OBJECTIVE:Piscine lactococcosis associated with Lactococcus petauri is a serious emerging threat to fish populations in the Americas. This bacterial disease commonly presents as a hemorrhagic septicemia, resulting in high mortality rates and substantial financial losses. There are no commercial vaccines in the United States, and treatment options are limited and understudied. Florfenicol (Aquaflor) is a broad-spectrum antibiotic approved for finfish aquaculture, and erythromycin (Aquamycin 100) is an investigational new animal drug. This study aimed to compare the effectiveness of florfenicol- and erythromycin-medicated feed against lactococcosis through cohabitation challenges in Rainbow Trout Oncorhynchus mykiss. METHODS:Shedder fish were intracoelomically injected with L. petauri (∼1.5 × 104 CFU) and introduced to naïve populations at 13°C or 18°C. Treatments were initiated immediately after observation of mortality, with fish receiving florfenicol at 15 mg/kg for 10 d, erythromycin at 100 mg/kg for 21 d, or the control diet. RESULTS:At 18°C, cohabitant survival was significantly higher in florfenicol-treated (100%) and erythromycin-treated (93%) tanks compared to untreated positive controls (60%). There were no mortalities at 13°C or in the negative control tanks. In sampled survivors, L. petauri was detected by quantitative PCR in 29% of positive control fish, 14% of erythromycin-treated fish, and 14% of florfenicol-treated fish at 18°C compared to 21, 7, and 0%, respectively, at 13°C. Culturable bacteria were only recovered from 14% of positive control fish at both temperatures. CONCLUSIONS:These results indicate that early intervention with florfenicol or erythromycin can limit the spread of L. petauri and that lower water temperatures reduce disease onset, improving options for managing lactococcosis in aquaculture.

ABSTRACT Piscine lactococcosis is a significant threat to cultured and wild fish populations worldwide. The disease typically presents as a per-acute to acute hemorrhagic septicemia causing high morbidity and mortality, recalcitrant to antimicrobial treatment or management interventions. Historically, the disease was attributed to the gram-positive pathogen Lactococcus garvieae . However, recent work has revealed three distinct lactococcosis-causing bacteria (LCB)— L. garvieae, L. petauri, and L. formosensis— which are phenotypically and genetically similar, leading to widespread misidentification. An update on our understanding of lactococcosis and improved methods for identification are urgently needed. To this end, we used representative isolates from each of the three LCB species to compare currently available and recently developed molecular and phenotypic typing assays, including whole-genome sequencing (WGS), end-point and quantitative PCR (qPCR) assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), API 20 Strep and Biolog systems, fatty acid methyl ester analysis (FAME), and Sensititre antimicrobial profiling. Apart from WGS, sequencing of the gyrB gene was the only method capable of consistent and accurate identification to the species and strain level. A qPCR assay based on a putative glycosyltransferase gene was also able to distinguish L. petauri from L. garvieae/formosensis . Biochemical tests and MALDI-TOF MS showed some species-specific patterns in sugar and fatty acid metabolism or protein profiles but should be complemented by additional analyses. The LCB demonstrated overlap in host and geographic range, but there were relevant differences in host specificity, regional prevalence, and antimicrobial susceptibility impacting disease treatment and prevention. IMPORTANCE Lactococcosis affects a broad range of host species, including fish from cold, temperate, and warm freshwater or marine environments, as well as several terrestrial animals, including humans. As such, lactococcosis is a disease of concern for animal and ecosystem health. The disease is endemic in European and Asian aquaculture but is rapidly encroaching on ecologically and economically important fish populations across the Americas. Piscine lactococcosis is difficult to manage, with issues of vaccine escape, ineffective antimicrobial treatment, and the development of carrier fish or biofilms leading to recurrent outbreaks. Our understanding of the disease is also widely outdated. The accepted etiologic agent of lactococcosis is Lactococcus garvieae . However, historical misidentification has masked contributions from two additional species, L. petauri and L. formosensis , which are indistinguishable from L. garvieae by common diagnostic methods. This work is the first comprehensive characterization of all three agents and provides direct recommendations for species-specific diagnosis and management.

