any hemolytic activity. Smooth cells of F. psychrophilum, in contrast to rough cells, showed a high ability to agglutinate erythrocytes and both hemagglutination and hemolytic activity was impaired by treatment of the cells with sialic acid. The hemolytic activity was reduced after proteolytic and heat treatment of the cells. The results from the present study suggest that the hemolytic activity in F. psychrophilum is highly expressed in the smooth phenotype, and that it is a contact-dependent, two-step mechanism that is initiated by the binding of the bacterial cells to the erythrocytes through sialic acid-binding lectins and then executed by thermolabile proteinaceous hemolysins.

Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a septicemic infection reported in a variety of fish species, which has historically been a major disease of salmonid fish reared in sea-cages in the south of Chile. In this work, the influence of two physicochemical variables (pH and temperature) on the viability of P. salmonis was assessed. Bacteria were cultured in the CHSE-214 cell line and harvested when monolayer reached approximately 80% of cytopathic effect. In order to test the pH effect, infectious supernatants were held at 17°C for 48 h at pH 5.5, 7.0, 8.5 and 10. Bacteria were then washed and resuspended with Eagle´s minimal essential medium (pH 7.2). Microbial suspensions were titrated by end-point dilution assays (Reed and Muench, 1938) in microplates with CHSE-214 cell monolayers. Titres were 10^5.8, 10^5.6, 10^5.7 and 10^1.2 TCID₅₀/mL to pH 5.5, 7.0, 8.5 and 10, respectively. In regard to the temperature effect, cultures were held at 17°C, 24°C, 31°C and 37°C for 48 h and then titrated as described for the pH experiment. There were not viable bacteria after the treatment with 31°C and 37°C. Titres were 10^5.4 and 10^3.4 TCID₅₀/mL to P. salmonis suspensions held at 17°C and 24°C, respectively. Results show that P. salmonis survivability is not reduced in a wide pH range (pH 5.5 to 8.5) but its viability is significantly diminished when the temperature of the environment is increased. Financed by grant 1080692 of the Chilean Fund for Science and Technology (Fondecyt).

Piscirickettsia salmonis is a facultative intracellular Gammaproteobacteria. This microorganism is the etiological agent of piscirickettsiosis, a disease particularly important in salmonid fish cultured in seashore areas of Southern Chile. In spite of the economic importance of this disease the virulence factors of P. salmonis are virtually unknown. In this communication, preliminary results of a study for detecting extracellular products (ECPs) of P. salmonis are presented. Two fresh P. salmonis isolates obtained from severe piscirickettsiosis outbreaks were used. The isolates were tested to confirm their virulence in controlled conditions by intraperitoneal inoculation of juvenile Atlantic salmon (Salmo salar). Both isolates caused the disease and cumulative mortalities were over 90% in the experimentally infected fish. Bacteria were cultured in the CHSE-214 cell line at 17 °C. Cell-free ultracentrifuged supernatants were analized by SDS-PAGE electrophoresis to look for ECPs presence. Both Coomassie blue and silver salts were used for gel staining. Results so far show no evidence of ECPs suggesting that exotoxins are not produced by P. salmonis. Financed by grant 1080692 of the Chilean Fund for Science and Technology (Fondecyt).

Characterization of Pseudoalteromonas spp. with Pathogenic Potential for Cultured Clam Larvae

AL Diéguez*¹, S Balboa¹, A Labella², D Castro², JJ Borrego² and JL Romalde¹

The aerobic heterotrophic bacteria of the genus Pseudoalteromonas comprise one of the most abundant groups of proteobacteria, widely distributed in the marine environments. Some species of this group are able to synthesize high molecular weight substances with autotoxic and antibiotic effects against Gram-positive and Gram-negative bacteria. Another species, like P. piscicida, shown a pathogenic potential for fish. Due to successive episodes of high mortalities in populations of cultured clams in Galicia (NW Spain), it is important to study the associated microbiota with the aim to detect and characterize potential new pathogens limiting the culture of this bivalve mollusc. In our study, sampling was performed for a period of 2 years in different geographic areas of Galicia. Biochemical and physiological characterization of the obtained isolates allowed the selection of those presenting an aerobic metabolism. Exoenzymatic activities associated to the extracellular products were studied using the API ZYM system, and the cytotoxic activities were assessed in the gilthead seabream cellular line SAF-1. A total of 32 isolates were selected by their activities and they were used in an experimental infection of clam larvae (Venerupis pullastra). Six isolates showed pathogenic potential for clam larvae, three of them belonging presumptively to the genus Pseudoalteromonas. The analysis of the fatty acids (FAME), the phylogenetic study on the basis of the 16S rRNA and gyrB genes, as well as the results of DNA-DNA hybridization experiments, suggest that these isolates could represent three new species of the genus Pseudoalteromonas.

Measurement of Viable Renibacterium salmoninarum Counts by Modified KDM-2 with Culture-spent Medium

KDM-2 is a microbiological growth medium widely used for isolation of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in fish. Use KDM-2 as a growth medium is limited because colonization of R. salmoninarum at low concentrations is difficult. In the present study we modified KDM by supplementation of the culture spent medium of R. salmoninarum in substitution for fetal bovine serum (FBS). No difference was observed in the growth rate of R. salmoninarum at ≥ 10³cells/mL in KDM broth with 1% FBS regardless of supplementation with culture spent media. Growth rate of R. salmoninarum decreased at 10¹ cells/mL of inoculation into KDM with 1% FBS, but it was recovered by supplementation of ≥ 1% culture spent medium into the growth media. The activity of culture spent medium in supporting bacterial growth was stable when treated at 60°C for 30 min and frozen at -20°C for 7 days. Inoculation of ≤ 300 CFU of R. salmoninarum, expected colony counts were obtained on the agar plates containing culture spent medium, while none or less than half of the bacteria was colonized on the agar plates without culture spent medium. Based on these results, we conclude that modified KDM by supplementation of culture spent medium, instead of FBS, is a convenient and inexpensive alternative for isolation of R. salmoninarum, especially at low concentration levels.

Pathogenicity mechanisms and environmental stressors in Streptococcal challenges using Nile tilapia (Oreochromis niloticus)

Diseases caused by streptococci have become a major problem in cultured freshwater, estuarine and marine fish species worldwide. Considerable morbidities and mortalities due to this pathogen have been reported, with estimated losses of $150 million annually. Among the streptococci, Streptococcus agalactiae and S. iniae have shown a broad host range, infecting both terrestrial and aquatic animals. S. iniae is responsible for a form of cellulitis resulting from a fish handling injury in humans, while S. agalactiae is the most common cause of neonatal sepsis. Nevertheless, zoonosis has only been demonstrated on the first case. The present study attempts to reveal the pathocenicity mechanisms underlying single and joint streptococcal infections, as well as the environmental factors involved. Challenge studies with Nile tilapia (Oreochromis niloticus) were carried out using different fish age, stocking densities and rearing conditions (i.e. held individually or in groups). Two S. iniae and two S. agalactiae strains recovered from natural outbreaks were tested at several concentrations and exposure times, using two different procedures to prepare the bacterial inoculums. No successful infection has been achieved so far, suggesting that environmental conditions and stressor agents might play a much more important role than expected in the onset of streptococcal infections. Further experimental challenges need to be performed, combined with in vitro trials, to investigate the major virulence factors in both bacterial species and determine the way they can be influenced.

Study on Manufacture Technology of Original Vibrio culture for Binary Inactivated Bacterin

Vibrio harvi strain SpGY020601 and Vibrio alginolyticus strain EpGS021001 were cultivated by vibrating culture and fermentation culture. The turbidity value and viable count were measured at different culture times. With different addition of formalin, the inactivation efficacy was also determined at different times. Under vibrating culture, the culture reached stationary growth stage after 18h of inoculation, the peak value was achieved within 20-24h, and the attenuation period occurred after 24h of inoculation. Culture of Vibrio could be influenced by the Defoamer GPE-1 if the final concentration of which was more than 0.2mL/L. Fermentation culture was conducted in fermentation vessel volumes of 5L, 50L and 500L. The culture reached stationary growth stage after 10h of culture at 28℃, and the peak value of 2.33×10¹⁰CFU/mL was achieved within the following 12-14h in 500L. Culture of Vibrio was most successful in the original medium under pH 7.5 without more control treatment during the course of fermentation. As the minimal inhibitory concentration of formalin was 0.025% for Vibrio, the suitable original culture could be obtained after inactivation with 0.3% formalin within 24h at 28℃.

A challenge for sustainable rainbow trout production is an increasing use of plant sources in fish feed due to shortage of marine protein and oil sources. Diets with high plant content are known to cause enteritis and injury to the intestine, which will affect the absorption of nutrients, affecting the overall health status and welfare of the fish. The result is a higher risk of disease following exposure to pathogenic microorganisms. The aim of this study was to elucidate how different feed types with varying amounts of marine versus organic plant protein and oil sources affected the survival of rainbow trout in connection with an infection. Enteric redmouth disease caused by Yersinia ruckeri is an economically important disease which causes problems in rainbow trout. Experimental infections (intraperitoneal injection of 150-200 g fish fed the different diets over a two month period) were done and mortalities in the different diet groups were recorded. Two weeks post challenge the mortalities in the different groups were between 50 and 89 %. The mortalities were lowest in the group fed the diet where half of the fish oil was replaced by organic plant oil. Unfortunately, the results were blurred due to a natural infection with Y. ruckeri that had occurred in the diet groups two months prior to the experimental infection (mortalities between 2 and 9 % in the different groups), and the half fish oil half plant oil group had had the highest cumulative mortality percentage in connection with this natural infection.

Multiple Independent Emergences of Biotype 2 Yersinia ruckeri in the United States and Europe

Biotype 2 (BT2) variants of the bacterium Yersinia ruckeri are an emerging disease problem in United States (US) and European salmonid aquaculture. The emergence of this biotype has been associated with an increased frequency of enteric redmouth disease (ERM) outbreaks in previously vaccinated salmonid fish. In this study, we have used a genetic complementation approach to identify the specific natural mutations that cause the concomitant loss of motility and secreted lipase activity (BT2 phenotype) in BT2 strains of Y. ruckeri from the US, the United Kingdom (UK) and mainland Europe. Four independent lineages of BT2 Y. ruckeri were identified each containing a unique mutation in a gene encoding an essential component of the flagellar secretion apparatus. Our results demonstrate the existence of independent mutations leading to the BT2 phenotype thus demonstrating that this phenotype has emerged separately at least four times. In addition, BT2 strains from the UK were shown to have the same mutant allele found in US BT2 strains suggesting a common origin of this BT2 lineage. This differentiation of distinct BT2 lineages is of critical importance for the development and validation of vaccination or other treatment strategies intended for the control of BT2 strains.

Haemocytic Neoplasia in Cultured Mediterranean Mussels (Mytilus galloprovincialis) in Slovene Sea

The Slovene Sea is part of the Gulf of Trieste in the northernmost portion of the Adriatic Sea, where the Mediterranean occurs furthest into the European continent. The sea is shallow with an average depth is only 17 meters with considerable variation in temperature (0^oC to 30^oC), salinity (20‰ to 40‰) and dissolved oxygen (6 mg/l to 11 mg/l O₂). Adult cultured Mediterranean mussels (n = 960; Mytilus galloprovincialis) were collected monthly over a one-year period in the Slovene Sea. Water temperature, dissolved oxygen and salinity were measured at each sampling and the shellfish farm was checked for mortality. One slide per mussel containing digestive gland, gonads and gills was stained with haematoxylin and eosin and microscopically examined for the presence of haemocytic neoplasia of mussels. No mortality occurred during our sampling. The prevalence of haemocytic neoplasia was 1.25%. Pleomorphic and anysocitotic neoplastic cells infiltrated connective tissue singularly, in small foci or diffusely. The number of mitosis was high. Necrosis and multifocal atrophy of digestive tubules were noticed in mussels with diffuse neoplasia whereas severe haemocytic infiltration of connective tissue was seen in mussels with single neoplastic cells. The haemocytic neoplasia was more frequently observed in spring and in autumn when the water temperature was between 11^oC and 20.3^oC, the dissolved oxygen below 7.1 mg/l and the salinity between 26‰ and 39‰.

