Experimental Infection of Australian Freshwater Fish with Epizootic Haematopoietic Necrosis Virus (EHNV)

Abstract

The ranavirus, epizootic hematopoietic necrosis virus (EHNV), is endemic to southern Australia with natural outbreaks resulting in mass mortality events in wild Redfin Perch Perca fluviatilis (also known as Eurasian Perch) and less severe disease in farmed Rainbow Trout Oncorhynchus mykiss. To further investigate the host range for EHNV, 12 ecologically or economically important freshwater fish species from southeastern Australia were exposed experimentally to the virus. A bath-challenge model at 18 ± 3°C was employed with limited use of intraperitoneal inoculation to determine if a species was likely to be susceptible to EHNV. Of the species tested, Murray–Darling Rainbowfish Melanotaenia fluviatilis and Dewfish Tandanus tandanus (also known as Freshwater Catfish) were considered to be potentially susceptible species. EHNV was isolated from approximately 7% of surviving Eastern Mosquitofish Gambusia holbrooki, indicating this widespread alien fish species is a potential carrier. The infection of Silver Perch Bidyanus bidyanus and Macquarie Perch Macquaria australasica and the lack of infection in Murray Cod Maccullochella peelii peelii and Golden Perch Macquaria ambigua ambigua after exposure to EHNV via water confirmed earlier data from Langdon (1989). Five other species of native fish were potentially not susceptible to the virus or the fish were able to recover during the standard 35-d postchallenge observation period. Overall, it appeared that EHNV was less virulent in the present experimental model than in previous studies, but the reasons for this were not identified.

Received May 21, 2012; accepted November 1, 2012     

Susceptibility of Australian Redfin Perch Perca fluviatilis Experimentally Challenged with Epizootic Hematopoietic Necrosis Virus (EHNV)

The ranavirus epizootic hematopoietic necrosis virus (EHNV) is endemic to Australia and is listed by the Office International des Epizooties. Clinical outbreaks have only been observed in wild populations of Redfin Perch Perca fluviatilis (also known as Eurasian Perch) and farmed populations of Rainbow Trout Oncorhynchus mykiss. The initial outbreaks of EHNV describe all age-classes of Redfin Perch as being susceptible and can lead to epidemic fish kills. Subsequently, experimental challenge studies using either cohabitation with the virus or injection exposures resulted in mixed susceptibilities across various age-groupings of Redfin Perch. We used an experimental bath challenge model to investigate the susceptibility of Redfin Perch collected from areas with and without a history of EHNV outbreaks. The median survival time for fish from Blowering Dam in New South Wales, a zone with a history of EHNV outbreaks, was 35 d, compared with fish from other areas, which had a median survival between 12 and 28 d postexposure. Redfin Perch from Blowering Dam demonstrated an increased mortality associated with epizootic hematopoietic necrosis up to approximately day 14 after exposure, and then there was a significantly reduced risk of mortality until the end of the trial compared with all other fish. Redfin Perch from Blowering Dam had markedly decreased susceptibility to EHNV, and less than 40% became infected following a bath challenge. In contrast, Redfin Perch from neighboring (e.g., Bethungra Dam and Tarcutta Creek) and distant water bodies (e.g., in Western Australia) with no previous history of EHNVdisplayed moderate to high susceptibility when given a bath challenge. Potential factors for the observed changes in the host–pathogen relationship include intense positive selection pressure for resistant fish following epizootic hematopoietic necrosis outbreaks and subsequent attenuation of the virulence of the virus in resistant fish.