Lactococcus petauri is an important emergent aquaculture pathogen in the USA. To better understand environmental conditions conducive to piscine lactococcosis and the susceptibility of fish species, laboratory-controlled challenges were used as models of infection. Rainbow trout Oncorhynchus mykiss maintained at 13 or 18°C were challenged by intracoelomic (ICe) injection with 10 1 , 10 3 or 10 5 colony-forming units per fish (CFU fish -1 ) and monitored for 21 d. At 13°C, trout experienced mortalities of 7, 7 and 0%, and bacterial persistence of 0, 20 and 0% in survivors, respectively. When exposed to the same bacterial doses, trout maintained at 18°C experienced mortalities of 59, 84 and 91%, and bacterial persistence of 60, 66 and 0% in survivors, confirming a significant role of temperature in the pathogenesis of lactococcosis. Additionally, the susceptibility of rainbow trout, Chinook salmon Oncorhynchus tshawytscha , white sturgeon Acipenser transmontanus , Nile tilapia Oreochromis niloticus , and koi Cyprinus carpio to infection by L. petauri was compared using ICe challenges at 18°C. Trout and salmon experienced 96 and 56% cumulative mortality, respectively, and 17% of surviving salmon remained persistently infected. There were no mortalities in the other fish species, and no culturable bacteria recovered at the end of the challenge. However, when surviving fish were used in further cohabitation trials, naïve trout housed with previously exposed tilapia exhibited 6% mortality, demonstrating that non-salmonids can become sub-clinical carriers of this pathogen. The data obtained provide useful information regarding temperature-associated virulence, fish species susceptibility, and potential carrier transmission of L. petauri that can be used in the development of better management practices to protect against piscine lactococcosis.

OBJECTIVE:The first objective of the study aimed to detect the presence of Lactococcus petauri, L. garvieae, and L. formosensis in fish (n = 359) and environmental (n = 161) samples from four lakes near an affected fish farm in California during an outbreak in 2020. The second objective was to compare the virulence of the Lactococcus spp. in Rainbow Trout Oncorhynchus mykiss and Largemouth Bass Micropterus salmoides. METHODS:Standard bacterial culture methods were used to isolate Lactococcus spp. from brain and posterior kidney of sampled fish from the four lakes. Quantitative PCR (qPCR) was utilized to detect Lactococcus spp. DNA in fish tissues and environmental samples from the four lakes. Laboratory controlled challenges were conducted by injecting fish intracoelomically with representative isolates of L. petauri (n = 17), L. garvieae (n = 2), or L. formosensis (n = 4), and monitored for 14 days postchallenge (dpc). RESULT:Lactococcus garvieae was isolated from the brains of two Largemouth Bass in one of the lakes. Lactococcus spp. were detected in 14 fish (8 Bluegills Lepomis macrochirus and 6 Largemouth Bass) from 3 out of the 4 lakes using a qPCR assay. Of the collected environmental samples, all 4 lakes tested positive for Lactococcus spp. in the soil samples, while 2 of the 4 lakes tested positive in the water samples through qPCR. Challenged Largemouth Bass did not show any signs of infection postinjection throughout the challenge period. Rainbow Trout infected with L. petauri showed clinical signs within 3 dpc and presented a significantly higher cumulative mortality (62.4%; p < 0.0001) at 14 dpc when compared to L. garvieae (0%) and L. formosensis (7.5%) treatments. CONCLUSION:The study suggests that qPCR can be used for environmental DNA monitoring of Lactococcus spp. and demonstrates virulence diversity between the etiological agents of piscine lactococcosis.