Hematopoietic Neoplasm in the Redfin Needlefish, Strongylura notata (Beloniformes: Belonidae) from Estuaries of Florida, USA

Y Kiryu*¹, M Bakenhaster¹, J Landsberg¹, M Tyler-Jedlund¹, P Wilson¹ and J Fournie²

Occurring along much of Florida’s coastline, the redfin needlefish exhibits a neoplastic lesion that is most prevalent in the Indian River Lagoon, an area reported for several tumor types in aquatic animals. Here we provide a gross and histological description of the lesion. Affected specimens were collected from two Florida Atlantic and two Gulf of Mexico estuaries over a 12-year period. Grossly, the lesion manifested as a single, large (20 mm x 15 mm to 50 mm x 20 mm), raised (approximately 10 mm high), white, creamy, or pinkish nodule, occurring on the flank, dorsal trunk, base of pectoral fin, or head. We also observed multiple small (< 5 mm diameter) nodules possessing poorly demarcated borders with neighboring tissues on the external jaw surface and at the base of the teeth. Histopathologically, neoplastic cells were found in the skin dermis, underneath the skeletal muscle, and in lacunae of the jaw bone. Neoplastic cells prominently invaded the area between the skeletal muscle and the myosepta. Parenchymal neoplastic cells had the basic characteristic features of atypical lymphocytes with a relatively high nuclear to cytoplasmic ratio. These, presumably fully matured, mononuclear cells tended to be necrotic. A moderate number of cells possessed 2 – 4 nuclei, or megakaryocyte-like structures. Neoplastic cells metastasized to the liver, spleen, and hematopoietic tissues in the kidney. Immuno-histochemical staining with Ki67 and pan Keratin demonstrated that neoplastic cells were mesenchymal origin. Here we tentatively diagnosed the redfin needlefish lesion as a hematopoietic neoplasm, lymphosarcoma, or other type of neoplasms.

Development and Evaluation of Real-time Loop-mediated Isothermal Amplification Assay for Detection of Disease Viruses in Penaeid Shrimp

T Itami*¹, T Mekata², R Sudhakaran¹, T Kono³, T Yoshida¹, Y Suzuki⁴, K Supamattaya⁵ M Inada⁶, S Okugawa⁶, J Nishi⁶, M Yoshimine⁶ and M Sakai¹

A one-step, single tube, real-time accelerated loop-mediated isothermal amplification (real-time LAMP) assay was developed separately to detect major shrimp viral diseases such as white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV), and yellow head virus (YHV). Real-time LAMP method is more sensitive than other conventional PCR, RT-PCR and LAMP methods. The applicability of this assay was validated with plenty of viral samples collected from Japan and Thailand. Highly conserved regions of each viral genome developed separately were used to design the real-time LAMP primers. The real-time LAMP assay reported in this study is simple and rapid, where specific amplification is obtained for WSSV, IHHNV, TSV and YHV in 60 minutes under isothermal conditions at 63°C employing six distinct sequences of the target gene. The quantification of viral load in the infected samples was determined from the standard curve based on their threshold time required for turbidity to occur in the reaction by precipitation of magnesium pyrophosphate. Sensitivity analysis revealed all of these viruses can be detected up to 100 copies of template DNA, rendering it tenfold more sensitive than conventional LAMP assay. This study was supported by Research and Development Program for New Bio-industry Initiatives and Japan Society for the Promotion of Science.

Real-time PCR Detection of Water-borne Pathogens From Commercial Catfish Ponds

MJ Griffin*¹, DJ Wise¹, MJ Mauel¹, LM Pote², AC Camus³, CM Doffitt^2,4, MC Yost⁴, TE Greenway¹ and LH Khoo¹

Real-time PCR assays were developed for pathogens commonly associated with the commercial production of channel catfish. The gram-negative bacterium Edwardsiella ictaluri, the myxozoan parasite Henneguya ictaluri, and the digenetic trematode Bolbophorus damnificus have all been implicated in significant reductions of net returns in commercial operations. Management practices designed to prevent losses from these organisms are contingent upon understanding the population dynamics of these pathogens in the pond environment. Field applications of the H. ictaluri assay have identified concentrations of the organism that correlate to risk of losing fish stocked into the system, especially following an outbreak where significant losses have occurred and water temperatures are permissive for the disease. Similar molecular techniques have been developed for the detection of the infective stage of B. damnificus in pond water. The current monitoring protocols for commercial farms are time consuming and labor intensive. Molecular analysis of pond water provides a low-cost expedient procedure applicable to industry scale sampling programs and will more accurately identify ponds that require treatments. These same molecular techniques have been applied to the detection of E. ictaluri in pond water. At present, the conditions that predispose a pond to experiencing an outbreak in its resident fish population remain unclear and the year-round prevalence of E. ictaluri in the pond environment is unknown. This assay will provide insight into the dynamics of E. ictaluri populations during an outbreak and throughout the year in hopes of providing information that can be used to more effectively manage for this disease.

Haptoglobin as a Candidate Biomarker of Domoic Acid Toxicosis in California Sea Lions

JA Ferrante*^1,2, B Neely¹, E Favre¹, JM Arthur^1,3, FMD Gulland⁴, E Andrews⁴, J Soper⁴ and MG Janech^1,2,3

Domoic acid toxicosis (DAT) has emerged as a health threat to California sea lions (Zalophus californianus) and is now recognized as a major cause of stranding along the west coast of the United States. Markers of DAT are limited and definitive diagnosis is often unattainable until necropsy. We utilized two-dimensional gel electrophoresis to compare plasma proteomes of California sea lions with DAT (n=5) to those without (n=9) following plasma protein normalization using ligand library beads. Proteins were identified using tandem mass spectrometry. A charge form of haptoglobin was 2.7 fold lower in sea lions with DAT than in non-DAT sea lions (p=0.009). Statistical performance measures were calculated to assess this charge form of haptoglobin as a biomarker, and a threshold level was selected based on minimal misclassification. Sensitivity and specificity was 90% and 80% respectively. Positive predictive value (PPV) and negative predictive value (NPV) were 90% and 80% respectively. These measures indicate one charge form of haptoglobin is a relatively good classifier of DAT. On the 2D gel, the identified haptoglobin spot belongs to chain that includes six additional spots, and are presumed to be charge forms of haptoglobin. Statistical performance measures were calculated for the combined seven spot intensities for each sea lion. Combined charge forms yield a sensitivity of 80% and specificity of 100%, with a PPV of 100% and an NPV of 90%. These data suggest haptoglobin is lower in sea lions with DAT and that dominant charge forms should be included as candidate biomarkers for qualification.

High School Classroom Modules to Discern Effects of Environmental Stress Using an Oyster Model

JK Stephens*¹,MJ Koroly^{2, 3}andAS Kane⁴

Laboratory experiences in public high school science classrooms that incorporate current technology and real-world connections to environmental research are often lacking or under-supported. This project describes a collaborative effort between Marion County (Florida, USA) high school teachers, and researchers and education specialists at the University of Florida. Students at North Marion High School are now studying the effect of climate change and other environmental factors by observing water filtration in American oysters, Crassostrea virginica. Water filtration by oysters is modified by a variety of factors including temperature and water quality, and students have an opportunity to utilize microscopy and obtain quantitative data to discern inter-individual differences in oyster filtration and gape behavior based on environmental change. Adjunct exercises include the application of basic microbiology techniques including serial dilution, colony counting, and bacterial growth response to temperature. This project has fostered the development of several education modules for Marion County high school classes. Students begin their research experience with modules that address oyster anatomy, their relevance in estuarine ecosystems, and how to “biologically establish” an aquarium prior to adding biomass. Water quality monitoring activities will help students develop competency in water testing, understanding environmental variables that affect the well-being of aquatic animals, collection of data, and interpretation of results. Students benefit by gaining an better understanding of real-world issues, environmental awareness, improved teamwork and problem solving, and technology utilization. Student research opportunities in the high school classroom, plus interaction with research scientists, will result in graduates better prepared for the rigors of post-secondary education and research opportunities.

The Aquatic Animal Health Program at the University of Florida (UF) is an interdisciplinary program that incorporates aspects of medical, biological, and environmental sciences. This non-traditional academic collaboration is ideally suited for delivery of educational programs that involve distinct units within the university. Goals of our program include education of veterinarians, veterinary medical students, graduate students, and related professionals working or interested in the field of aquatic animal health. Current programs include a Certificate in Aquatic Animal Health which is a 15 credit hour elective program for veterinary students enrolled at the University of Florida. We hope to adapt this program over the next few years so that veterinary students from other institutions would be able to participate. In addition we offer several summer short courses to veterinary and graduate students. These training programs are also available to working professionals and non-UF students for continuing education credit. Recognized examples include Diseases of Warm Water Fish and Sea Vet. Graduate training is available at the MS and PhD level from a number of units on campus including the College of Veterinary Medicine, the Whitney Laboratory for Marine Bioscience, the Program in Fisheries and Aquatic Sciences and the Department of Biology. Graduate programs must be negotiated with individual faculty. Finally, we are in the process of developing a series of on-line distance education classes which will be taught at the undergraduate and/or graduate level. These are available to non-UF students and UF academic credit is provided following successful completion.

In recent years the catfish aquaculture industry in the Mississippi Delta has observed sudden mortalities of apparently healthy market and brooder sized catfish in the spring and fall with no associated infectious etiology. When sera from affected fish were injected into sentinel catfish fingerlings they caused clinical signs of this disease. Because of the apparent toxic etiology and associated lesions of catfish viscera, this disease was given the name visceral toxicosis in catfish (VTC). Using antibody neutralization and endopep mass spectroscopy assays, VTC was shown to be caused by the botulinum neurotoxin serotype-E (BoNT-E). Botulinum neurotoxin causes paralysis by preventing release of acetylcholine at the neuromuscular junction. Because VTC is economically devastating to catfish farmers, our ultimate goal is to evaluate methods to prevent and/or treat VTC. Therefore, in this study we focused on establishing an experimental challenge model using purified BoNT-E. Because BoNT is one of the most potent naturally occurring neurotoxins, working with this toxin required special safety and containment precautions. We ran two median lethal dose trials by intracoelomically injecting catfish fingerlings with gradient dilutions of purchased purified BoNT-E holotoxin activated by trypsin digestion. We found that channel catfish and hybrid catfish LD₅₀ values were approximately 4.8 ng/kg and 4.3 ng/kg, respectively. The result shows that channel catfish and hybrid catfish have similar sensitivities to BoNT-E. Our current research includes development of an immunoassay to detect BoNT-E antibodies in fish and identification of risk factors for VTC outbreaks.

In the last two years, a novel pathological condition was studied in sole (Solea senegalensis) juveniles cultured in farms along the European Atlantic coast. Affected fish present abscess-like nodules in the muscle, liver, kidney, heart, intestine and ovary. Nodules display a chronic inflammatory response surrounding a large core of homogeneous necrotic tissue. A zone with abundant macrophages harbouring spherical plasmodial parasitic organisms is observed in most cases as a peripheral ring between the external inflammatory reaction and the necrotic tissue. The morphology and ultrastructure point toward an amoeba or related organism, although unequivocal diagnostic characters are not present. In order to ascertain the identity of these parasites, DNA was extracted from ethanol-preserved muscle lesions. Several sets of universal and taxon-specific PCR primers targeting the eukaryotic SSU rRNA gene were used to amplify parasitic DNA. A non-metazoan fragment was successfully amplified using primers 18S-EUK581-F and 18S-EUK1134-R. Because this fragment was not amplified from uninfected sole samples, the product was sequenced and internal primers were designed and used in combination with modified universal primers, completing the SSU rDNA sequence. Preliminary characterization of the sequence confirms a parasitic amoeba as the aetiological agent of this pathology. The closest relatives found in Blast searches against the Genbank database are archamoebids such as Endolimax and Mastigamoeba. The relationship of this sole parasite to other amoebiasis from marine fishes is uncertain due to absence of comparative data.

The AquaPathogen X database is a blank database template freely available for recording information on individual isolates of aquatic pathogens. This database can accommodate the nucleotide sequence data generated in molecular epidemiological studies along with the myriad of abiotic and biotic traits associated with isolates of various pathogens (e.g. viruses, parasites, bacteria, etc.) from multiple aquatic animal host species (e.g. fish, shellfish, shrimp, etc.). The AquaPathogen X database can be used in surveillance of emerging aquatic animal diseases, elucidating key risk factors associated with pathogen incursions into new water systems, and facilitate monitoring of aquatic zoonotic pathogens. The AquaPathogen X template database is available for download at <http://wfrc.usgs.gov>. An application of the template database that stores the epidemiological profiles of fish virus isolates, called Fish ViroTrak, has also been developed. Currently, Fish ViroTrak catalogs isolates of three aquatic rhabdoviruses; infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), and spring viremia of carp virus (SVCV). Exported records of IHNV and VHSV isolates from the Fish Virotrak database were also used to create two web-accessible databases, Molecular Epidemiology of Aquatic Pathogens (MEAP) IHNV <http://gis.nacse.org/ihnv>, and MEAP VHSV <http://gis.nacse.org/vhsv>. These web-enabled IHNV and VHSV databases provide on-line access to a reference catalog of fish virus isolates for fish health managers, researchers, and fish producers. The databases provide a baseline of epidemiological data that can be used in studying disease emergence in aquatic wildlife and domestic fish populations of North America.