Received August 22, 2015; accepted February 13, 2016     

Influence of environmental temperature on experimental infection of redfin perch (Perca fluviatilis) and rainbow trout (Oncorhynchus mykiss) with epizootic haematopoietic necrosis virus, an Australian iridovirus

SUMMARY Experimental transmission of epizootic haematopoietic necrosis virus (EHNV) to adult redfin perch Perca fluviatilis and juvenile rainbow trout Oncorhynchus mykiss was undertaken at different water temperatures using intraperitoneal (IP) and bath inoculation. Redfin perch were highly susceptible to EHNV by both routes of infection. Bath inoculation with as few as 0.08 TCID₅₀. _mL^-1 was lethal. The incubation period in redfin perch was about 11 days at a water temperature of 19–21°C but was longer at colder temperatures and disease did not occur at temperatures below 12°C. The longest incubation period recorded in redfin perch was 28 days. Rainbow trout were not susceptible to infection by bath inoculation but the disease was reproduced after IP inoculation with 10^5.6 TCID₅₀ at water temperatures ranging from 8–21°C. The incubation period was 3–10 days at 19–21°C, but was up to 32 days at 8–10°C. Persistent infection with EHNV was detected by virus isolation in a clinically unaffected rainbow trout after 63 days. The implications of these findings in the understanding of the epidemiology of EHNV infection are discussed.     

The epizootic haematopoietic necrosis virus (Iridoviridae) induces apoptosis in vitro

The epizootic haematopoietic necrosis virus (EHNV) is an iridovirus causing severe disease in different fish species. We investigated the induction of apoptosis during EHNV infection of the epithelioma carp papulosum (EPC) cell line. Apoptosis reveals several characteristic morphological changes, such as chromatin condensation, nuclear fragmentation, cytoplasm membrane disorientation, or mitochondrial changes. During EHNV infection of EPC cells the occurrence of apoptosis was analysed using a fluorescein-isothiocyanate (FITC) conjugate of annexin-V to detect phosphatidylserines that have changed cytoplasm membrane localization. Annexin-V labelling was obvious 12 h after infection. At 54 h after EHNV infection 39% of the investigated EPC cells exhibited fluorescence. Furthermore, EHNV-infected cells were stained with 4'-6'-diamidino-2-phenylindole (DAPI) to detect pycnotic nuclei. Appearance of DAPI-positive nuclei was found beginning at 18 h after infection. At 54 h after EHNV infection approximately 56% of the EPC cells showed fragmented nuclei. Assays to inhibit a protein kinase-dependent (e.g. double-stranded RNA-dependent protein kinase) apoptosis pathway with 2-aminopurine revealed a reduction of EHNV titres, e.g. titres were reduced 1000-fold in the presence of 100 and 200 mM 2-aminopurine. Apoptosis takes place during iridovirus infection in vitro and it seems to involve the activation of protein kinases.     

Quarantine requirements for the importation of black rhinoceros from Zimbabwe into Australia

SUMMARY The proposal by the Zoological Parks Board of New South Wales to import 10 southern black rhinoceros (Diceros bicornis) from Zimbabwe as part of an international project for conservation of the species presented the Australian Quarantine and Inspection Service (AQIS) with a unique challenge. This importation is, at least in the modern era, the first importation of live herbivores from the African continent. Many of the serious animal diseases in the world are endemic in parts of Africa. Knowledge of which of these diseases infect wild species and may be transmitted from the wild species to domesticated species, is limited. This paper describes the strategies adopted by AQIS to facilitate the importation of rhinoceros while maintaining protection of Australian consumers, rural industries, domestic livestock and fauna against the entry and spread of unwanted pests and diseases.     

Global trade in ornamental fish from an Australian perspective: the case for revised import risk analysis and management strategies