Lactococcus petauri is an important emergent bacterial pathogen of salmonids in the USA. The purpose of this study was to evaluate the protection conferred to rainbow trout (Oncorhynchus mykiss) against L. petauri by formalin-killed vaccines in immersion and injectable forms, as well as the enhanced protection afforded by booster vaccination. In the first challenge, fish were immunized via intracoelomic injection (IC) or immersion (Imm) routes alone. Approximately 418 degree days (Temperature in degree Celsius × days post-immunization) (dd) Imm, or 622 dd IC post-vaccination, fish were challenged via IC with wild-type L. petauri. In the second experiment, initial Imm vaccination was followed by booster vaccination via Imm or IC routes 273 dd post-immunization along with appropriate PBS controls. The various vaccination protocol efficacies were evaluated by challenging fish with L. petauri by cohabitation with diseased fish 399 dd post-booster administration. A relative percent survival (RPS) of 89.5% and 28% was recorded in the IC and Imm single immunization treatments, respectively. In the second study, an RPS of 97.5%, 10.2%, 2.6% and −10.1% plus approximately 0%, 50%, 20%, and 30% bacterial persistence was recorded in the Imm immunized + IC boosted, Imm immunized + mock IC boosted, Imm immunized + Imm boosted, and Imm immunized + mock Imm boosted treatments, respectively. Only the Imm immunized + IC injection boosted treatments provided significant protection when compared to unvaccinated and challenged treatments (p < 0.05). In conclusion, although both Imm and IC vaccines appear safe for trout, the inactivated Imm vaccines seem to provide only mild and temporary protection against lactococcosis; whereas IC immunized trout develop a significantly stronger protective response in both challenges.

Piscine lactococcosis is an emergent bacterial disease that is associated with high economic losses in many farmed and wild aquatic species worldwide. Early and accurate detection of the causative agent of piscine lactococcosis is essential for management of the disease in fish farms. In this study, a TaqMan quantitative polymerase chain reaction (qPCR) targeting the 16S-23S rRNA internal transcribed spacer region was developed and validated. Validation of the qPCR was performed with DNA of previously typed L. petauri and L. garvieae recovered from different aquatic hosts from distinct geographical locations, closely related bacterial species and common pathogens in trout aquaculture. Further diagnostic sensitivity and specificity was investigated by screening of fish, water and faecal samples. The developed qPCR assay showed high specificity, sensitivity and accuracy in detection of L. petauri and L. garvieae with lack of signals from non-target pathogens, and in screening of rainbow trout (Oncorhynchus mykiss) posterior kidney and environmental samples. The detection limit of the qPCR was four amplicon copies. Moreover, the sensitivity of the qPCR assay was not affected by presence of non-target DNA from either fish or environmental samples. The robustness, specificity and sensitivity of the developed qPCR will facilitate fast and accurate diagnosis of piscine lactococcosis to establish appropriate control measures in fish farms and aquaria.

Lactococcus garvieae is an emergent bacterial pathogen of salmonid fish in North America that causes acute infections particularly at water temperatures above 15°C. During 2020, L. garvieae was detected in rainbow trout, Onchorhyncus mykiss, cultured in Southern California and the Eastern Sierras. Infected fish exhibited high mortalities and nonspecific clinical signs of lethargy, erratic swimming, dark skin pigmentation, and exophthalmia. Macroscopic changes included external and internal hemorrhages, mainly in the eyes, liver, coelomic fat, intestine, and brain. Histological examination revealed splenitis, branchitis, panophthalmitis, hepatitis, enteritis, and coelomitis, with variable degrees of tissue damage among evaluated fish. Pure colonies of L. garvieae were isolated from infected trout and specific PCR primers for L. garvieae confirmed the preliminary diagnosis. Multilocus sequence analysis showed that the strains recovered from diseased trout represent a novel genetic group. Isolates were able to form biofilms within 24 h that increased their resistance to disinfection by hydrogen peroxide. Laboratory challenge methods for inducing lactococcosis in steelhead trout, O. mykiss, were evaluated by intracoelomic injection with serial dilutions of L. garvieae. The median lethal dose 21 days post challenge was ∼20 colony-forming units/fish. Experimentally infected trout presented similar clinical signs, gross changes, and microscopic lesions as those with natural disease, fulfilling Koch's postulates and demonstrating the high virulence of the recovered strains.