Decompression as a negative factor in pot fisheries of crabs in the Okhotsk Sea

TV Ryazanova*

Red Paralithodes camtschaticus and blue P. platypus king crabs, followed by the tanner crab Chionoecetes bairdi and the snow crab Ch. opilio, are commercially valuable crab species in the Okhotsk Sea. The damage to populations of crabs caused by pots is minimal in comparison to other fishing gears. The most appreciable injuries in the crabs are broken shells and lost limbs. Nevertheless, lifting of the pots from deep waters can have even more substantial effects on the crabs. The influence of this factor on the population of the red and blue king crabs, tanner crabs and snow crabs from the north-east Okhotsk Sea was studied. Gas bubble disease (gas embolism) and remarkable change of composition of the microflora of the haemolimph were noticed in crabs of all examined species after sink-lifting experiment. Gas bubbles were visible in the gills, heart and muscles of the dissected animals. Histological studies demonstrated that decompression had a negative influence on organs and tissues of crabs. Gas embolism in gill lamellae and their deformations were found in crabs with external pathological features (shell disease and/or others) and in apparently healthy crabs. Aggregation of hemocytes in heart and vacuolization of R-cell cytoplasm in hepatopancreas were also demonstrated. Regardless of the fact that some damages are reversible the part of released crabs died and losses due fisheries are more considerable.

SC Yarnell*¹, JFX Wellehan², Y Qiu^3,4, J Blackburn^3,4, JG Morris^4,5, M Lauzardo^1,4,6, D Ashkin^6,7, JA Johnson^1,4 and AS Kane^4,8

Environmental mycobacteria are associated with colonization and disease in a range of aquatic animals including fish, turtles and mammals. Environmental mycobacteria are also the causative agents of non-tuberculous mycobacteria (NTM) diseases in humans; Mycobacterium avium, M. kansasii, M. xenopi, M. scrofulaceum, and M. marinum are associated with pulmonary disease, bone and soft tissue lesions, and disseminated infections. The incidence of NTM disease in humans caused by environmental mycobacteria has dramatically increased over the past 20 years, with the number of cases exceeding that of tuberculosis cases within the United States. Disseminated infection with Mycobacterium avium complex (MAC) is the most common opportunistic bacterial infection in adults infected with HIV-1 in the developed world, and is second only to AIDS Wasting Syndrome as the most common cause of death in these patients. The incidence of NTM disease is also increasing in otherwise healthy subjects, with the majority associated with environmental sources. The primary route of NTM infection in humans is believed to be via ingestion or inhalation of aerosolized water containing mycobacteria. We hypothesize that the chemistry of different surface waterways influence the number and diversity of mycobacteria, and therefore alters the relative risk for NTM infections. This presentation discusses NTM disease and the application of novel sequence-based qPCR probes to discern mycobacteria at the genus level, as well as MAC, from environmental samples. Use of non-culture based molecular probes is critical since existing methods for NTM identification rely on the ability to culture isolates in the laboratory, and many environmental mycobacteria are not readily cultured or exhibit very slow growth. These efforts are expected to lead to species-specific methods for the rapid and accurate detection of environmental mycobacteria associated with NTM disease directly from clinical and environmental samples.

Several toxicological studies into the effects of aquatic pollutants on the liver of teleost fish exist in literature. The focus on the liver in these studies is predicated on its central nature in the scheme of biotransformation and excretion of xenobiotics following exposure in polluted water bodies. As a consequence of the latter primary role of the liver in these processes it is regarded as a predilective site for the sub lethal effects of xenobiotics on the organism usually detectable at histological level. Hepatic histopathology recorded in livers from feral populations of the brackish water catfish Chrysichthys nigrodigitatus from locations on the Lagos lagoon complex with significant anthropogenic inputs from denizen populations and industries are presented. Liver sections from sixty specimens from two locations on the Lagos lagoon complex (Badagry lagoon: 6°24’N, 2°56’E; and Lagos lagoon: 6°29’N, 3°22’E) were analyzed. Observed pathologies included hydropic degeneration (58%), portal/sinusoidal congestion (33%), hepatic necrosis (26%), hemosiderosis (12%), and foci of cellular alterations (FCA’s). No obvious oncologic features were observed. The presence of the hydropic vacuolation lesion was taken as prelude to the development of neoplasms.

No Observable Adverse Effects Levels (NOAELs) for Fish and Pigs Fed Melamine and Cyanuric Acid

CB Stine*, J Ward, C Gieseker, ER Evans, T Mayer, N Hasbrouck, E Tall, C Nochetto and R Reimschuessel

Following reports of renal failure in cats and dogs in 2007, the practice of adulterating feed ingredients with melamine, a high nitrogen content compound, to boost protein levels was discovered. Laboratory studies have shown that cats, pigs and fish form renal crystals following administration of melamine and the related s-triazine, cyanuric acid. Here we report studies in fish and pigs to determine No Observable Adverse Effects Levels (NOAELs) for melamine-cyanurate crystal formation. Catfish and trout were exposed simultaneously to identical doses of melamine and cyanuric acid at 0.1, 0.5, 1, 2.5, 5, 10 and 20 mg/kg bw/day. The NOAELs in catfish for crystal formation were 10 mg/kg bw (single dose), 2.5 mg/kg bw/day for 4 daily doses and 0.5 mg/kg bw/day for 14 daily doses. The NOAELs in trout for crystal formation were 2.5 mg/kg bw (single dose), 2.5 mg/kg bw/day for 4 daily doses and 0.5 mg/kg bw/day for 14 daily doses. Furthermore, trout produced crystals when cyanuric acid was administered two weeks following the melamine dose (20 mg/kg bw for each compound). Additionally, pigs were used as a mammalian model of renal crystal formation. The NOAELs reported herein support the ‘levels of no concern’ included in FDA risk assessments.

The St. Lucie Estuary, located in Southeast Florida, is threatened by increasing residential and commercial development, urban drainage projects, industry and agriculture. Effects of these anthropogenic changes are also associated with the distribution, quality, and volume of freshwater entering the estuary. This study reports observations of physical deformities from fish collected in the St. Lucie River and adjacent waterways from 2006 to 2008. Thirty-four fish with deformities were digitally photographed and radiographed to generate observational data. Eleven species of fish, representing 7 taxonomic families, were observed; black margate and pinfish were the two most common species. Deformities primarily included missing or deformed dorsal fin spines or pyterygiophores, with or without concave soft tissue defects along the dorsal surface. The prevalence of these anomalies in the field-sampled population in the St. Lucie system was 0.18%. This is the first study to report multiple fish species with similar, dorsal hard structure deformities from a single locale. These deformities may be genetic, developmental, and/or associated with a variety of other etiologies including trauma, parasites, infection or environmental chemical exposure. There is no direct evidence, however, making such a link at this time. A website has been developed to provide details of the study for other investigators and to allow input from complimentary disciplines [http://aquaticpath.epi.ufl.edu/deformities]. Ongoing studies are focusing on histopathology, as well as examination of water and sediment data to explore relationships between deformities within the different fish species and various environmental stress agents.

Fish Health in a Florida Estuarine System in Relation to Canal Discharges and Water Quality

Indicators of environmental quality are essential for assessing change in the support value of an estuary for fish and wildlife and for evaluating the effectiveness of restoration. The prevalence of fish with gross abnormalities may be used as an index of environmental quality. In an analysis of a 12+ year dataset, we examined the prevalence of fish with any externally visible abnormality (ANY) in relation to potential causal factors, namely canal discharge. Fish were sampled weekly by rod and reel from multiple sites within the St. Lucie estuary and inlet. Fish were scored as normal or having one of sixteen externally observed abnormalities. Freshwater discharge and water quality were obtained from the South Florida Water Management District and summarized by different time lags for analysis. We used mixed-effects regression models with species as a random effect to account for species variation. Our results suggest that hydrologic variables affected ANY in the estuary and in the inlet but with different time lags. All water quality variables were significantly related to ANY in the estuary, the inlet or both. In general, higher color, visibility, chlorophyll-a, and salinity were associated with a reduction in ANY, and higher total and volatile suspended solids were associated with an increase in ANY. In general, longer-lagged variables had stronger effects. The suggested beneficial effect of color on fish health may be due to the sequestering of heavy metals such as copper. We conclude that both discharge volumes and water quality affect fish health in the St. Lucie system.

Linking individual to population level responses in the Eastern Mosquitofish (Gambusia holbrooki): Are populations in Floridian watersheds impacted by endocrine disruptors?

Endocrine disrupting chemicals (EDCs) cause abnormal secondary sexual characteristics and decreased reproductive output in wildlife, but a connection between individual endpoints and population health is not well elucidated. The goal of this project is to identify populations of Eastern Mosquitofish (Gambusia holbrooki) in Florida waterways with effects at the individual level and determine if this data can be linked to population effects. The types of pollutants of interest in this study are paper mill effluent, organochlorine pesticides, and dairy farm effluent. Anal fin lengths, gonadosomatic / hepatosomatic indices (GSI/HSI), reproductive stages, brood sizes and embryo weights were used to determine individual reproductive health. Data on sex ratios, size distributions, and the number of females in each reproductive stage indicate population-level effects. Results indicate GSI is a more sensitive indicator of exposure to EDCs than HSI, as GSI values were significantly higher in male fish from all contaminated sites as compared to the respective control sites. One site had females with masculinized anal fins at a ratio that was statistically significant. This site also had a larger proportion of mature males than mature females. Males were significantly larger at two of the sites and females were significantly larger than the control site females at one contaminated site. There were no major differences between the proportions of females in each reproductive stage. This study will serve as a foundation for future research into developing G. holbrooki as a bioindicator organism for EDCs and how population-level effects can be elucidated using individual-level endpoints.

Goiter has long been recognized as a common anomaly observed in captive elasmobranchs. Some species, such as the spotted eagle ray (Aetobatus narinari), seem particularly susceptible. Traditionally, elasmobranch husbandry protocols include dietary supplementation with iodine as a means of preventing development of clinically obvious signs of goiter. In advanced stages of the disease affected animals have a readily observable swelling in the “throat” region, ventral midline and caudal to the mandible. Recent studies have demonstrated that elevated nitrate (>70 mg/L NO3-N) was goitrogenic in juvenile male white spotted bamboo sharks (Chiloscyllium plagiosum ) exposed for 29 days. These concentrations are not unusual in modern aquaria using re-circulating technologies. Further, clinical evidence suggests that ozonation may exacerbate the condition by decreasing available environmental iodide (I-), required for synthesis of thyroid hormone in these species. Ozonation oxidizes iodine, forming iodate (IO3) in lieu of I-. The combination of elevated nitrate, which decreases the physiological ability to import I- into the thyroid gland, and ozonation, which decreases available environmental I-, may be contributing to increases in clinical and subclinical goiter in captive elasmobranchs. Thyroid hormone (T4) analysis may aid in the diagnosis of subclinical disease, but our evidence suggests that at least in the short term (29 days), significant decline in thyroid hormone concentration may not occur. Better methods of detecting this condition early in the course of disease are warranted.

Antimicrobial peptides (AMPs) play significant role in innate immunity of shrimp in the forms of anti-lipopolysaccharide factors (ALFs), crustins, penaeidins and lysozymes. AMPs have broad spectrum of antimicrobial activities such as killing or neutralizing bacteria, fungi, parasites and viruses. Innate immunity is the primary defense mechanism of invertebrates against harmful agents, hence AMPs are crucial for invertebrates to defend themselves from pathogenic microorganisms. ALF genes have been found in different species of shrimp includes Penaeus monodon (PmALF), Fenneropenaeus chinensis, Litopenaeus setiferus, L. vannamei, L. stylirostris and Marsupenaeus japonicus (MjALF1). More than one isoform has been identified in P. monodon, and representation of MjALF1 was up-regulated upon stimulation with LPS in vitro. In the present study, we cloned a new ALF isoform from hemocytes of kuruma shrimp M. japonicus (MjALF2). The full-length MjALF2 gene consists of 558 bp with a 363 bp open reading frame, encoding 121 amino acids, which contains a putative signal peptide of 22 amino acids. The deduced amino acid sequence of MjALF2 showed 83.3% identity with sequences of PmALF2. Phylogenetic analysis revealed MjALF2 had been placed in the group closer to PmALF1 and PmALF2 then to that of MjALF1. Transcriptional analysis revealed that MjALF2 was constitutively expressed in brain, gill, heart, hemocytes, hepatopancreas, intestine, lymphoid organ, muscle, nerve, stomach. This study was supported by Research and Development Program for New Bio-industry Initiatives and Japan society for the promotion of science.