Over 1 billion ornamental fish comprising more than 4000 freshwater and 1400 marine species are traded internationally each year, with 8-10 million imported into Australia alone. Compared to other commodities, the pathogens and disease translocation risks associated with this pattern of trade have been poorly documented. The aim of this study was to conduct an appraisal of the effectiveness of risk analysis and quarantine controls as they are applied according to the Sanitary and Phytosanitary (SPS) agreement in Australia. Ornamental fish originate from about 100 countries and hazards are mostly unknown; since 2000 there have been 16-fold fewer scientific publications on ornamental fish disease compared to farmed fish disease, and 470 fewer compared to disease in terrestrial species (cattle). The import quarantine policies of a range of countries were reviewed and classified as stringent or non-stringent based on the levels of pre-border and border controls. Australia has a stringent policy which includes pre-border health certification and a mandatory quarantine period at border of 1-3 weeks in registered quarantine premises supervised by government quarantine staff. Despite these measures there have been many disease incursions as well as establishment of significant exotic viral, bacterial, fungal, protozoal and metazoan pathogens from ornamental fish in farmed native Australian fish and free-living introduced species. Recent examples include Megalocytivirus and Aeromonas salmonicida atypical strain. In 2006, there were 22 species of alien ornamental fish with established breeding populations in waterways in Australia and freshwater plants and molluscs have also been introduced, proving a direct transmission pathway for establishment of pathogens in native fish species. Australia's stringent quarantine policies for imported ornamental fish are based on import risk analysis under the SPS agreement but have not provided an acceptable level of protection (ALOP) consistent with government objectives to prevent introduction of pests and diseases, promote development of future aquaculture industries or maintain biodiversity. It is concluded that the risk analysis process described by the Office International des Epizooties under the SPS agreement cannot be used in a meaningful way for current patterns of ornamental fish trade. Transboundary disease incursions will continue and exotic pathogens will become established in new regions as a result of the ornamental fish trade, and this will be an international phenomenon. Ornamental fish represent a special case in live animal trade where OIE guidelines for risk analysis need to be revised. Alternatively, for countries such as Australia with implied very high ALOP, the number of species traded and the number of sources permitted need to be dramatically reduced to facilitate hazard identification, risk assessment and import quarantine controls.     

Transmission of Cyprinid herpesvirus-3 (CyHV-3) from goldfish to naïve common carp by cohabitation

Cyprinid herpesvirus-3 (CyHV-3) has spread worldwide and has had a major impact on koi and common carp production. Previous studies on the host range of the CyHV-3 found that fish species other than koi and common carp are fully resistant to natural virus exposure. Recently, CyHV-3 was detected in goldfish (Carassius auratus auratus) that were in contact with CyHV-3 infected koi. In the present study, a specific RT-PCR product was amplified from the viral thymidine kinase gene in gills, intestine and brain tissues of CyHV-3 infected goldfish. This implied that CyHV-3 replicated in these goldfish. Also, in the presence of a stress factor such as temperature fluctuation, the CyHV-3 infected goldfish transmitted the virus to cohabitated naïve SPF common carp. CyHV-3 DNA was detected in the cohabitated naïve carp tissues by PCR. The results of this study demonstrate that goldfish is a carrier for CyHV-3, permit virus propagation, and disseminate the virus to susceptible carp causing the disease.     

Effectiveness of Praziquantel Bath Treatments against Bothriocephalus acheilognathi in Grass Carp

Abstract

Many states ban the importation of fish infected with the Asian tapeworm Bothriocephalus acheilognathi. Treatment with an effective tapeworm parasiticide prior to shipment would allow fish to be imported that might otherwise be rejected. In this study, extended praziquantel bath treatments of infected grass carp Ctenopharyngodon idella were tested to determine their efficacy against Asian tapeworms. Preliminary praziquantel treatments of 0.7 mg/L or more for an exposure period of 24 h at grass carp densities of 69 g/L were effective in eliminating all tapeworms from 22-g fish. When the treatment duration was lowered to 12 h, a praziquantel concentration of 2.8 mg/L was required for effective treatment. In replicated studies, significantly reduced tapeworm numbers (0–5 tapeworms per treated fish compared with 30+ tapeworms per control fish) were observed following 24-h exposures to praziquantel at 0.5, 0.7, 1.0, and 1.5 mg/ L. Tapeworm elimination occurred only at the maximum concentration of 1.5 mg/L when 88-g grass carp were stocked at a density of approximately 6 g/L. When fish density was raised to 60 g/L, the 1.5-mg/L treatment did not eliminate all tapeworms: 4 of 30 fish contained a single tapeworm.     