Flavobacterium columnare is the causative agent of columnaris disease. Previous work has demonstrated a high degree of genetic variability among F. columnare isolates, identifying 4 genetic groups (GGs) with some host associations. Herein, a total of 49 F. columnare isolates were characterized, the majority of which were collected from 15 different locations throughout the US Pacific Northwest. Most isolates were collected from 2015-2018 and originated from disease outbreaks in salmonid hatcheries and rearing ponds, sturgeon hatcheries and ornamental fish. Other isolates were part of collections recovered from 1980-2018. Initial identification was confirmed by F. columnare species-specific qPCR. Study isolates were further characterized using a multiplex PCR that differentiates between the 4 currently recognized F. columnare GGs. Multiplex PCR results were supported by repetitive sequence-mediated PCR fingerprinting and gyrB sequence analysis. F. columnare GG1 was the most prevalent (83.7%, n = 41/49), represented by isolates from salmonids (n = 32), white sturgeon (n = 2), channel catfish (n = 1), ornamental goldfish (n = 1), koi (n = 3), wild sunfish (n = 1) and 1 unknown host. Six isolates (12.2%, n = 6/49) were identified as GG3, which were cultured from rainbow trout (n = 3) and steelhead trout (n = 3). Two isolates were identified as GG2 (4.1%, n = 2/49) and were from ornamental fish. No GG4 isolates were cultured in this study. The biological significance of this genetic variability remains unclear, but this variation could have significant implications for fish health management. The results from this study provide baseline data for future work developing strategies to ameliorate columnaris-related losses in the US Pacific Northwest.

The Gram-negative bacterium, Flavobacterium psychrophilum, is endemic to California, USA, where it is an important pathogen in salmonid aquaculture, especially in rainbow trout (Oncorhynchus mykiss). Disease outbreaks caused by F. psychrophilum in rainbow trout fingerlings can approach 90% mortality, resulting in millions of dollars of economic losses annually. The focus of this study was to investigate the genetic diversity of 49 F. psychrophilum isolates collected from disease outbreaks in 17 salmonid hatcheries in California, USA, from 2015 to 2018 using multilocus sequence typing. Results suggest California F. psychrophilum isolates are diverse, representing 11 distinct sequence types (STs), three of which were previously undescribed. Still, the majority of genotyped isolates (n = 41) belonged to a single clonal complex (CC), CC-ST10, which is the largest CC worldwide and has been linked to disease outbreaks on several continents. Results of this study provide evidence of marked intraspecific genetic diversity of F. psychrophilum from California. The biological significance of this genetic variability is unclear but could have implications for future vaccine development and treatments. Further studies investigating the virulence, antigenic, and antimicrobial susceptibility profiles of F. psychrophilum are warranted to better understand the epizootiology of this pathogen in the Western United States.

hinook Salmon (Oncorhynchus tshawytscha) are increasingly vulnerable to anthropogenic activities and climate change, especially at their most southern range in California’s Central Valley. There is considerable interest in understanding stressors that contribute to population decline and in identifying management actions that reduce the effects of those stressors. Along the west coast of North America, disease has been linked to declining numbers of salmonids, and identified as a key stressor that results in mortality. In the Central Valley, targeted studies have revealed extremely high prevalence of infectious agents and disease. However, there has been insufficient monitoring to understand the effect that disease may have on salmon populations. To inform future research, monitoring, and management efforts, a two-day workshop on salmon disease was held at the University of California, Davis (UC Davis) on March 14-15, 2018. This paper summarizes the science presented at this workshop, including the current state of knowledge of salmonid disease in the Central Valley, and current and emerging tools to better understand its effects on salmon. We highlight case studies from other systems where successful monitoring programs have been implemented. First, in the Klamath River where the integration of several data-collection and modeling approaches led to the development of successful management actions, and second in British Columbia where investment in researching novel technologies led to breakthroughs in the understanding of salmon disease dynamics. Finally, we identify key information and knowledge gaps necessary to guide research and management of disease in Central Valley salmon populations.