Shrimp is one of the main aquaculture species and is economically important worldwide. However, pathological problems are increasing in the vast majority of the shrimp producing countries. White spot syndrome virus (WSSV), a member of the virus family, Nimaviridae, is the most virulent pathogen in shrimp. Comprehensive genomic information about shrimp, as a host for WSSV, is essential for understanding further shrimp infection mechanism and biodefenses, but is yet to be completed. To gain more information about the shrimp genome, we constructed a bacterial artificial chromosome (BAC) library for kuruma shrimp (Marsupenaeus japonicus). Among the predicted Open Reading Frames (ORFs) in the sequences of the BAC clones, we found that some of these showed significant similarity to the WSSV ORFs, with functions that were still unknown. By full-sequencing one of the BAC clones, 7 ORFs were significantly similar to the WSSV ORFs; by BAC-end-sequencing the BAC library, 8 ORFs were significantly similar to the WSSV ORFs. These predicted ORFs showed 20 % to 30 % of amino acid identities to the WSSV ORFs. A total of 10 of the 15 genes were considered to be within 200kb in the shrimp genome sequences. All mRNAs of the predicted ORFs were detected in various tissues of kuruma shrimp. Two ORFs were detected in all tissues, and 13 ORFs were marginally detected in certain tissues. Following WSSV infection, mRNAs of the low level group were increased, except for 1 ORF.

White Spot Disease Virus (WSDV) is the most serious viral pathogen of kuruma shrimp in the worldwide. It is an important issue to establish a remedy against WSDV as soon as possible. In this study we administered imiquimod and polyI:C which have the ability to produce interferon in vertebrates, and examined the protective effect in kuruma shrimp against WSDV infection. As a result, the protection of kuruma shrimp treated with imiquimod and polyI:C was increased against WSDV. Moreover, we examined the expression of innate immune-related genes in kuruma shrimp after administration of imiquimod and polyI:C. The expression of Rab7, crustin, lysozyme and penaeidin in kuruma shrimp treated with imiquimod and polyI:C was up-regulated. These results suggest that imiquimod and polyI:C can increase the protection against WSDV and the innate immune response in kuruma shrimp. This study was supported by the grant from “Research and Development Program for New Bio-industry Initiatives.”

To date, the subunit vaccines targeting envelope proteins have been studied, and these vaccines show good protection against WSDV. These vaccines have reported effectiveness against WSDV. However, there are few reports on the immune-related genes that are expressed by their treatment.In this study, we developed subunit vaccine encoding VP28 envelope protein in kuruma shrimp (Marsupenaeus japonicus) by using E. coli and wheat germ cell-free protein synthesis. Furthermore, we analyzed the expression of antimicrobial peptide genes, and confirmed its potential of protection against WSDV infection. We injected these two types of vaccine into the abdominal muscle; a week after, we performed WSDV infection. As a result, the vaccination of wheat germ protein synthesis was effective to WSDV (Relative Percent Survival: RPS 93.2%). But the vaccination of E. coli was not effective (RPS 15%). Then, rVP28 protein that was produced by wheat germ cell-free protein synthesis was mixed into the feed for oral vaccination. We fed the rVP28-feed for a week, then WSDV infection experiments were carried out. The result confirmed the effectiveness of the vaccine (RPS 80.6%). In addition, the change in the expression level of antimicrobial peptide genes was observed upon oral vaccination. These results indicate that subunit vaccine will help to increase immune responses in kuruma shrimp and become potential arms to combat WSDV in shrimp. This study was supported by Research and Development Program for New Bio-industry Initiatives.

Penaeid shrimps, like other invertebrates, are thought to possess only an innate immune system. To date, information on their immune system is limited and for the most part from model organisms such as nematode, fruit fly and horseshoe crab. However, recent advances in scientific technology enabled us to analyze the molecular functions of genes in shrimp. Here, using RNA interference (RNAi), we elucidated the function of lysozyme, one of the major antimicrobial peptides which can hydrolyze β-1, 3 bound of peptidoglycan of Gram-positive bacteria. We constructed a lysozyme-specific double stranded RNA (dsRNA-LYZ) and injected it into the shrimp body at 3µg/g body weight. Interestingly, within a week after injection, the lysozyme deficient group suffered 100% mortality and showed an increase in the number of bacteria in the haemolymph without any microbial infection. 16s ribosomal DNA sequence analysis showed that most of the bacteria were identified to be Vibrio sp. Furthermore, to elucidate the relationship between lysozyme and Gram-negative bacteria, we constructed a GFP carrying E. coli and injected it into the shrimp body. Subsequent fluorescent microscopy showed that the number of GFP expressing E. coli in lysozyme-deficent shrimp increased which in contrast, normal shrimp were able to eliminate readily. These results and the previous experiment which revealed the lytic activity against Gram-negative bacteria of recombinant lysozyme, clearly imply that shrimp lysozyme takes an important role in the regulation of Gram-negative bacteria in the haemolymph.

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

In vertebrates, cytokines have been known to regulate hematopoiesis and immune responses. In invertebrates however, information about cytokines or cytokine-like factors concerning the hematopoiesis was very few, although the importance of the hemocytes has been well known in crustacean immune responses. Astakine, the first cytokine-like factor, found in crustacean can induce the shrimp hematopoietic stem cell differentiation. We present here the entire cDNA sequence (1,589 bp) of the kuruma shrimp Marsupenaeus japonicus astakine (Mjastakine), generated using the RT-PCR and 5′- and 3′- rapid amplification PCRs. The open reading frame of Mjastakine encoded a protein of 124 amino acids with an estimated mass of 13.3 kDa. Sequence homology of Mjastakine were 79.8% and 52.4% to these of the tiger shrimp Penaeus monodon and freshwater crayfish Pacifastacus leniusculus. Amino acid sequence and 11 cysteines were highly conserved in prokineticin domain of crustaceans. In healthy shrimp, Mjastakine mRNA was highly expressed in the brain and hemocytes. On the other hand, Mjastakine mRNA was low level expression in hepatopancreas and ovary. When white spot syndrome virus (WSSV) was injected into the kuruma shrimp, Mjastakine expression in the hemocytes reached its peak one day and decreased to its normal level 5 days after the virus injection. This study was supported, in-part, by the Research and Development Program for New Bio-industry Initiatives and by the University of Miyazaki's Program for the Support of Women in the Sciences, and the Japan Society for the Promotion of Science.

Although apoptosis, programmed cell death, is known as a defense mechanism against virus infection in animals, the apoptosis mechanisms of shrimp are not well understood. In mammals, c-Jun N-terminal kinase (JNK) is activated by various stresses, such as ultraviolet (UV), heat shock and osmotic shock, and is known as an essential and key factor for UV induced apoptosis. In this study, we cloned JNK gene from kuruma shrimp Marsupenaeus japonicus (MjJNK) and this is the first report about JNK gene in crustacean. The full length cDNA of MjJNK consists of 2,388 bp with a 94 bp 5’-untranslated region (UTR), a 884 bp 3’-UTR and a 1,410 bp open reading frame (ORF) encoding 458 amino acids. The deduced amino acid sequence of MjJNK showed 87.5 % and 79.2 % identity with sequences of fruit fly (Drosophila melanogaster) JNK and human (Homo sapiens) JNK. MjJNK has S_TKc domain (Serine/Threonine protein kinases, catalytic domain), that has an ATP binding activity. Phylogenetic analysis revealed that MjJNK is included in group of insects. Expression of MjJNK in the healthy kuruma shrimp was confirmed in muscle, hematopoietic tissue, intestine, nerve and lymphoid organ. The highest gene expression was found in a muscle and it was 30-hold higher than that in the ovary. This study was supported, in-part, by Research and Development Program for New Bio-industry Initiatives, the University of Miyazaki’s Program for the Support of Women in the Sciences and Japan Society for the Promotion of Science.

In order to identify the genes induced by different bacterial cells which may contain different types of pathogen associated molecular patterns (PAMPs), high-throughput gene expression analyses using a microarray constructed for Japanese flounder Paralichthys olivaceus were conducted. The mRNA levels of 67 genes were significantly changed in the kidneys of the fish treated with formalin-killed cells (FKC) of four different bacteria, including two Gram-negative, Edwardsiella tarda and Vibrio anguillarum, and two Gram-positive, Lactococcus garvieae and Streptococcus iniae. The genes significantly induced by the treatments included well-known immune-related genes such as granulocyte-colony stimulation factor, haptoglobin and hepcidin. Among the genes studied, the mRNA level of deionase type 3 (dio3) was most significantly induced by the treatments with E. tarda, V. anguillarum and L. garviaea FKCs, but only slightly by S. iniae FKC. It was noted that the changes of the other mRNA levels after S. iniae FKC treatment were also less significant than those of the other FKCs. Dio3 is an enzyme catalyzing tri-iodothyronine (T3) and thyroxine (T4) and producing their inactivated metabolites, 3,3'-diiodothyronine and 3,3',5'-triiodothyronine, respectively. In mammals, the mRNA level of dio3 was increased during bacterial infection, resulting in reduction of serum T3 and T4 levels. A dio3 knockout mouse showed increasing bacterial load in tissues suggesting that it is involved in the clearance of bacteria. Changes in serum T4 levels after FKC treatments are now being evaluated along with further gene expression analyses.

Antibiotic intervention: Timing and the effect on the generation of Edwardsiella ictaluri specific antibody

T Greenway*, M.Griffin, D Wise, T.Byars, and J Walker

The use of medicated feed is a common management practice used to control acute losses associated with enteric septicemia of catfish (ESC). While effective in curtailing mortality, the timing of treatment is critical to the success of therapy. Under commercial fish-farming conditions, it is not uncommon for fish populations to require multiple antibiotic treatments to control mortality. In such instances, disease reoccurrence is usually not associated with the development of bacterial resistance to the antibiotic, suggesting the lack of developing immunity within the infected population of fish. Timeline investigations examining the temporal relationship between antibiotic intervention and the stage of infection were conducted. To simulate the various stages of infection occurring within populations of fish during an ESC epizootic, laboratory fish were fed bactericidal and bacteriostatic antibiotics starting 2 d before (pre-infection), 2 d after (early infection) and 5 d days after (late infection) challenge. Fish were exposed to a virulent field isolate of E. ictaluri under static water flow conditions for 30 minutes followed by the resumption of water flow. The bactericidal drug was shown more effective in treating infection but was shown to interfere with the development of acquired immunity if used prior to infection. Fish fed the bactericidal drug starting 2 days prior infection had 100% survival but were not shown to develop protective immunity against subsequent exposure. Antibody avidity indices served as a measure of antibody maturation and function and indicated early antibiotic intervention can truncate antigen exposure preventing the development of a protective immune response.

We have isolated several temperature tolerant Edwardsiella ictaluri strains (TTS) from diseased fish. Bacterial identification was confirmed by biochemical reactions (API-test strips) and PCR. Unlike other field isolates, bacterial growth was shown to occur at higher temperatures considered non-permissive to the virulent field isolates. We investigated whether prior exposure to these temperature tolerant E. ictaluri strains alter the kinetics and magnitude of anti- E. ictaluri immune responses as well as protection from challenge with the more typical E. ictaluri temperature restricted virulent (TRV) field isolates. Fish were exposed to graded doses of TTS and TRV isolates at concentrations ranging from 5x10⁺⁵ to 7x10⁺⁶ CFU/ml to evaluate virulence and antibody production. Temperature tolerant strains differed markedly in their ability to confer protection as well as induce a lethal infection. Regardless of dose, no fish died following exposure to the TTS isolate. In contrast, a dose response ranging between 6.7 and 100% mortality was generated following exposure to the TRV isolate. Fish subjected to exposure from the TTS mutants exhibited virtually no E. ictaluri specific antibody production (against TTS and TRV antigen preps) as determined by ELISA. Exposure of fish to the typical virulent strain resulted in antibody production reactive to both TTS and TRV antigen preparations. The temperature tolerant strains afforded no protection upon rechallenge with the virulent field isolate. Prior exposure to temperature tolerant strains however did not seem to affect the subsequent generation of specific antibodies upon re-exposure to virulent field strains.

This study investigates the kinetics of antibody responses generated following natural exposure to E. ictaluri in the pond environment. Whether through vaccination or natural exposure to infectious agents, protective efficacies are often associated with antibody production. Infection by E. ictaluri is known to elicit antibody responses and to a certain extent this antibody response provides protection against reinfection. Studies performed in our laboratory have shown a distinct correlation between levels of E. ictaluri specific antibody and susceptibility to enteric septicemia of catfish (ESC). Channel catfish fingerlings were collected weekly from a commercial farm, with initial sampling beginning prior to what is considered the optimal temperature window for E. ictaluri disease outbreaks. Serum samples were analyzed for E. ictaluri specific antibody and antibody avidity assays were used to determine the strength of antibody binding. At various periods, cohorts from the sample collection were brought to the laboratory and challenged with a virulent field isolate of E. ictaluri, with fish from each pond consisting of an E. ictaluri exposed and non-exposed control group. Following challenge, three distinct mortality patterns developed where a majority of the subsampled fish were 1) in the recovery stage of infection and refractive to subsequent infection, 2) immunologically naïve with a low subclinical infection rate and 3) immunologically naïve with high subclinical infection rate. Comparison of challenge mortality to antibody profiles, indicates a strong correlation between sero-positive populations of fish and increased disease resistance.