Re: Analysis of the risk of introduction and spread of porcine reproductive and respiratory syndrome virus through importation of raw pigmeat into New Zealand

Extract

In the paper by EJ Neumann, RS Morris and M Sujau published in the New Zealand Veterinary Journal 55, 326–336, 2007, entitled, “Analysis of the risk of introduction and spread of porcine reproductive and respiratory syndrome virus through importation of raw pigmeat into New Zealand”, the authors sought to analyse the risk of the introduction and spread of porcine reproductive and respiratory syndrome (PRRS) virus through importation of raw pigmeat. As cautioned by the International Committee of the World Organisation for Animal Health (OIE), any epidemiological model ultimately depends for its validity on the accuracy and completeness of the data underpinning it.     

Comparative pharmacokinetics and tissue distribution of quinocetone in crucian carp (Carassius auratus), common carp (Cyprinus carpio L.), and grass carp (Ctenopharyngodon idella) following the same experimental conditions

The pharmacokinetics and tissue distribution of quinocetone (QCT) in crucian carp (Carassius auratus), common carp (Cyprinus carpio L.), and grass carp (Ctenopharyngodon idella) were compared after oral administration of QCT (50 mg/kg body weight) at water temperature of 24 ± 1 °C. Similar QCT plasma concentration–time profiles were found in the three species of cyprinid fish at the same dosage regimen and water temperature, which were all fitted two‐compartment open pharmacokinetic model. However, different pharmacokinetic parameters were observed in crucian carp, common carp, and grass carp. The absorption rate constants (K_a) of QCT were 1.65, 1.40 and 1.74/h, respectively and absorption half‐lives (t_1/2kα) were 0.42, 0.49, and 0.40/h, respectively. The distribution half‐life (t_1/2α) was 2.83, 0.67, and 0.88 h, respectively, and elimination half‐lives (t_1/2β) of QCT were 133.97, 63.55, and 40.76 h, respectively. The maximum concentrations (C_max) of QCT in plasma were 0.315, 0.182, and 0.139 μg/mL and the time to peak concentrations (T_p) were 1.45, 0.96, and 1.08 h, respectively. The area under the plasma concentration‐time curves (AUC) were 12.35, 5.99, and 4.52 μg·h/mL, respectively. The distribution volumes (V_d/F) of QCT were calculated as 117.81, 128.71, and 220.10 L/kg, respectively. The tissue analysis showed that a similar regularity was obtained in the three species of cyprinids with a single dose of 50 mg/kg body weight after oral administration at the same water temperature. The tissue concentration of QCT in each fish was in order of liver>kidney>muscle, while the residues of QCT in the three species of cyprinid fish were in order of crucian carp>common carp>grass carp.     

Detection of Infectious Pancreatic Necrosis Virus from the Leeches Hemiclepsis marginata and Hirudo medicinalis

Leeches have been reported to harbor several important fish pathogens, including spring viremia of carp virus, infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV), and also may contain blood protozoa. In the present study, leeches were collected from water bodies located in Kurdistan province, Iran. The specimens were tested for IHNV, VHSV, and infectious pancreatic necrosis virus (IPNV) using the PCR method. The results showed that two different species of leeches, Hemiclepsis marginata and Hirudo medicinalis, were infected by IPNV among the seven species studied. The infected leeches were found in areas that were polluted with untreated sewage coming from upstream fish farms culturing Rainbow Trout Oncorhynchus mykiss. In addition, the fish at fish farms in the vicinity had been infected with IPNV 9 months previously. Our results showed that the virus causing infectious pancreatic necrosis is present in the leeches H. marginata and H. medicinalis, suggesting that leeches are a potential source of IPNV in fish farms.