Chinook salmon are a keystone fish species of great ecological and commercial significance in their native northern Pacific range and in regions to which they have been introduced. Threats to salmon populations include habitat degradation, climate change, and infectious agents, including viruses. While the first isolation of a flavivirus from wild migrating salmon may indicate an emerging disease threat, characterization of the genome provides insights into the ecology and long evolutionary history of this important group of viruses affecting humans and other animals and into an expanding group of recently discovered aquatic flaviviruses.

Event Abstract Back to Event Spatio-temporal evaluation of Flavobacterium psychrophilum genotypes recovered from salmonid hatcheries in California, US Uzonna C. Uba1*, Beatriz Martínez-López2, Fernanda Sebastiao2, Thomas P. Loch3, Christopher Knupp3, Kaveramma Mukkatira4, Tresa Veek4, Christine Richey4, Mark Adkison4, Matt J. Griffin5 and Esteban Soto2* 1 University of California, Davis, United States 2 Department of Medicine & Epidemiology, School of Veterinary Medicine, University of California, Davis, United States 3 Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, United States 4 California Department of Fish and Wildlife, United States 5 Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, United States INTRODUCTION: Flavobacterium psychrophilum (1), is a gram-negative, filamentous, bacterium belonging to the family Flavobacteriaceae, and a major bacterial pathogen of cultured Salmonid aquaculture worldwide. It is the etiologic agent of Bacterial Cold-Water Disease (BCWD) and rainbow trout fry syndrome (RTFS) in salmonids, causing substantial economic losses (2). Juvenile fish are mostly affected, typically resulting high mortalities. Recent studies suggest that several genotypes of Flavobacterium psychrophilum can be found in California. However, the spatio-temporal distribution of this pathogen is unknown. This study investigated spatio-temporal distribution of different Flavobacterium psychrophilum genotypes recovered from outbreaks in cultured salmonids in 2015-2019. METHOD: First, we evaluated descriptively the spatio-temporal evolution of Flavobacterium psychrophilum isolates that were collected from Bacteria cold water disease outbreaks from 17 Salmon fish hatcheries spread across California from 2015-2019. Then, we used multiple correspondence analysis (MCA) to analyze the association between the different F. psychrophilum clonal complexes (CC) with water temperature as well as other spatio-temporal dependent features such as month and year, hatchery and fish species. RESULTS: Fifty-one Flavobacterium psychrophilum were recovered from BCWD outbreaks from 17 Salmon fish hatcheries in California from 2015-2019. The highest number of cases were concentrated in spring 2018, summer 2017 and summer 2018. When grouped by number of individual cases, CC 10 (which consists of sequence types 10,78, 85, 319) had the highest frequency of 30 (58.82%). When grouped by hatchery, CC10 had the highest number of occurrences of 21, CC296 and CC310 had only one occurrence. Sequence type 85 was present in 21 out of the 51 isolates. Clonal complex 10 occurred between temperature (5ºC to 18.8ºC) mean 12.79ºC, CC296 occurred at temperature 8ºC; CC310 occurred at 11.6ºC. The Clonal complex Singleton occurred at 8.8ºC-15.5 ºC mean 12.74ºC. CC296 was associated with a temperature lower than 10ºC, while CC10 had the highest temperature range inferring increased adaptation capability. ST85 had the widest temperature range 5ºC to 18.8ºC with a mean of 12.61ºC. ST 333 had a temperature range (8.8ºC to 15ºC). Darrah spring hatchery had the highest temperature range (5ºC -13.8ºC) with ST85 and CC10 present. While American River Hatchery had a temperature range of 8ºC - 16ºC but had ST296 and CC296 present in one of its cases at 8 ºC. CONCLUSION: The spatial analysis of BCWD in California confirms the genetic diversity of F. psychrophilum in North America. It further revealed that CC 10 and Sequence type 85 was the major driver of the outbreak. The results suggest that Flavobacterium psychrophilum outbreaks present different spatio-temporal patterns due to the different environmental conditions (e.g. water temperatures, hatchery management practices, etc.) and host species. Future studies need to further evaluate the different tropism to species and temperature variations. Spatio-temporal analysis of genetically diverse bacteria has been shown to be a valuable tool to understand disease dynamics that can be useful to develop better health management practices for this important disease in Salmonid aquaculture. Acknowledgements We acknowledge all who contributed to the success of this project. References 1. Bernardet, J.-F., Segers, P., Vancanneyt, M., Berthe, F., Kersters, K., Van damme, P., 1996. Cutting a Gordian knot: emended classification and description of the genus Flavobacterium, emended description of the family Flavobacteriaceae, and proposal of Flavobacterium hydatis nom nov. (basonym, Cytophaga aquatilis Strohl and Tait 1978). Int. J. Syst. Evol. Microbiol. 46, 128–148. 