During the last several years we have developed a number of antigen specific antibody assays to a variety of pathogens affecting catfish. Antibodies generated to a number of bacterial pathogens including Edwardsiella ictaluri, E. tarda, Flavobacterium columnare, Aeromonas hydropllia as well as to certain lifestages of the trematode parasite Bolbophorus sp. and the myxozoan Henneguya ictaluri have been examined. The amount of antibody cross-reactivity to a variety of antigens, in both immunized and specific pathogen free (spf) fish, points to the polyreactive nature of natural antibodies. In other immunization studies, fish were also shown to react to sheep red blood cells, goat IgG, DNA fragments and synthetic short chain peptide fragments. Care must be taken with the interpretation of antibody dilution curves to insure accurate reporting of antigen specific antibody titers (as oppose to an O.D. value). Since polyreactive antibodies are generally considered low affinity antibodies, avidity measurements used in conjunction with antibody titer may be a better indicator of the quality of antigen specific acquired antibody production.

In the first line of host defence against pathogens, Toll-like receptors (TLRs) recognize specific pathogen-associated molecular patterns (PAMPs) triggering innate and acquired immune responses. In mammals, 13 types of TLRs (TLR1~TLR13) have been identified. Interestingly, teleost fish possesses mammalian TLR orthologs as well as fish-specific novel TLRs, however the latter were not identified from mammals (for example TLR5 soluble, TLR14, TLR21 and TLR22). Hence, understanding the TLRs in teleost fish could provide important information about the perceived uniqueness of teleost fish immunity due to genetic diversification. Therefore, we identified a total of 11 types of TLRs (TLR1, TLR2, TLR3, TLR5, TLR5 soluble form, TLR7, TLR8, TLR9, TLR14, TLR21 and TLR22) from the commercially important marine fish species Japanese flounder (Paralichthys olivaceus) using degenerate PCR and Expressed Sequence Tag (ESTs) annotation. All the Japanese flounder TLRs consisted of functionally important structural features such as extracellular leucine rich repeats and intracellular Toll/interleukin receptor domain. Phylogenetic analysis of cloned Japanese flounder TLRs indicated that they were classified with their respective counterparts in other fish and mammals. It is evident that various types of immune-related gene expression and immunity are triggered following TLR ligand stimulation in teleost fish. The loci of Japanese flounder TLR gene family were determined by linkage map. Most of the Japanese flounder TLR genes were expressed in immune-related organs including kidney, spleen and peripheral blood leukocytes.

Recombination activation gene 1 deficient (rag1^-/-) mutant zebrafish lack T cell receptor (TCR) and immunoglobulin (Ig) transcript expression and cannot perform T or B lymphocyte driven responses. However, when re-exposed to the same bacterial pathogen, they mount a response that provides long-term specific protection and adoptive cell transfers demonstrate that there is a cellular component to this response. We utilized RNA based gene expression microarray analyses to determine if innate immune cells that mediate enhanced specific memory, or secondary responses demonstrate signature gene expression profiles that are distinct from innate immune cells following primary exposure. The three following treatment groups were compared to the control (SS), and to each other: 1) primary bacterial (Edwardsiella ictaluri) exposure, secondary bacterial (Edwardsiella ictaluri) exposure, or EE; 2) primary bacterial exposure, secondary sham exposure, or ES (represents a persistent primary response); and 3) primary sham, secondary bacterial exposure or SE (represents a primary response). Common and unique genes among different treatments were identified. Controls were compared with SE, EE and SE using a 1.5 fold change in the gene expression cutoff. Early immune response proteins in Zebrafish were discovered in ES compared with control. Up-regulation (greater than 1.5 fold) of transcripts involved in complement components, iron homeostasis and transport proteins, was observed. When EE was compared with SE, 191 up-regulated and 83 down-regulated transcripts were found that included genes involved in myeloid and lymphoid proliferation and differentiation.

Carp kidney leukocytes co-cultured with supporting cell layer resulted in the rapid proliferation of various types of leukocytes including with immature leukocytes. Expressions of marker genes for multiple blood cell developments were observed in the primary culture. However, after several passages, the proliferating cells expressed only T cell and macrophage marker genes (Katakura et al. Dev. Comp. Immunol., 2009 and 2010). Further RT-PCR analysis revealed that the proliferating cells expressed TCR constant regions (Cα, Cβ, Cγ, Cδ), CD3γ/δ and CD4L-1, but not CD8α and CD8β. In situ hybridization analysis showed that the majority of proliferating cells expressed Cα, Cβ, Cγ, Cδ and CD4L-1. Additionally, 5’-RACE sequences of TCR variable regions (Vα, Vβ, Vγ, Vδ) revealed that the proliferating cells contained polyclonal T cell repertoire, and most of Vα and Vβ sequences were functional, but Vγ and Vδ sequences were non-functional with frame shift and stop codons. Taken together, these results indicated that the proliferating cells after serial passages predominantly contained CD4+ αβT cells which co-expressed non-functional γδTCR at same time. To obtain T cell clones, single cells were derived from the bulk culture, and seeded into 96-well plates. T cell colonies were formed from single cells. These colony cells were expressed Cα, Cβ, Cδ and CD4L-1, and weekly expressed Cγ, but not expressed CD8α, CD8β and CD4L-2. Taken together, these clonal T cells resemble a subpopulation of mammalian helper T cells.

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.

Myxosporeans (Myxozoa) are a speciose group of microscopic, multicellular, spore-forming parasites of aquatic invertebrates and vertebrates. Life cycles are known for some 50 of 2,500 species, and all comprise development of myxospore and actinospore stages in obligate vertebrate and invertebrate hosts, respectively. As part of my PhD work, I characterized myxozoans from wild caught and cage-exposed fish as well as annelid worms. Fish yielded at least 80 myxospore infections from 10 genera: Myxobolus, Myxidium, Henneguya, Chloromyxum, Sphaerospora, Myxobilatus, Ceratomyxa, Parvicapsula, Zschokkella and Unicauda. Oligochaete and polychaete worms had at least 75 actinospore infections from 8 morphological collective groups: Triactinomyxon, Aurantiactinomyxon, Echinactinomyxon, Raabeia, Guyenotia, Antonactinomyxon, Tetractinomyxon and Siedleckiella. I found actinospores with unusual morphologies: hair-like, barb-like, or finger-like protrusions on their caudal processes. I discovered 2 actinospores almost 1 mm across which are the largest known members of Raabeia and Triactinomyxon, which demonstrates that myxosporeans can range across 3 orders of magnitude in size (10-1000 μm). I sequenced and aligned the small subunit ribosomal RNA gene from 82 samples (34 myxospores, 48 actinospores) to identify putative conspecific life stages. Eleven actinospore sequences matched myxospores from my dataset or GenBank, four of which were already known: Myxobolus cerebralis, Ceratomyxa shasta, Parvicapsula minibicornis and Myxidium truttae. Novel life cycles were inferred for Myxobilatus gasterostei, Chloromyxum auratum, Sphaerospora oncorhynchi, the ‘CKX’ organism, Chloromyxum sp. NH02, Myxobolus sp. MWR03 and Myxobolus sp. MMR01. Spore imagery, phylogenetic data and inferred life cycle connections will be presented.

Spironucleus salmonis is a diplomonad flagellate parasite common in the intestinal lumen of young farmed rainbow trout, Oncorhynchus mykiss. In dense infections, the fish weigh less than uninfected fish, and there are increased mortalities. Although we know that transmission occurs via the resistant cyst stage in the water, much of the life cycle remains unknown. During in vitro culture of S. salmonis, we noticed that in one culture media, not only was there transformation of the individual swimming trophozoites to cysts as expected, but also that trophozoites attached to each other via the tip of the two posterior flagella, and subsequently the whole of the posterior flagella, and part of the main cell surface, eventually leading to clusters of cysts. This attachment process was highly organized, and observed in multiple replicate cultures, strongly suggesting that this process was not artifactual. Our fortuitious observation of this stage of S. salmonis raises interesting questions including under what conditions does this occur in nature, and what is the advantage of the clusters of cysts? We suggest that attachment is triggered by environmental conditions that are sub-optimal for the trophozoites, such as death of the host. Clustering of cysts would be an advantage for transmission, since clusters may be buoyant, and help ensure large numbers of parasites are ingested at one time by a fish, likely exceeding minimum infective dose. New treatments could be developed, targeted at inhibition of the flagellar attachment that preceeds formation of clusters of cysts, thus interrupting part of the life cycle.

From 2000 until the present, we have examined 2,569 fish of 51 species for parasites, and recorded their prevalence, using light microscopy techniques. Among the 145 taxa of fish parasites recorded in Romania, 61 cause diseases. In aquarium fish, the most damaging parasites are Ichthyouris bursata and Neocapillaria pterophylli in Symphisodon discus; ichthyophthiriosis, dactylogyrosis and gyrodactylosis in goldfish and koi carp; and Euclinostomum heterostomum in Trichopsis vittatus (imported from Thailand). The most important diseases in carp and trout aquaculture were: ichthyophthiriosis (30%), chilodonellosis (fingerlings, spring, 80%), carp spherosporosis (10%), carp dactylogyrosis (90%), trout and carp gyrodactylosis (90 %), carp khawiosis (spring, 40%), and argulosis and lerneosis (20-30 %). In wild fish, the most significant infections were: clinostomosis in roach, chub and gudgeon (Motru river, 90%), Ancyrocephalus paradoxus in pike-perch (Danube, 95%), Silurodiscoides vistulensis in wells (Danube, 90%), carp and pike-perch diplostomosis (Danube delta, 50%), eustrongylidosis in pike-perch, perch, wells and asp (Danube, 30-50%), posthodiplostomosis in cyprinids (Danube delta, 75%), acanthocephalosis in brown trout (Olt river), chub (Motru river), wels and sterlet (Danube river), and Achtheres percarum in pike-perch (west Danube, 50 %). Some 25 fish parasites are important for food safety: (i) zoonotic species include Clinostomum complanatum (Motru river), Apophallus donicum (Bega, Timis and Danube), Metagonimus yokogawai (Nera river), Anisakis sp., Eustrongylides sp. and plerocercoids of Diphyllobothrium latum; (ii) those affecting appearance include metacercaria of four species in sturgeons, and Argulus sp., and Lernaea sp. To avoid spreading of Gyrodactylus salaris to Atlantic salmon Salmo salar, monitoring of trout and grayling must be obligatory.

The ornamental fish trade constitutes a significant portion of the worldwide trade in aquatic animals. Turkey imports various tropical aquarium fishes from Southeast Asian countries; the imported species are now increasing in scale and number. The potential for transboundary transfer of disease agents through the live fish trade is internationally recognized. Despite legislation restricting and regulating non-indigenous species, the dangerous introduction of previously unknown parasites is occurring. Platies (Xiphophorus maculatus), goldfish (Carassius auratus) and guppies (Poecilia reticulata) imported into Turkey between January 2010 and April 2010 were examined for ectoparasites using routine methods. We found 2 monogeneans (Dactylogyrus sp.; Gyrodactylus sp.), 1 ciliates (Tetrahymena sp.) and 1 trematode (metacercarial cysts of Centrocestus formosanus) from the fish examined. Of these, the metacercarial cysts of Centrocestus formosanus was the most common parasite with a prevalence of 27%. The overall prevalence of Dactylogyrus sp. was 17%, Gyrodactylus sp. 16% and Tetrahymena sp. 13%, however the prevalence varied depending on fish species. It is concluded that ornamental fish pose a significant threat to native host populations in importing countries. Considering that fact, much more emphasis should be given to studies to assess the unidentified hazards and quarantine practices affecting the ornamental fish trade.