Received October 14, 2015; accepted June 1, 2016 Published online September 29, 2016     

A SURVEY OF ANTIBODY TO AINO VIRUS IN CATTLE AND OTHER SPECIES IN AUSTRALIA

SUMMARY A serological survey of healthy cattle in Australia showed that antibodies to Aino virus were present in serums from cattle in northern Australia and down the east coast as far as central New South Wales in 1975, 1976 and 1977, but occurred with a lower frequency than antibodies to Akabane virus. in contrast to the findings with Akabane virus, no neutralising antibodies to Aino virus were detected in serums from camels, dogs or horses. Antibodies to both viruses were detected in buffaloes and sheep, but not in humans or any of the Australian indigenous species so far tested. All positive serums originated from within the known range of Culicoides brevitarsis.     

MRI of the live fish brain at 3 Tesla: Feasibility,technique and interspecies anatomic variations

Advances in aquatic animal medicine and continued growth of the fish hobbyist and aquaculture communities have led to a developing interest in antemortem diagnostic imaging of aquatic species. The aims of this prospective, pilot study were to determine whether advanced neuroimaging can be safely achieved in live fish using clinically available equipment, to optimize imaging parameters, and to develop a comparative MRI atlas of a few fish species of economic or research value. Two each of channel catfish (Ictalurus punctatus), koi (Cyprinus rubrofuscus), and grass carp (Ctenopharyngodon idella) of at least 30 cm in length were individually anesthetized for 3 Tesla (3T) magnetic resonance imaging (MRI) of the brain. All fish achieved an adequate anesthetic level for prolonged immobilization during imaging. Diagnostic quality images were obtained for all subjects; however, the spatial resolution was maximized with larger fish. Imaging protocols were optimized for standard neuroimaging sequences. Additionally, inversion times for fluid-attenuation inversion recovery (FLAIR) sequences were adapted to the naturally high protein content of fish pericerebral fluid. Following imaging, the fish successfully recovered from anesthesia, were humanely euthanized, and were immediately processed to assess brain histopathology. Necropsy confirmed the sex and health status of each fish. A limited comparative MRI atlas was created of the brains of these species for clinical reference. Findings from the current study supported the use of 3T MRI as an adjunct diagnostic test for fish with suspected neurologic disease and provided a limited anatomic atlas of the teleost brain for use as a reference.     

Experimental Transmission of Walleye Dermal Sarcoma to Yellow Perch

Abstract

Walleye dermal sarcoma was transmitted under experimental conditions to yellow perch Perca flavescens. Fish (20 weeks posthatch) were challenged with cell-free tumor filtrates by topical application on the right flank and then held for observation for 25 weeks in 15°C dechlorinated municipal water. Additional treatment groups included yellow perch challenged with cell-free filtrates of walleye discrete epidermal hyperplasia, yellow perch discrete epidermal hyperplasia, normal yellow perch skin (control), and normal walleye skin (control). Walleye dermal sarcoma was first observed on yellow perch at 20 weeks postexposure, at which time the tumors were small (1–2-mm), slightly raised masses on the right flank of the fish. At the end of the 25-week study, walleye dermal sarcoma was grossly observed in 42% (22 of 53) of the remaining fish and was confirmed by microscopic examination in an additional 29% (9 of 31). Gross and microscopic evaluation of fish at 25 weeks postexposure did not reveal development of lesions in any of the other treatment groups. This study extends the host range of experimentally transmitted walleye dermal sarcoma virus to include the yellow perch.     

Cytochemical characterization of peripheral blood cell populations of two Cyprinidae,Carassius auratus and Ctenopharyngodon idellus