2. Dalsgaard I. (1993) Virulence mechanisms in Cytophaga psychrophila and other Cytophaga-like bacteria pathogenic for fish. Annual Review of Fish Diseases 3, 127–144. 3. Nematollahi, A., Decostere, A., Pasmans, F., Haesebrouck, F., 2003. Flavobacterium psychrophilum infections in salmonid fish. J. Fish Dis. 26,563–574. 4. Kulldorff M. (2001) Prospective time-periodic geographical disease surveillance using a scan statistic. J R Stat Soc A Stat Soc. ; 164:61–72 5. Rebecca J. E., Lars Eisen 2008. Spatial modelling of Human Risk of Exposure to Vector-borne pathogens Based on Epidemiological Versus Athropod Vector Data. J. Med. Entomol. 45(2): 181-192 6. Wakabayashi H, Horinouchi M, Bunya T, Hoshiai G. 1991. Outbreaks of cold-water disease in coho salmon in Japan. Fish Pathol. 26:211–212. http://dx.doi.org/10.3147/jsfp.26.211 Keywords: Flavobacterium psychrophilum, Spatio-Temporal Analysis, Genotype, cultured salmon aquaculture, water temperature Conference: GeoVet 2019. Novel spatio-temporal approaches in the era of Big Data, Davis, United States, 8 Oct - 10 Oct, 2019. Presentation Type: Student Poster-no session Topic: Spatio-temporal phylogenetic approaches, phylogeography and phylodynamics Citation: Uba UC, Martínez-López B, Sebastiao F, Loch TP, Knupp C, Mukkatira K, Veek T, Richey C, Adkison M, Griffin MJ and Soto E (2019). Spatio-temporal evaluation of Flavobacterium psychrophilum genotypes recovered from salmonid hatcheries in California, US. Front. Vet. Sci. Conference Abstract: GeoVet 2019. Novel spatio-temporal approaches in the era of Big Data. doi: 10.3389/conf.fvets.2019.05.00103 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 22 Jun 2019; Published Online: 27 Sep 2019. * Correspondence: Dr. Uzonna C Uba, University of California, Davis, Davis, United States, ucuba@ucdavis.edu Dr. Esteban Soto, Department of Medicine & Epidemiology, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616, United States, sotomartinez@ucdavis.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Uzonna C Uba Beatriz Martínez-López Fernanda Sebastiao Thomas P Loch Christopher Knupp Kaveramma Mukkatira Tresa Veek Christine Richey Mark Adkison Matt J Griffin Esteban Soto Google Uzonna C Uba Beatriz Martínez-López Fernanda Sebastiao Thomas P Loch Christopher Knupp Kaveramma Mukkatira Tresa Veek Christine Richey Mark Adkison Matt J Griffin Esteban Soto Google Scholar Uzonna C Uba Beatriz Martínez-López Fernanda Sebastiao Thomas P Loch Christopher Knupp Kaveramma Mukkatira Tresa Veek Christine Richey Mark Adkison Matt J Griffin Esteban Soto PubMed Uzonna C Uba Beatriz Martínez-López Fernanda Sebastiao Thomas P Loch Christopher Knupp Kaveramma Mukkatira Tresa Veek Christine Richey Mark Adkison Matt J Griffin Esteban Soto Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Myxobolus cerebralis is a microscopic metazoan parasite (Phylum Myxozoa: Myxosporea) associated with salmonid whirling disease. There are currently no vaccines to minimise the serious negative economical and ecological impacts of whirling disease among populations of salmonid fish worldwide. UV irradiation has been shown to effectively inactivate the waterborne infective stages or triactinomyxons of M. cerbralis in experimental and hatchery settings but the mechanisms by which the parasite is compromised are unknown. Treatments of triactinomyxons with UV irradiation at doses from 10 to 80mJ/cm2 either prevented (20–80mJ/cm2) or significantly inhibited (10mJ/cm2) completion of the parasite life cycle in experimentally exposed juvenile rainbow trout (Oncorhynchus mykiss). However, even the highest doses of UV irradiation examined (80mJ/cm2) did not prevent key steps in the initiation of parasite infection, including attachment and penetration of the epidermis of juvenile rainbow trout as demonstrated by scanning electron and light microscopy. Furthermore, replication of UV-treated parasites within the first 24h following invasion of the caudal fin was suggested by the detection of concentrations of parasite DNA by quantitative PCR comparable to that among fish exposed to an equal concentration of untreated triactinomyxons. Subsequent development of parasites treated with an 80mJ/cm2 dose of UV irradiation however, was impaired as demonstrated by the decline and then lack of detection of parasite DNA; a trend beginning at 10days and continuing thereafter until the end of the study at 46days post parasite exposure. Treatments of triactinomyxons with a lower dose of UV irradiation (20mJ/cm2) resulted in a more prolonged survival with parasite DNA detected, although at very low concentrations, in fish up to 49days post parasite exposure. The successful invasion but only short-term survival of parasites treated with UV in rainbow trout resulted in a protective response to challenges with fully infective triactinomyxons. Prior treatments of juvenile rainbow trout with UV-treated triactinomyxons (10 and 20mJ/cm2) resulted in a reduced prevalence of infection and significantly lower concentrations of cranial myxospores (two direct measures of the severity of whirling disease) compared with trout receiving no prior treatments when assessed 5months post parasite exposure to fully infective triactinomyxons.