In recent years the change in eating habits, involving the consumption of some raw, cold smoked or marinated freshwater fish, has resulted in re-emerging and emerging fish-borne parasitic zoonoses in Italy. In particular, a recrudescence of human Diphyllobothriasis by Diphyllobothrium latum (Cestoda) has been observed in Como Lake area, with over 30 cases diagnosed in the last eight years, and some outbreaks of human Opistorchiasis by Opisthorchis felineus (Trematoda) have been described in central Italy after consumption of marinated lacustrine fish. On the basis of these data, and in order to understand the epidemiology of these parasites, a parasitological survey in fish populations from Italian lakes was undertaken in 2009. Preliminary results of the survey indicate a wide distribution of D. latum plerocercoid larvae in European perch (Perca fluviatilis) from Como Lake with prevalence values >20%, while in Maggiore Lake none of the fish examined were positive for the same parasite. Concerning O. felineus metacercariae in tench (Tinca tinca), almost all of the fish coming from lakes Vico, Bracciano, and Bolsena (Lazio region) were positive, while the parasite was never detected in fish from Trasimeno lake (Umbria region). The recent trends towards consumption of raw perch (“carpaccio of perch”) and marinated tench in the areas under study represent the main risk factor for consuming viable infective stages of D. latum and O. felineus, respectively. Further studies are in progress to assess the additional biotic and abiotic risk factors influencing the epidemiology of these zoonotic helminths in Italy. Research funded by Italian Ministry of University and Research (MIUR), PRIN08.

Metacercariae of Clinostomum spp. (Platyhelminthes: Digenea) parasitize fish and amphibians and mature in piscivorous birds. The taxonomy of this cosmopolitan genus has been repeatedly revised due to a lack of morphological characters that reliably diagnose species. A growing number of studies of digeneans employ molecular data for the identification and discovery of species. Most authors have used sequences from internal transcribed spacer (ITS) regions of nuclear rDNA and/or a fragment of the mitochondrial gene cytochrome oxidase I (COI). In this study, sequences of ITS and the DNA barcode region of COI were obtained from a total of 50 specimens collected from four continents and more than 20 host species, including representatives of Clinostomum complanatum, C. marginatum, C. cutaneum, C. phalacrocoracis and unidentified Clinostomum species. Interspecific divergence in ITS sequences ranged from 0.9% to 10.4% over 459 bp, while divergence in COI ranged from 11.9% to 25.5% over 433 bp. Thus, Clinostomum species can be more easily distinguished by sequences of COI than ITS, although this approach to species identification should include morphological study. Separate phylogenies based on COI and ITS sequences both placed Clinostomum species from the old world in a clade that is basal to and clearly separate from new-world species.

Digenetic trematodes of the genus Bolbophorus are commonly associated with channel catfish (Ictalurus punctatus) aquaculture in the southeastern United States. Bolbophorus damnificus has been implicated in significant losses to the industry, while the effect Bolbophorus type II. sp. has on catfish production is not well understood. Whole pond treatments of copper sulfate pentahydrate (CSP; CuSO_4·5₂O) have been shown to drastically reduce snail populations in catfish ponds at concentrations ranging from 3-5 ppm. Unfortunately, in mid-summer when water temperatures are optimal for release of Bolbophorus cercaria, the phytotoxic effects of CSP at these concentrations can lead to lethal oxygen depletions. In addition, copper toxicity to fish increases with temperature. Therefore, at increased water temperatures copper itself can be toxic to catfish, especially at concentrations required for snail eradication. This called for the development of alternative treatment strategies to reduce the impact of trematode infestations when temperatures are not permissive for CSP applications sufficient to kill snails. Lower concentrations (0.5-3.0 ppm) of CSP were tested for their ability to kill Bolbophorus sp. cercaria. All concentrations were successful in killing cercariae of both species within 24 hours. This suggests that in the incidence of trematode infestations during periods of high water temperatures, CSP could still be applied at low concentrations to reduce cercaria numbers within the pond. By periodically reducing the number of infectious agents within the pond, the incidence of infection is indirectly reduced, thereby improving overall pond production until higher CSP concentrations targeting the snail host can be safely administered.

A duplex real-time polymerase chain reaction (qPCR) assay was developed to simultaneously differentiate between Bolbophorus damnificus and Bolbophorus type II sp. cercariae. Both trematode species are prevalent throughout the commercial catfish industry, as both infect the ram’s horn snail Planorbella trivolvis, which is commonly found in catfish ponds. Identification of cercaria to species is important in catfish disease challenge experiments as only B. damnificus has been shown to have negative impacts on channel catfish. Oligonucleotide primers and fluorescence resonance energy transfer (FRET) hydrolysis probes were designed to amplify the 18S small subunit ribosomal DNA gene of each species. The quantification cycle (C_q) indicative of the number of cercariae in the sample preparation was determined and standard curves correlating to cercaria numbers were established. For both species, the assay was found to be highly repeatable and reproducible, with a linear dynamic range covering 7 orders of magnitude. The sensitivity limit of the assay was ~1/256^th of a cercaria, regardless of species, and there was no remarkable interference between the two assays when run simultaneously within the same reaction. In a field study, identification of cercaria by the duplex qPCR assay was in complete agreement with previously established end-point PCR protocols, demonstrating the assay to be a more rapid, quantifiable means of parasite identification.

The Uruguayan Atlantic coast is characterized by a system of sandy beaches and coastal lagoons. In these ecosystems live a large number of shellfish species which are targets of artisanal fisheries and involve communities of fishermen. In a framework of integrated coastal management and an ecosystem-based management approach, diseases, abnormalities and seafood quality are noted as ecological indicators of ecosystems health. In 2009 for the first time, Aquatic Organisms Pathology and Marine Science laboratories began a survey of parasites and epibionts affecting Uruguayan Atlantic coast shellfish. This assessment provides information about absence/presence of protozoan parasites notifiable to the OIE and of dinoflagellates as a cause of mortalities in commercial decapod crustaceans. Coordinated efforts with DINARA (National Aquatic Resources Institution) have allowed the histopathological examination and hemolymph analysis of blue crabs Callinectes sapidus specifically at the Rocha Lagoon. Other crustacean and molluscan species were sampled; most of them are the target of artisanal fisheries (Farafantepenaeus paulensis, Mytilus edulis) or are likely reservoirs of several pathogens (Ostrea puelchana, Ostreola equestris, Cyrtograpsus angulatus). An interdisciplinary approach characterizes this research integrating a team of biologists, veterinarians, oceanographers, sociologists and anthropologists. Its focus is also to incorporate local community knowledge to enable a holistic view of problems thus promoting the recovery of seafood (certification and traceability) as a process to promote sustainable management of the coast and its resources with the active participation of coastal communities.

NOAA’s International Registry of Coral Pathology (IRCP) serves as a research tool and resource of voucher materials for the coral research community. Diseases of coral have increased significantly in frequency and distribution over the last decade; however, the etiologies of many coral diseases remain uncertain. An important function of the coral registry is to facilitate the sharing of histology materials and related information among coral pathologists worldwide to better understand the causes and mechanisms of disease in corals. More than 2600 specimens of healthy and diseased scleractinian and soft corals, representing over 30 warm water species from 22 geographic locations, have been accessioned resulting in a collection of over 9000 microscope slides. This unique resource allows coral researchers the opportunity to apply new diagnostic techniques to coral disease investigations. Tissues of corals collected during surveys of Florida’s coral reefs during the 1970’s to 1990’s provide a valuable baseline of historical data for emerging disease issues. IRCP’s archives are currently being expanded to include tissues of cold water and deep sea corals. Information gained from the utilization of IRCP products is useful to researchers, students, and managers and provides a better understanding of the causes and mechanisms of coral disease and the measures needed to preserve and protect coral reef ecosystems.

Extensive collections of histopathology materials from studies of marine and freshwater fish, mollusks, crustaceans, echinoderms, and other organisms are archived in the Registry of Tumors in Lower Animals (RTLA), the U.S. Environmental Protection Agency, NOAA’s National Marine Fisheries Service, and other agency or academic institutions. These collections are valuable resources for scientists seeking to understand health and disease in diverse species, train new aquatic pathologists, predict risks from biotic and abiotic stressors (e.g., toxicant impacts on organisms in multiple locations), determine disease status through DNA extraction and analysis, supply data for historical reconstructions (e.g., when a virus first affected a host species), examine trends in parasite distribution and prevalence, and improve interpretation of host and parasite population fluctuations for modeling ecosystems. However, they are in danger. For example, RTLA’s collection now at Experimental Pathology Laboratories, Sterling, VA, formerly funded by the National Cancer Institute, lacks current funding for maintenance or processing of additional case submittals. To ensure future availabilities of these irreplaceable resources, online databases with cross-linking records of materials for search and retrieval — as is being developed for the EPA’s Atlantic Ecology Division's collections — can provide access, but these collections need cross-agency support to improve their database capabilities, maintain histoslides, and provide hands-on examination and study.

Alabama is a biodiversity focus for freshwater mussels (Bivalvia: Unionidae), holding 178 species of 43 genera. Because historical classification schemes for these bivalves have focused almost exclusively on external shell morphology, their internal soft structures remain understudied and poorly documented. As a result, we have little specific information about how unionid tissues appear when normal or when lesioned. To aid ongoing conservation and management efforts on behalf of the Alabama Department of Conservation and Natural Resources as well as to broaden our basic understanding of the general biology of freshwater mussels, we are developing a histological atlas for freshwater mussels based on wild and hatchery-reared specimens of the Alabama rainbow, Villosa nebulosa. A total of 48 specimens of wild V. nebulosa (25–60 mm shell length) were hand sampled by snorkeling the south fork of Terrapin Creek (Coosa River, Cleburne County, AL) during May and August 2010, transported alive to the laboratory in an aerated cooler filled with ambient creek water, opened by cutting the adductor muscles and gently refracting the shells, immersed for 48 hrs in 10% neutral buffered formalin, processed routinely for paraffin embedding, sectioned at 4 mm, and stained using hematoxylin and eosin. In addition, 13 specimens of hatchery-reared V. nebulosa (9–12 mm shell length) were obtained from the Alabama Aquatic Biodiversity Center (Marion, AL) and processed as above. Digital photomicrographs of histological sections comprising mantle, labial palp, gill, gonad, foot, digestive gland, and adductor muscle were analyzed and compiled into a preliminary photographic atlas.

This study used NMR and GC-MS to conduct metabolic studies using flounder serum. Spectroscopic data analysis was carried out to investigate a relationship between flounder serum metabolite profiles and vaccination. The compound for response to vaccination, formalin-killed Edwardsiella tarda, was analyzed by spectral profiling of multivariate analysis. In ¹H-NMR profiling, relative lipid region was distinguished between pre- and post-vaccine treatment. Lipid was extracted from serum and analyzed by GC-MS. Through principal component analysis of GC-MS data, identified compounds were detected and used as an indicator to develop adjuvant for Edwardiella vaccine.

Edwardsiella tarda is known as an intracellular bacterial pathogen. There is annual serious economic loss in Japanese flounder farming due to the outbreak of this bacterium. Hence, development of efficient E. tarda vaccine is an urgent matter. Induction of particular immune responce by live vaccine is believed to be crucial for the protection against intracellular bacterial pathogens. To confirm this, we evaluated the tissue persistence and live vaccine efficacy of five avirulent E tarda strains (E22, SU100, SU117, SU138, and SU244) isolated from the Japanese eel (Anguilla japonica) and from the environment. The live vaccines, containing a single strain, were injected intraperitoneally into Japanese flounder (Paralichthys olivaceus). Viable bacteria from all the strains (excluding SU100) were recovered from trunk-kidney tissue 28 d post-injection. Japanese flounder inoculated with E22 had the highest relative percentage survival (RPS = 45%) in an artificial challenge with virulent E. tarda (NUF806). The serum of E22-vaccinated fish had a significantly higher agglutination titer against NUF806. In contrast, there was little or no increase in the agglutination titer of the fish that were inoculated with the remaining avirulent strains. Injection with avirulent E. tarda increased the expression of cytokine genes, including interleukin-1β (IL-1( β)), type 1 interferon (IFN), and IFN-γ in head-kidney 28 d post-injection. Taken together, production of specific antibody and induction of IFN-mediated immune responses are suggested to be important for protection against E. tarda infection.

In this study, we evaluated survivability of KHV in natural environmental water by using cell lines and fish. KHV infectivity remained for more than 7 days in autoclaved or filtered (0.45μm) environmental water by using cell lines. However, significant reduction in the infectious titer of KHV was observed within 3 days in untreated environmental water. Same results were observed by using fish. The results suggest that in the absence of hosts, KHV can be rapidly inactivated in environmental water. And it is considered that rapid KHV inactivation in natural environmental water is due to bacteria in environmental water. Then, we isolated anti-KHV bacteria from environmental water and intestinal contents of common carp. Twelve of 269 (4.5%) bacterial isolates from environmental water and 14 of the 161 (8.7%) isolates from intestinal contents showed anti-KHV properties in the culture filtrates, respectively. It is considered that these bacteria are playing a major role in KHV inactivation in environmental water. Furthermore, we made a model of biological processing of fish breeding effluent water that used anti-KHV bacteria and preliminarily examined running condition of model. When anti-KHV bacteria was cultured together with starfish bone, approximately 10⁸ CFU/g of anti-KHV bacteria was adsorbed to starfish bone. It is considered that starfish bone is useful to model biological processing of fish breeding effluent water. In future, we’ll examine whether this model is useful for the inactivation of KHV in fish breeding effluent water.