Fish are the most diverse species of all vertebrate groups, and their blood cells have shown variable characteristics in terms of morphology. Cytochemical staining for enzyme activity in blood leukocytes will help assess the immune function of fish. We characterize blood cells from crucian carp (Carassius auratus) and grass carp (Ctenopharyngodon idellus) by using a Diff‐Quick stain as well as different cytochemical methods. Blood specimens obtained from crucian carp and grass carp were evaluated after cytochemical staining for acid phosphatase (ACP), alkaline phosphatase (ALP), naphthol AS chloroacetate esterase (AS‐DNCE), naphthyl acetate esterase (NAE), α‐naphthyl butyrate esterase (NBE), peroxidase (MPO) and periodic acid–Schiff's reaction (PAS) using commercial kits. Blood cell types were evaluated based on their morphological characteristics and the presence or absence of specific chromogen. The expression pattern of enzymes was similar between the two Cyprinidae and was also broadly consistent with other fish species. However, there were some interesting differences detected between crucian carp and grass carp, including naphthol AS chloroacetate esterase activity in monocytes, peroxidase activity and location in thrombocytes. The ACP, ALP and MPO expressions of different leukocytes of the two Cyprinidae were evaluated by Image Pro Plus and were analysed for statistical significant differences. This investigation provides basic haematology and enzyme activity analyses for crucian carp and grass carp and serves as an approach to evaluating the immune response of fish.     

ANTIBODIES TO AKABANE VIRUS IN AUSTRALIA

SUMMARY Neutralising antibody to Akabane virus was shown to develop in cattle in northern Australia throughout the year and also on the east coast of New South Wales in the summer during 1975/1976. Other species found to have antibody to Akabane virus were buffaloes, horses, camels and sheep, but no antibody was found in domestic chickens, ducks, wallabies or man. The biting midge Culicoides brevitarsis has been detected in all the major areas where antibody was demonstrated in this study.     

Gill Reaction to Pollutants from the Tamiš River in Three Freshwater Fish Species,Esox lucius L. 1758, Sander lucioperca (L. 1758) and Silurus glanis L. 1758: A Comparative Study

The study evaluated the effects of waterborne pollutants from the Tami? River on gill histology and possible differences in gill reaction patterns between three freshwater fish species, pike Esox lucius L. 1758, pike‐perch Sander lucioperca (L. 1758) and wels catfish Silurus glanis L. 1758 from the Tami? River. Gills from analysed fish species showed moderate to intense histopathological alterations. The most frequent progressive alteration was hyperplasia of epithelium, whereas the most frequent regressive alteration was epithelial lifting. Circulatory disturbances were most often manifested in the form of hyperaemia. During comparative analysis, differences in gill indices, reaction and alteration indices, as well as in gill and filament prevalence between analysed species, were observed. Although all analysed fish species did show both progressive and regressive alterations, there was a significant difference in the level of expression of these reaction patterns. Gill index obtained for pike clearly stands out as the lowest. Wels catfish showed the highest progressive reaction index, significantly higher in comparison with the other two species (P < 0.05), while pike‐perch showed the highest regressive reaction index, also significantly higher in comparison with the other species (P < 0.001). These results may implicate species‐specific gill reactions and thus present a useful tool for better understanding toxic mechanisms of various pollutants.     

Utilization of dipeptide/protein based diets in larval and juvenile Koi carp – post‐prandial free amino acid levels