Significant differences in cytokine transcription were found between Oncorhynchus mykiss euthanized using the pharmacological agents MS-222 v. benzocaine and also when contrasting death induced by carbon dioxide asphyxiation v. physical methods (cervical dislocation). This study highlights the need to consider the potentially confounding effect of euthanization method on gene expression data.

White seabass Atractoscion nobilis surviving experimental exposure to Piscirickettsia salmonis harbored the bacterium for periods up to at least 123 d post injection (dpi). Intraperitoneal injections of juvenile white seabass with 1.26 x 10(2) TCID50 P. salmonis fish(-1) resulted in a 29% cumulative mortality over a 27 d period. Both molecular and histologic methods provided evidence for persistence of the bacterium in fish sampled sequentially from the surviving population. Throughout the period of acute mortality, the bacterium was detected in all impression smears of liver tissue stained with Giemsa and was reisolated in cell cultures from all dead fish sampled. Polymerase chain reaction (PCR) assays detected P. salmonis-specific DNA in 13.3 to 50% of the fish sampled at time points between 28 and 123 dpi, while cell culture reisolation was largely ineffective in detecting the bacterium. An enzyme-linked immunosorbent assay (ELISA) detected serum anti-P. salmonis antibodies in 48 of 59 white seabass exposed to P. salmonis but not in fish which were not exposed to the bacterium. At the end of the 4 mo experiment, microscopic lesions consisting of single to multiple and coalescing granulomas were found in liver and kidney tissues of 9 of 10 fish examined from the exposure group, while no lesions were detected in the fish from the control group. Immunohistochemical staining with anti-P. salmonis polyclonal antibodies detected bacterial antigens in some but not all granulomas examined from the exposure group at 4 mo. This study demonstrates that P. salmonis may persist among white seabass following infection, and thus provide a potential reservoir of infection contributing to transmission both within and between fish species in the marine environment.

Pathogen-free rainbow trout (Oncorhynchus mykiss) aged 735 degree days were experimentally exposed to a low dose of infectious Myxobolus cerebralis (20 triactinomyxons fish(-1)). Three time periods were chosen for sampling that included 10 days (d), 67 d, and 5 months (mo) post exposure. Five diagnostic assays were used: (1) conventional single-round polymerase chain reaction (PCR), (2) nested PCR, (3) real-time TaqMan PCR, (4) pepsin-trypsin digest, and (5) histopathology. M. cerebralis was detected among individual rainbow trout by all of the PCR diagnostic tests employed at each of the three sampling time points. This result demonstrates that any of these three diagnostic approaches are capable of detecting the parasite from infected fish tissues under the conditions tested. Real-time PCR provided good biological evidence that parasite replication increases temporally as shown by quantification values that were significantly different (P < 0.0001) at 10 d as compared to 67 d and 5 mo postexposure. Although sampling at 10 d by real-time PCR may be too early to accurately predict quantities of the parasite that will be present at 5 mo, it does forecast the proportions of fish that are likely to be infected at 67 d and 5 mo postparasite exposure. Real-time PCR could potentially be used as a quantitative diagnostic PCR tool to predict parasite load and outcome of M. cerebralis infection.

In September of 1998 our laboratory was asked to investigate epidemics characterized by high mortality occurring in populations of koi (Cyprinus carpio) in both the U.S.A. and Israel. Either live fish or frozen tissues arrived from both locations in September and November of 1998. In addition, tissues fixed for both light and electron microscopy from fish in Israel and the U.S.A. were obtained for examination. The virus observed and then isolated in 1998 from these initial samples has become the focus of a worldwide concern for the health and welfare of both captive and wild populations of common carp and ornamental koi. In this short report we review the initial isolation and characterization of the herpes-like virus referred to as koi herpesvirus or KHV. We also discuss the important role of temperature on in vivo and in vitro infections with the virus, recent comparisons of KHV to other herpes- like viruses from fish and lastly review current methods to detect the virus or evidence that fish have been exposed to the virus.

Rainbow trout are highly susceptible to whirling disease, whereas brown trout and coho salmon have been shown to be considerably more resistant. Field and laboratory studies suggest that some strains of cutthroat trout and the German Hofer strain of rainbow trout are more resistant to whirling disease than North American strains of rainbow trout. We have incorporated these species into an in-depth temporal histopathology study and are comparing their responses to the disease. A qualitative analysis of the data from the initial phases of the infection suggests that brown trout resistance may be due to different mechanisms than that in coho salmon and cutthroat trout. The use of the 18S rDNA gene as a genetic marker has greatly enhanced the identification of M. cerebralis. The presence of the 18S rDNA gene, as identified by the polymerase chain reaction (PCR) has allowed the rapid, sensitive and specific identification of all developmental stages of the parasite within the oligochaete and fish host. PCR technology has recently been improved with the development of real-time TaqMan PCR technology, which allows relative and absolute quantification by measuring the PCR product accumulation through a dual-labeled fluorogenic probe. In this study we used real-time quantitative TaqMan PCR technology to track the rate of replication of the parasite in the resistant and susceptible species over the first two months of infection, as it traveled through the 3 major sites of replication, the skin, the nerves and the cartilage, in the resistant species and compared it to the rate of replication in the susceptible domestic rainbow trout. We