Mold infection is one of the most important problems in aquaculture. In the past decade interest on the topic of antimicrobial plant extracts has been extended. Furthermore, some studies on antibacterial and antifungal activities of essential oils and herbal extracts on aquatic animal have been reported. The aim of the present study was to evaluate acute toxicity of a new antifungal combination of four essential oils (Eucalyptus globulus, Mentha piperita, Salvia officinalis and Thymus vulgaris) were assessed by calculating of LC50 (48,96hours) and compared to malachite green. The composition of the CEO was determined by gas chromatography (GC) and by GC coupled with mass spectrometry (MS). Toxicity tests, LC50 (Lethal Concentration 50) 24, 48, and 96 hours were carried out according to OECD guideline for testing of chemicals (OECD guidline:203,1992). Six aquariums were filled with aired water. For adaptation, fifteen fingerlings of rainbow trout (Mean weight 3± 0.2 grams) were transferred to aquaria for 96 hours. After 24, 48 and 96 hours, the mortality rate was counted and recorded. In this study, calculated LC50 48 and 96 hours of CEO were 34.98 ppm. There was not any difference between LC50, 48, 72 and 96 hours and after 48 hours, mortality rate was fixed. Based on the concentration of LC50, 48 hours which were obtained from this study the combined essential oils is an agent with moderate toxicity but according other reports malachite green is a chemical agent with high toxicity. As results, the combined essential oils can be a substitute of chemical agents for controlling fungal and mold infection diseases in aquaculture.

Bacterial fish pathogens are a worldwide problem in aquaculture and antimicrobial treatments have been applied for many years. Use of antimicrobials, however, has unwanted side effects such as contamination and development of antimicrobial resistance. The prevention and treatment of bacterial diseases by applying natural products from seaweed appears to be a possible alternative. Thirty-seven species of common seaweeds from the coast of Korea have been screened for antimicrobial activity. The seaweed extracts were tested in laboratory assays against Streptococcus iniae, a common bacterial fish pathogen. Growth was inhibited by the methanol extracts of the seaweed of Ecklonia cava, Hizikia fusifome, Chondrus pinnulatus and water-soluble extract of Sargassum thunbergil, Undaria pinnatifida and Enteromorpha prolifera. Streptococcus iniae was especially inhibited by Ecklonia cava methanol extracts. The antimicrobial substance was isolated by silica gel chromatography using ethyl acetate and methanol as the eluent and reverse phase C8 column using HPLC. Chromatography with a linear gradient showed a single peak for the antimicrobial compound with 60% acetonitrile. The chemical constituents possessing antimicrobial activity were characterized using gas chromatography and nuclear magnetic resonance techniques.

The marine white spot, caused by the protozoan Cryptocaryon irritans, is a disease that produces a large number of mortalities every year in marine fish culture. In the present study, we explored the usage of two quinine-based compounds (quinacrine hydrochloride and chloroquine diphosphate), and two peracetic acid-based compounds (Proxitane® 5:23 and 15% peracetic acid [PAA]), against the C. irritans theront stage in a series of in vitro trials. Both quinine-based products have been used before to control infections by Plasmodium spp., the causative agent of malaria in humans. In the current study, quinacrine hydrochloride was the most effective treatment against the C. irritans theront stage, resulting in approximately 90% theront mortality when applied for 1.5 h at 20, 50, or 80 mg/L. Cloroquine diphosphate application for 1.5 hr resulted in 96% theront mortality at 40 mg/L, and 87% and 63% mortality at 10 and 5 mg/L, respectively. PAA-based compounds have been widely used as broad antimicrobial agents. In the current study, Proxitane® 5:23 application for 1 hr produced a moderate effect of 54% theront mortality at 10 mg/L, and greater effects of 84% and 94% mortality at 50 and 250 mg/L, respectively. PAA (15%) application for 1 hr also produced a significant dose-dependent effect, with theront mortality rates ranging from 79 to 94% when applied at 10, 50, and 250 mg/L. These results demonstrated the high potential of the compounds tested to control C. irritans infections; nonetheless, further research is required to optimize their administration in farm systems.

Marine turtle fibropapillomatosis (MTFP) is a neoplastic disease recognized since 1938, and is characterized by external and visceral tumours. In the last decades, an increased prevalence has been observed in different marine turtle species, and due to the extension of external and internal lesions impairing the survival of the affected animals, MTFP is currently considered as an emerging panzootic disease. Although MTFP etiology is still undetermined, the detection of Cheloniidae Fibropapilloma-associated Herpesvirus (CFPHV) genomic DNA is a constant factor in fibropapillomas of affected animals. In order to investigate the involvement of CFPHV sequences in MTFP lesions from green turtle Chelonia mydas of Príncipe Island, Gulf of Guinea, West Africa, genomic DNA was extracted from infected tissues. A 462 bp fragment included in the pol gene of CFPHV was amplified by conventional PCR, cloned and sequenced. A phylogenetic tree was constructed based on the genetic distances between the viral sequences, and three main clusters were observed grouping sequences amplified from different marine turtle species and from distinct geographic locations. Although included in one of the main clusters the African sequences grouped individually in a separate branch supported by high bootstrap values. Our results confirm the association of viral sequences with MTFP lesions and describe for the first time the phylogenetic distribution of CFPHV sequences from West Africa. The understanding of the phylogeographic patterns of new viral sequences will certainly contribute for a better understanding of the virus evolutionary pattern and involvement in the MTFP etiology.

Water temperature is considered to be one of the most important environmental factors affecting outbreaks of white spot disease (WSD), the most serious disease for shrimp farming worldwide. Here, we investigated the effect of water temperature on the progress of WSD and evaluated the effect of low temperature on viral infection and replication of WSSV, the causative agent of WSD in kuruma shrimp (Marsupenaeus japonicus). Shrimp were maintained at 4 different temperatures 15^oC, 20^oC, 25^oC and 33^oC for 24h before challenge with WSSV. Shrimp kept at 25^oC displayed the earliest and highest mortality pattern, culminating with 100% cumulative mortality at 6 d.p.c. Lower temperatures (15^oC, 20^oC) reduced mortalities and about 80% of shrimp maintained at 15^oC survived until the end of the experiment. High mortalities were also observed in challenged-shrimp maintained at 33^oC, but a similar observation was detected for control group injected with PBS and maintained at 33^oC. To evaluate the effect of low temperature (15^oC), shrimp were maintained at 25^oC before challenge and 15^oC afterwards, or maintained at 15^oC before challenge and 25^oC afterwards. The delayed and reduced mortalities were observed when shrimp was transferred from 25^oC to 15^oC compared to shrimp held at 25^oC before and after challenge. In contrast, increased mortalities were determined in shrimp shifted to 25^oC when compared to mortalities of shrimp continuously held at 15^oC. These results showed that WSSV infection is temperature dependent and further suggested that low temperature (15^oC) reduced rather than stopped viral replication.

Infectious hematopoietic necrosis virus (IHNV) is a pathogen that infects many species of salmonids. Especially susceptible to this pathogen are sockeye salmon Oncorhyncus nerka, chinook salmon O. tschawytscha, and rainbow trout Oncorhyncus mykiss. However, the results of virological examinations of over 20,000 wild and cultured salmonids (Oncorhyncus nerka, O. tschawytscha, O. keta, and O. kisutch) from Kamchatka during 1996-2010, revealed IHNV only in sockeye salmon populations. We have done two experimental inoculations of fingerling chinook salmon with IHNV isolated from adult Kamchatka sockeye, to determine their sensitivity to the virus. There were experimental and control groups of 40 fish in every experiment, and inoculations were performed by standard bath method. None of the fish died, nor did we see any signs of pathology, in either group of chinook 30 days post-inoculation. IHNV was not detected in the fish, demonstrating that Kamchatka chinook is not sensitive to IHNV. When we repeated this experiment under the same conditions with sockeye and coho fingerlings, the fish showed signs of pathology and died, and the virus was re-isolated from them. Genetic typing of Russian virus isolates by sequence analysis of partial glycoprotein and nucleocapsid genes showed that they were genetically homologous to each other, and to common isolates from the USA and fell within the U genogroup of IHNV. Thus Kamchatka and American stocks of chinook salmon most likely have a large genetic diversity, and it would be very interesting to investigate their immune status. Such work may lead to the creation of a more effective vaccine against IHNV.

Spring viremia of carp (SVC) which has not yet been reported in Japan is one of the notorious diseases being predicted to have a lasting negative impact on both the carp and gold fish industries. The distribution of the virus that causes SVC has not been sufficiently examined. The present study aims at determining which organ is the target for initial viral multiplication prior to systemic infection. Nineteen fancy carp were infected with SVCV isolate 980548 by immersion and 13 fish were sampled at day 1 post-infection (dpi) and 3 fish were sampled at 3 and 22 dpi, respectively. The skin, gill, intestine, blood, kidney and brain were excised from the sampled fish and the viral titer in each tissue was estimated by cell culture. At 1 dpi, the virus was detected from 12 fish where the titers in the skin ranged from 2.3 to 5.6 Log TCID₅₀/100 mg tissue. The other tissues of those fish had no detectable virus except for the gills of six fish and the blood of one fish where titers of 1.9 to 2.3 were detected. The viral titer of the skin was higher than that of gill in each fish. At 3 and 22 dpi titers of 2.8 to 6.13 were recorded in the most tissues except the brain where the virus was undetectable in six fish. The present study indicates that the skin is the major target organ for initial viral multiplication in SVC.

This poster emphasizes the impact our research has had on emergency regulations to control viral hemorrhagic septicemia virus (VHSV) IVb outbreaks and possible spread, as well as disease surveillance policies. VHSV has recently become established in the Great Lakes. Fish kills caused by VHSV have alarmed the public and provoked government attention to pathogen introductions into US waters. The seasonality of VHSV expression has been documented. VHSV is present in fish in the greatest numbers in the spring during the months of March – May. The development of a quantitative reverse transcriptase – polymerase chain reaction assay (qRT-PCR) for VHSV has provided a diagnostic and research tool that is more rapid and sensitive than the current cell culture technique. The qRT-PCR serves as an initial negative screen for surveillance and research samples. Major surveillance efforts in 2008-2009 demonstrated that pathogen distribution information could be developed quickly and is clearly needed for aquatic ecosystem conservation, management of affected populations, and informed regulation of the trade in aquatic animals. We have determined the relative susceptibility of various fish species to infection by this virus. These fish include species of importance to aquaculture and sport fishery management. We are testing whether VHSV is transmitted vertically from parent to progeny as an external contaminant on the outside of the egg or as an internal infection within the egg. Efficacy of iodophore egg treatments is also being examined during walleye egg exposure to VHSV.

The Guangdong Evergreen Group Company was established in 1998, to provide healthy feed and fingerlings to fish farmers in the rapidly growing aquaculture community in southern China. After ten years of development, Evergreen Group has successfully established an integrated industrial chain providing fingerlings and juveniles (fish and shrimp) for stocking, feed production, aquaculture demonstration, aquatic product processing, and domestic and international trade to provide safe, nutritious and healthy food for customers. Evergreen Group has built large-scale training facilities to provide outreach on breading techniques, providing technical information, and pathogen examination of both broodstocks and juveniles. The 863 Research Center (Ocean Seedling Breeding Project - Southern Base of Evergreen Group), is ranked top for its scale and equipment in China due to the great and persistent support from the government, from within the company, and scientific collaboration with Sun Yat-Set University, Ocean University of China and Guang Dong Ocean University. The system for healthy juvenile and fingerlings has been established, including high quality broodstock, water treatment, pathogen-free food and reasonable management. Guangdong Evergreen Group is also one of the biggest produce processing and export enterprises in China; it has acquired ISO9001 and will obtain 2000 International Quality System certificate, American HACCP certificate, and EU certificate.

The United States marine mammal stranding network responded to over 37,000 stranded marine mammals from 2001-2007 representing 10,090 cetaceans and 27,620 pinnipeds (excluding walrus). The U.S. stranding network consists of 117 stranding network organizations and other federal, state and local government agencies. Over this time period, 32 rehabilitation facilities have rehabilitated and released 41 cetaceans and 6,807 pinnipeds. Since 1992, the National Marine Fisheries Service’s Marine Mammal Health and Stranding Program (MMHSRP) has provided coordination, standards, and oversight of the national stranding network. The MMHSRP has closely worked with the stranding network to develop performance requirements for stranding organizations and best practices for rehabilitation facilities and release determinations following rehabilitation. Over the past several years, the demand to determine the risk and impact of human activity on marine mammal populations has dramatically increased. As a result, the expectations of the stranding network are changing to include a wide range of skill sets to effectively respond and investigate stranding events. For example, more stranding network participants are seeking training in advance necropsy techniques to identify signs of human interactions (vessel collision, fishery entanglement, acoustic trauma, etc). Since 2001, the MMHSRP has provided approximately $33 million in Prescott grants to the national stranding network; however, this has not fully funded the stranding network and there is still a critical need for more support. This presentation will review U.S. stranding data from the past two decades by geographical distribution and discuss the challenges that the U.S. stranding network is facing as new concerns arise with climate change, increased human utilization of the marine environment and changing demographics. The emphasis will be on how to better prepare the U.S. stranding network to face the new challenges through increased coordination and capacity.