We have shown previously that diets in which the nitrogen portion is based on synthetic dipeptides (PP) resulted in weight gain by rainbow trout alevins when free amino acid (FAA) based diets did not. However, the protein‐based diet used as a control in the previous study resulted in a significantly better performance of fish than peptide‐ or FAA based diet fed fish. Therefore, the objectives of our study were (i) to test how stomachless fish respond to peptide‐based diets, (ii) to evaluate PP and protein‐PP mixture diets and (iii) to examine if post‐prandial response to FAA concentrations in the fish body can be used as an indicator of the availability of dietary amino acid sources. The first experiment was conducted with a 4‐day old Koi carp (Cyprinus carpio) larvae and included groups fed a casein–gelatin (CG) based diet, a commercial diet and live Artemia nauplii. In the second experiment, fish fed live Artemia for 3 weeks (69 ± 12 mg) were placed in glass aquaria in triplicate per dietary treatment. Four diets provided equivalent amounts of nitrogen in the form of a CG, a PP, a 50% PP and 50% CG (PP50) mix and as FAA. Fish were fed at 1 and 3 h intervals, and the survival and growth were monitored during the second and third weeks of experiments 1 and 2 respectively. Following the completion of feeding, juvenile carp were sampled prior to feeding and 3 h after a meal. Whole body FAA analyses were carried out. We observed marginal suitability of the CG diet for larval Koi carp, as earlier indicated in common carp. Juvenile Koi carp fed the CG diet achieved 236 ± 19 mg, whereas PP50, PP and FAA diet fed fish grew to 140 ± 37, 70 ± 8 and 73 ± 5 mg respectively. Free amino acids in the fish body, and in particular indispensable amino acids (IDAA), were excellent indicators of dietary availability. The present experiment shows that a dietary 1:1 ratio of protein to synthetic PPs results in better growth, survival and whole body IDAA concentrations in relation to FAA‐ and PP‐based diets confirming earlier reports on the use of synthetic PP‐based diets, although this diet does not match the performance of complete protein, CG‐based diet.     

Investigation of serology for diagnosis of outbreaks of botulism in cattle

Serology has been used to diagnose retrospectively types C and D outbreaks of botulism in cattle in Australia and this study has investigated whether the approach would be applicable in England and Wales. Three hundred sera from routine surveillance submissions in England and Wales were used as a negative control population. Some stored sera were available from a small number of clinical cases of botulism and 125 samples were collected from cohort groups of clinical cases in four new outbreaks of botulism. Three of these outbreaks were identified as being caused by type D Clostridium botulinum toxin. Sera were tested by antibody ELISA in laboratories in Australia and Germany. There was no increase in the proportion of animals seropositive to type C or D antibody in the botulism-associated cattle. The proportion of samples which were seropositive to type D antibodies was <2% in both the negative control and outbreak populations. It was concluded that single time serology is unlikely to be helpful for retrospective diagnosis of outbreaks of type D botulism in England and Wales.     

Aphanomyces invadans and Ulcerative Mycosis in Estuarine and Freshwater Fish in Florida

Abstract

In the spring of 1998, the Florida Fish and Wildlife Research Institute received numerous reports of lesioned or ulcerated fish primarily from the St. Lucie Estuary on the southeast coast of Florida, an area known since the late 1970s for lesions of the ulcerative mycosis (UM) type. From these and archived reports, as well as others received from different areas of Florida, we documented that diseased specimens had randomly distributed skin ulcers (usually reddened or hemorrhagic) with raised irregular margins and, in some cases, deeply penetrating hyphae in the surrounding muscle tissue. Since 1998, 256 fish (comprising 18 species) with ulcerative lesions (from 15 different locations) were confirmed with hyphae in fresh squash preparation or by histological evaluation. Squash preparations revealed nonseptate, sparsely branching, thick-walled hyphae; histological sections revealed mycotic granulomas in the dermis that occasionally penetrated into the skeletal muscle. These pathological characteristics were consistent with UM caused by the oomycete Aphanomyces invadans in Southeast Asia, Japan, Australia, and the United States. For specific identification, six isolates from ulcerated fish were cultured and prepared for molecular characterization using established diagnostic methods. Ribosomal RNA gene sequence analysis identified three isolates as Aphanomyces invadans, one as the oomycete Achlya bisexualis, and two as the ascomycete Phialemonium dimorphosporum. A more extensive survey of 67 ulcerated skin samples from fish collected between 1998 and 2003 was performed using a polymerase chain reaction assay specific for Aphanomyces invadans. Of these, 26 (38.8%) samples from seven fish species and nine collection locations were positive. Confirmation of UM associated with Aphanomyces invadans represents new host records in Florida for the sheepshead Archosargus probatocephalus, striped mullet Mugil cephalus, white mullet Mugil curema, silver perch Bairdiella chrysoura, black drum Pogonias cromis, largemouth bass Micropterus salmoides, and American shad Alosa sapidissima.