¹	Faculdade de Medicina Veterinária de Lisboa, Universidade Técnica de Lisboa. Rua da Universidade Tècnica, Alto da Ajuda, 1300-477 Lisboa, Portugal. (MSc student). lauracns@gmail.com
²	Instituto Nacional de Recursos Biológicos / INRB,I.P.-LNIV, Estrada de Benfica 701, 1549-011 Lisboa, Portugal. teresa.albuquerque@lniv.min-Agricultura.pt
³	Faculdade de Medicina Veterinária, Universidade Lusófona de Humanidades Tecnologias Campo Grande 376, 1749 - 024 Lisboa, Portugal. mbraganca@gmail.com
^⁴	Oceanário de Lisboa, Esplanada D. Carlos I, 1990-005 Lisboa, Portugal. nbaylina@oceanario.pt
⁵	Oceanário de Lisboa, Esplanada D. Carlos I, 1990-005 Lisboa, Portugal. npereira@oceanario.pt

¹	USGS Western Fisheries Research Center, eemmenegger@usgs.gov, tmthompson@usgs.gov, gkurath@usgs.gov
²	Oasis Group, Shoreline, WA evan@portagebay.com
³	NW Alliance for Computational Science & Engineering, pittams@nacse.org, ryanadam@nacse.org, keon@nacse.org
⁴	USGS National Biological Information Infrastructure Program, Denver, CO jcarlino@usgs.gov
⁵	Department of Microbiology, Immunology & Pathology, Colorado State University, CO ryan.troyer@colostate.edu
⁶	Fisheries and Oceans Pacific Biological Station, Nanaimo, BC, kyle.garver@dfo-mpo.gc.ca
⁷	School of Aquatic and Fisheries Sciences, University of Washington, WA, rbjmax@u.washington.edu

¹	Interdisciplinary Program in Biomedical Sciences, University of Florida, Gainesville, FL
²	Department of Small Animal Clinical Sciences, University of Florida, Gainesville, FL
³	Department of Geography, College of Liberal Arts and Sciences, University of Florida, Gainesville, FL
⁴	Emerging Pathogens Institute, University of Florida, Gainesville, FL
⁵	Department of Epidemiology and Health Policy Research, University of Florida, Gainesville, FL
⁶	Southeast National Tuberculosis Center, University of Florida, Gainesville, FL
⁷	AG Holly State Tuberculosis Hospital, and Florida Department of Health, Lantana, FL
⁸	Department of Environmental & Global Health, University of Florida, Gainesville, FL

¹	Aquatic Pathobiology Laboratory, Department of Aquaculture, Universidad Católica del Norte, Coquimbo, Chile cdmirand@ucn.cl
²	Centro de Estudios Avanzados (CEAZA), Coquimbo, Chile

¹	Department of Biology, University of Oporto, Portugal jasousa@fc.up.pt
²	Interdisciplinary Centre for Marine and Environmental Research (CIIMAR), Portugal
³	A. Coelho e Castro Lda., Praça Luís de Camões, Portugal acoelhocastro@mail.telepac.pt

¹	USGS, Leetown Science Center, National Fish Health Research Laboratory, diwanowicz@usgs.gov, cottinger@usgs.gov
²	USGS, Columbia Environmental Research Center, Columbia, MO, USA, kechols@usgs.gov
³	USGS, Oregon Water Science Center, Portland, OR, USA tmwood@usgs.gov
⁴	USGS, Klamath Falls Field Station, Klamath Falls, OR, USA 97603 scott_vanderkooi@usgs.gov
⁵	USGS, Florida Integrated Science Center, Orlando, Florida, brosen@usgs.gov

¹	College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USA dgibbs@cvm.msstate.edu
²	Thad Cochran National Warmwater Aquaculture Center, College of Veterinary Medicine, Mississippi State University, Stoneville, MS, USA mauel@cvm.msstate.edu
³	Thad Cochran National Warmwater Aquaculture Center, College of Veterinary Medicine, Mississippi State University, Stoneville, MS, USA griffin@cvm.msstate.edu
⁴	College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USA lawrence@cvm.mstate.edu

¹	College of Veterinary medicine, Mississippi State University, MS, USA
²	MSA Genomics Laboratory, USDA-ARS-CGRU, Stoneville, MS, USA

¹	Departamento de Microbiología y Parasitología. CIBUS-Facultad de Biología. Universidad de Santiago de Compostela. Spain.
²	Departamento de Microbiología. Facultad de Ciencias. Universidad de Málaga. Spain.

¹	Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan yosimizu@fish.hokudai.ac.jp
²	Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Kamiura, Japan matsui-takanori@pref.oita.lg.jp
³	Department of Aqualife Medicine, Chonnam National University, Yeousu, Korea jjnishi@chonnam.ac.kr
⁴	Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan, hisae@fish.hokudai.ac.jp
⁵	Nagano Prefectural Fisheries Experimental Station, Akashina, Japan kohara-masakazu@pref.nagano.jp

¹	Institute of Aquaculture, Pathfood Building, University of Stirling, Stirling, UK meritxell.diezpadrisa@stir.ac.uk
²	Institute of Aquaculture, Pathfood Building, University of Stirling, Stirling, UK mc3@stir.ac.uk

¹	National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, US Department of Agriculture, Kearneysville, West Virginia
²	Cefas Weymouth laboratory, Weymouth, Dorset, UK

¹	Institute of Pathology, Forensic and Administrative Veterinary Medicine, Veterinary Faculty University of Ljubljana, Slovenia mitja.gombac@vf.uni-lj.si
²	National Veterinary Institute, Unite Nova Gorica, Veterinary Faculty University of Ljubljana, Slovenia rosvita.sitar@vf.uni-lj.si
³	Institute for Breeding and Health Care of Game, Fish and Bees, Veterinary Faculty University of Ljubljana, Slovenia vlasta.jencic@vf.uni-lj.si

¹	Department of Medicine, Medical University of South Carolina, Charleston, SC, USA neely@musc.edu, favre@musc.edu, arthurj@musc.edu, janechmg@musc.edu
²	Grice Marine Laboratory, College of Charleston, Charleston, SC, USA JasonAFerrante@gmail.com
³	Research Service, Ralph H. Johnson VA Medical Center, Charleston, SC, USA
⁴	The Marine Mammal Center, Sausalito, CA, USA Gullandf@TMMC.org,soperj@tmmc.org

¹	XRAq (Generalitat de Catalunya). Universitat Autònoma de Barcelona, Barcelona, Spain maria.constenla@uab.es, francesc.padros@uab.es
²	Instituto de Acuicultura de Torre la Sal (CSIC). Castellón, Spain oswaldo@iats.csic.es

¹	Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, St. Petersburg, Florida, USA
²	US EPA, Gulf Ecology Division, Gulf Breeze, Florida, USA

¹	Faculty of Agriculture, University of Miyazaki, Japan itamit@cc.miyazaki-u.ac.jp
²	National Research Institute of Aquaculture, Fisheries Research Agency, Japan mekata@affrc.go.jp
³	Interdisciplinary Research Organization, University of Miyazaki, Japan tkono@cc.miyazaki-u.ac.jp
⁴	Faculty of Engineering, University of Miyazaki, Japan suzuki@civil.miyazaki-u.ac.jp
⁵	Faculty of Natural Resources, Prince of Songkla University, Thailand
⁶	Graduate School of Agriculture, University of Miyazaki, Japan

¹	Thad Cochran National Warmwater Aquaculture Center, Mississippi State University, MS
²	Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, MS
³	Department of Pathology, College of Veterinary Medicine, University of Georgia, Georgia
⁴	Department of Biological Sciences, Mississippi State University, MS

¹	North Marion High School, Citra, Florida USA jill.stephens@marion.k12.fl.us
²	Center for Precollegiate Education & Training, University of Florida, Gainesville, FL USA korolymj@cpet.ufl.edu
³	Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL USA
⁴	Aquatic Pathobiology Laboratory, University of Florida, Gainesville, FL USA kane@ufl.edu

¹	Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL
²	Program in Fisheries and Aquatic Sciences, University of Florida, Gainesville, FL
³	Tropical Aquaculture Laboratory, Program in Fisheries and Aquatic Sciences, University of Florida, Ruskin, FL

¹	Department of Basic Science, College of Veterinary Medicine, Mississippi State University.
²	Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University

¹	Department of Large Animal Clinical Sciences, College of Veterinary Medicine, P.O. Box 100136, Gainesville, FL
²	Program in Fisheries and Aquatic Sciences, University of Florida, Gainesville, FL
³	Department of Biology, University of Florida, Gainesville, FL

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

¹	Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Konan 4-5-7, Minato, 108-8477, Tokyo, Japan hirono@kaiyodai.ac.jp
²	Department of Biological Sciences, University of Santo Tomas, Espana, Manila, 1008 Philippines marybethmaningas@yahoo.com

¹	School of Chemistry and Molecular Biosciences, The University of Queensland, QLD 4072, Australia
²	Department of Microbiology, Oregon State University, Corvallis, Oregon, USA

POSTER SESSION ABSTRACTS

¹	Department of Molecular and Comparative Pathology, Johns Hopkins University School of Medicine, Room 807, Broadway Research Building, USA spoynton@jhmi.edu
²	Leibniz Institute for Freshwater Ecology and Inland Fisheries, Müggelseedamm 310, Berlin D 12587, Germany (former address)

¹	Department of Veterinary Public Health & Animal Pathology, Faculty of Veterinary Medicine, Alma Mater Studiorum, University of Bologna, Ozzano Emilia (BO) Italy monica.caffara@unibo.it
²	State Veterinary Institute of Piemonte, Liguria e Valle d’Aosta, Torino, Italy

¹	Department of Veterinary Public Health & Animal Pathology, Faculty of Veterinary Medicine, University of Bologna, Ozzano Emilia (BO) Italy monica.caffara@unibo.it
²	St. Lawrence Centre, Environment Canada, Montréal, Canada

¹	Área Acuicultura y Patología de Organismos Acuáticos, Instituto de Investigaciones Pesqueras, Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay.
²	Área Ciencias del Mar, Instituto de Investigaciones Pesqueras, Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay.
³	Centro de Manejo Costero Integrado del Cono Sur, Universidad de la República, Montevideo, Uruguay.

¹	NOAA National Ocean Service, National Centers for Coastal Ocean Science, Center for Coastal Environmental Health and Biomolecular Research, Cooperative Oxford Laboratory, 904 S. Morris Street, Oxford, MD USA shawn.mclaughlin@noaa.gov, molly.billmyre@noaa.gov, dorothy.howard@noaa.gov
²	Maryland Department of Natural Resources, Fisheries Service, 904 S. Morris Street, Oxford, MD USA styler@dnr.state.md.us

¹	U.S. EPA, NHEERL, Atlantic Ecology Division, Narragansett, RI USA borsay.dodi@epa.gov
²	Dept of Environmental Science and Policy, George Mason University, Fairfax, VA USA epeters2@gmu.edu
³	State of Connecticut, Department of Agriculture, Bureau of Aquaculture, Milford, CT USA
⁴	Experimental Pathology Laboratories, Sterling, VA USA JWolf@epl-inc.com

¹	Department of Fisheries and Allied Aquacultures (FAA), Aquatic Parasitology Laboratory, Auburn University, Auburn, azm0034@auburn.edu
²	FAA, Aquatic Microbiology Laboratory, crarias@auburn.edu

¹	Department of Marine Biotechnology, Soon Chun Hyang University, Korea , jycho@sch.ac.kr
²	Pathology division, National Fisheries Research and Development institute, Korea mskim@nfrdi.go.kr

¹	Graduate school of Fisheries Sciences, Hokkaido University, Hakodate, Japan nyosida@fish.hokudai.ac.jp
²	Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan hisae@fish.hokudai.ac.jp
³	Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan yosimizu@fish.hokudai.ac.jp

¹	Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran, zargarm@ut.ac.ir
²	Department of Fisheries, Faculty of Marine Natural Resources, Khoramshahr Marine Sciences and Technology University, Khoramshahr, Iran
³	Department of Horticulture, Faculty of Agriculture, University of Tarbiat Modarres, Tehran, Iran
⁴	Department of Food Control & Hygiene, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran