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Objective: We sought to evaluate accurate and reproducible detection of Myxobolus cerebralis (Mc), the causative agent of whirling disease, by using nested polymerase chain reaction (nPCR) and three previously established real-time quantitative PCR (qPCR) assays: K18S (Kelley 18S), C18S (Cavender 18S), and Hsp70 (heat shock protein 70). We used a "fit for purpose" approach combined with intra- and interlaboratory testing to identify a molecular testing method that would be equivalent to the currently accepted nPCR procedure for Mc., Methods: Assay performance was compared using a combination of intra- and interlaboratory testing that used synthetic gBlocks along with naturally and experimentally infected fish tissue. North American isolates representing geographically distinct locations were also tested using all three assays., Result: The K18S and C18S assays exhibited high assay sensitivity, intra- and interlaboratory repeatability of sample replicates, and reproducible identification of all test samples across multiple laboratories. In contrast, the Hsp70 assay failed to detect several positive samples at low DNA concentrations during intra- and interlaboratory testing. The K18S assay was the only procedure that demonstrated perfect detection accuracy when testing geographically distinct Mc isolates. Results demonstrated the K18S assay is robust under variable test conditions, is more accurate than the C18S and Hsp70 assays, and provides detection capabilities equivalent to those of the currently accepted nPCR confirmation assay "gold standard" that is described in the American Fisheries Society-Fish Health Section (AFS-FHS) Blue Book., Conclusion: The "fit for purpose" approach and preliminary completion of the World Organization for Animal Health validation pathway demonstrate that the K18S assay provides an alternate method for Mc testing. This work provides the foundation for acceptance of the K18S assay into the AFS-FHS Blue Book as a standardized test procedure for Mc., (© 2024 American Fisheries Society.)

Objective: The Largemouth Bass Micropterus salmoides is an important freshwater fish that is native to the southeastern United States and is cultured for conservation, food, and for the sports fishing industry. Francisella orientalis is a globally distributed bacterial pathogen of warmwater fish species and is associated with granulomatous inflammation and high mortalities. Outbreaks of piscine francisellosis in the United States have been reported in only a few fish species. This study describes three case presentations of francisellosis in Largemouth Bass from a public display system in north-central Florida. Additionally, laboratory-controlled immersion challenges using an F. orientalis isolate from tilapia Oreochromis spp. evaluate susceptibility of Largemouth Bass fingerlings to F. orientalis infection and mortality through this exposure route., Methods: Necropsy, histologic examination, immunohistochemistry, bacterial recovery and culture, and quantitative polymerase chain reaction were used as diagnostic tools to evaluate both the affected display fish and the immersion-challenged fingerlings., Result: Although the display fish and immersion-challenged fingerlings presented with nonspecific clinical signs, gross and histological changes were indicative of granulomatous disease. Immunohistochemical and molecular testing methods confirmed F. orientalis infection in affected fish., Conclusion: The three case presentations described here mark the first reporting of naturally occurring piscine francisellosis in Largemouth Bass that were held in a public display exhibit. Additionally, causality was proven in the Largemouth Bass fingerlings through the immersion challenges. These findings demonstrate susceptibility through immersion-based exposure and assert that francisellosis should be considered among the list of differential diagnoses for Largemouth Bass with granulomatous disease., (© 2023 American Fisheries Society.)

Histopathological assessments of young-of-the-year (age-0) Smallmouth Bass Micropterus dolomieu in the Susquehanna River drainage identified a high prevalence of the myxozoan Myxobolus inornatus. This myxozoan infects the connective tissue of the muscle below the skin but is sometimes observed in the esophagus and buccal cavity. In some instances, shallow infections cause breaks in the skin, which could increase the chance of opportunistic bacterial infections. Several microbial pathogens, including Flavobacterium columnare, Aeromonas spp., and Largemouth Bass virus, have also been cultured from clinically diseased young of year. A multiplex fluorescence in situ hybridization (FISH) assay was developed to determine potential colocalization of M. inornatus, Flavobacterium spp., and Aeromonas spp. infections. With FISH, 75% of age-0 Smallmouth Bass exhibited M. inornatus infections, 10% had Aeromonas spp. infections, and 5% had Flavobacterium spp. infections, while 3% had coinfections with both bacterial species and M. inornatus. The results of the multiplex FISH assay revealed a low occurrence of coinfections of Flavobacterium spp. and/or Aeromonas spp. with M. inornatus in randomly sampled individuals., (Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Journal of Aquatic Animal Health published by Wiley Periodicals LLC on behalf of American Fisheries Society.)

Viral hemorrhagic septicemia virus (VHSV) is an ongoing cause of disease and mortality in freshwater fishes across the Great Lakes region of the Midwestern United States. Antibody detection assays such as enzyme-linked immunosorbent assay (ELISA) are nonlethal serological methods that can have significantly shorter turnaround times than the current validated viral detection diagnostic methodology for VHSV: cell culture with confirmation by polymerase chain reaction (PCR). This study evaluated an ELISA that detects nonneutralizing antinucleocapsid antibodies to VHSV in Northern Pike Esox lucius. Juvenile Northern Pike were experimentally infected with VHSV by intraperitoneal injection. The infected fish were monitored for 12 weeks for signs of disease, and weekly serum samples were obtained. An analysis of the survival data showed that mortality occurred significantly more quickly in inoculated fish than in control fish. Fish that were infected by injection showed a significant increase in antibody response by 2 weeks postinfection. However, variation in the rate and pattern of antibody response among the infected fish was high at any given point. The optimum window for detecting antibodies in Northern Pike is 2-12 weeks postinfection, which generally follows the median time to appearance of clinical signs (21 d postinfection). The receiver-operating characteristic curve analysis showed the ELISA to have a sensitivity of 80.5% and a specificity of 63.2% in Northern Pike, but these values can be adjusted by choosing different percent inhibition cutoffs, which may facilitate the use of the test for specific management goals. The results of this study offer insights into the disease progression and immune kinetics of VHSV, including interindividual variation, which will aid in the management of this economically important virus., (© 2020 American Fisheries Society.)

Molecular tools, such as high-throughput quantitative polymerase chain reaction (HT-qPCR), are useful for monitoring multiple infectious agents in wild animal populations (i.e., broad-based screening). If destructive tissue samples cannot be obtained due to experimental design requirements (e.g., bio-telemetry; holding with repeated biopsy) or the conservation status of host species, then nonlethally sampled tissues can be substituted. However, infection profiles have been found to differ between nonlethally and destructively sampled tissues. We present a comparative analysis of nonlethal (gill and blood) and destructive (pool of internal and external tissue) approaches for broad-based infectious agent screening of adult Chinook Salmon Oncorhynchus tshawytscha. Of a possible 47 agents, 16 were detected overall by nonlethal and destructive methods. Our results indicated moderate differences in infection profiles among tissues, with limitations of each tissue type dependent on the ecology of each agent. The gill was the most comprehensive screening tissue, as more infectious agents were detected overall in gill (n = 16) than in blood (n = 12) or multi-tissue pools (n = 15). The agreement in the estimated agent prevalence between tissue types ranged from poor to excellent, while overall agent community structure (the combined prevalence of all agents) showed low agreement between tissue types. Two agents occurred at 100% prevalence in all tissue types. Nine agents, including types of bacteria and gill parasites, were more prevalent in gill than in blood, while five agents, including one virus and several microparasites, were more prevalent in blood. Future studies should pair microscopy and histopathology with HT-qPCR to better characterize host health and disease development relative to molecular detection of agents across tissue types., (© 2019 American Fisheries Society.)

The objectives of this study were to describe a minimally invasive coelomic fluid sampling technique in elasmobranchs, to characterize the coelomic fluid composition in clinically normal and abnormal animals, and to compare findings from wild and managed populations. Fluid was collected via the coelomic pore in 89 individuals from 16 species spanning clinically normal and abnormal patients within a managed population (n = 54), a semi-managed open-lagoon population (n = 18), and a wild population (n = 17). Biochemical and cytological fluid analyses were performed on all samples, and bacterial and fungal culture, protein electrophoresis, and cholesterol electrophoresis were performed on a subset of samples. The presence of a variable volume of colorless to white and clear to slightly turbid coelomic fluid was consistent with a normal finding; however, the cytological and chemical makeup of coelomic fluid was found to provide additional clinically relevant information. The coelomic fluid from some of the abnormal samples (n = 37) contained white blood cells (n = 15) and concurrent bacteria (n = 7), the latter suggestive of bacterial coelomitis. Yolk was identified in both clinically normal and abnormal females. Of the biochemical parameters tested, calcium, chloride, cholesterol, osmolality, phosphorus, salinity, sodium, specific gravity, total protein, and urea nitrogen have clinical utility. Abnormal samples were mostly associated with reproductive disease, but to a lesser extent with coelomitis and hemocoelom. The wild and semi-managed groups had biochemical differences presumably reflective of the higher salinity of ocean water compared with that in the managed habitat. Aerobic bacteria were identified in normal (n = 7) and abnormal (n = 11) animals. Positive bacterial culture without inflammation may be normal. This study contributes to a further understanding of elasmobranch coelomic fluid analysis and its use as a diagnostic modality for the evaluation of elasmobranch health., (© 2019 American Fisheries Society.)

The present study supports the usefulness of ancillary techniques, such as immunohistochemistry, as a valid diagnostic tool in the field of fish oncology. The immunohistochemical patterns observed in four neoplasms on four individual teleosts belonging to different species are described. Cytokeratin, vimentin, actin, S100, calretinin, and Melan-A antibodies were used. Diagnoses of papilloma in a Bream Abramis brama, fibroma in a Sand Steenbras Lithognathus mormyrus, schwannoma in a Crucian Carp Carassius carassius, and melanoma in a spontaneously inbred Xiphophorus hybrid were made. Diagnosis of tumors in fish is not always easy to carry out, and the tool provided by antibodies used on mammalian tissue is essential for obtaining definitive, unambiguous, and inexpensive identification., (© 2018 American Fisheries Society.)

Conventional PCR is an established method to detect Tetracapsuloides bryosalmonaeDNA in fish tissues and to confirm diagnosis of proliferative kidney disease (PKD) caused by T. bryosalmonae. However, the commonly used PKX5f-6r primers were designed with the intention of obtaining sequence information and are suboptimal for determining parasite DNA presence. A new PCR assay to detect T. bryosalmonae 18s rDNA, PKX18s1266f-1426r, is presented that demonstrates specificity, repeatability, and enhanced sensitivity over the PKX5f-6r assay. The limit of detection of the PKX18s1266f-1426r assay at 95% confidence was 100 template copies, and the new primers detected parasite DNA more consistently at template concentrations below 100 copies than did PKX5f-6r. The PKX18s1266f-1426r also achieved 100% detection at sample DNA concentrations one order of magnitude lower than PKX5f-6r. Out of 127 salmonid fish with unknown T. bryosalmonae infection status, PKX5f-6r detected 35 positive samples, while the new assay detected 43. The discrepancy in T. bryosalmonae detection between the two primer sets may be attributed to several differences between the assays, including oligonucleotide melting temperatures, the use of a touchdown PCR thermal cycle, and amplicon length., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

Myxobolus cerebralis (Mc) is a myxozoan parasite causing whirling disease in hatchery- and natural-origin salmonids. To minimize spread of this parasite and the incidence of its associated disease, fish health professionals routinely screen fish for Mc before stocking or moving the fish to Mc-free waters. Sample collection for Mc traditionally entails lethal sampling of cranial tissue followed by pepsin-trypsin digest (PTD) and screening of the sample for mature myxobolid myxospores (PTD method); however, nonlethal sampling methods would be advantageous in some circumstances, such as when dealing with rare or otherwise valuable fish. Accordingly, we compared Mc detections in cranial cartilage by using the PTD method with PCR assays of fin biopsies collected from juvenile Chinook Salmon Oncorhynchus tshawytscha and adult steelhead O. mykiss. Cranial samples were also analyzed using PCR methods for comparative purposes. Results indicated that Mc could be detected by PCR in fin clips, but the results generated by this approach differed significantly from those associated with PTD- and/or PCR-based analysis of cranial cartilage samples. Polymerase chain reaction-based analysis-of individual head samples and head digest pools in both species as well as fins in steelhead-yielded more positive detections than PTD analysis alone. The PCR-based analysis of head and fin tissues yielded different Mc detection rates in both species, but the nature of the detection disparity varied depending on the species and/or life stage of the fish. We conclude that for lethal cranial samples, neither PTD nor PCR should be used alone, but using these techniques in concert may provide the most complete and accurate estimation of Mc presence in a group of salmonids. If imperiled or highly valuable fish are in question, nonlethal fin samples may be used to generate some information regarding Mc status, with the understanding that parasite DNA detections do not necessarily signify mature infections or disease., (© 2017 American Fisheries Society.)

A 480-g flowerhorn cichlid (an ornamental hybrid) with severe bilateral abdominal swelling, bulla-like structures on the skin, bilateral exophthalmia, and a prolapsed intestine was presented. Radiographs showed compression of the posterior part of the swim bladder and abdominal distention. Ultrasonography of visceral organs revealed a heterogeneous mass with hypoechoic to anechoic polycystic parenchyma and free fluid in the abdominal cavity. At necropsy, free fluid in the abdominal cavity and a large polycystic mass originating from the posterior kidney were observed. Histologically, the mass was composed of more cystic growth of tubules. The renal architecture was replaced by tubules, often irregular in shape, lined by simple to lightly stratified layers of neoplastic and pleomorphic cuboidal to columnar epithelial cells and the absence of glomeruli. Birefringent crystals were observed with polarized light within the lumen of some tubules. The apical border of the neoplastic cells was periodic acid-Schiff positive. Immunohistochemically, the neoplastic cells were positive for cytokeratin AE1/AE3 and proliferating cell nuclear antigen and were negative for p53 (tumor suppressor protein). Microscopic metastasis was seen in the spleen. The metastatic tumor was classified as a cystic adenocarcinoma of the kidney, originating from the proximal tubules. Received October 7, 2016; accepted June 18, 2017.

Nonlethal sampling techniques have previously been evaluated for detection of a variety of viral salmonid pathogens. However, many of these studies have used molecular assays in lieu of widely accepted cell culture techniques to evaluate the sampled tissues. Samples were collected from female steelhead Oncorhynchus mykiss broodstock using three potential nonlethal sampling methods (mucus/skin scrape, pectoral fin clip, and gill tissue biopsy) and evaluated for the presence of infectious hematopoietic necrosis virus (IHNV) via cell culture techniques. The results were compared with those from samples collected using a standard lethal sampling method (pooled anterior kidney and spleen tissues) applied to the same fish. Of the three nonlethal sampling techniques that were evaluated, fin clipping was the easiest and least invasive method. Furthermore, fin tissue was as sensitive as or more sensitive than kidney/spleen tissue for detecting IHNV in this population of fish. However, with the exception of gill tissue, the nonlethal samples did not appear to be appropriate surrogates for lethally collected tissues with regard to identifying an active infection in a particular fish. Nevertheless, nonlethal sampling coupled with cell culture appears to be suitable for helping to define the IHNV status of a steelhead population. Received July 27, 2016; accepted December 11, 2016.

Methods for a plaque neutralization test (PNT) were optimized for the detection and quantification of viral hemorrhagic septicemia virus (VHSV) neutralizing activity in the plasma of Pacific Herring Clupea pallasii. The PNT was complement dependent, as neutralizing activity was attenuated by heat inactivation; further, neutralizing activity was mostly restored by the addition of exogenous complement from specific-pathogen-free Pacific Herring. Optimal methods included the overnight incubation of VHSV aliquots in serial dilutions (starting at 1:16) of whole test plasma containing endogenous complement. The resulting viral titers were then enumerated using a viral plaque assay in 96-well microplates. Serum neutralizing activity was virus-specific as plasma from viral hemorrhagic septicemia (VHS) survivors demonstrated only negligible reactivity to infectious hematopoietic necrosis virus, a closely related rhabdovirus. Among Pacific Herring that survived VHSV exposure, neutralizing activity was detected in the plasma as early as 37 d postexposure and peaked at approximately 64 d postexposure. The onset of neutralizing activity was slightly delayed in fish reared at 7.4°C relative to those in warmer temperatures (9.9°C and 13.1°C); however, neutralizing activity persisted for at least 345 d postexposure in all temperature treatments. It is anticipated that this novel ability to assess VHSV neutralizing activity in Pacific Herring will enable retrospective comparisons between prior VHS infections and year-class recruitment failures. Additionally, the optimized PNT could be employed as a forecasting tool capable of identifying the potential for future VHS epizootics in wild Pacific Herring populations. Received November 7, 2016; accepted January 14, 2017.

Historically, serological tests for finfish diseases have been underused when compared with their use in terrestrial animal health. For years the nonspecific immune response in fish was judged to make serology unreliable and inferior to the direct measurement of agent analytes. We conducted a systematic review of peer-reviewed publications that reported on the development, validation, or application of serological tests for finfish diseases. A total of 168 articles met the screening criteria; most of them were focused on salmonid pathogens (e.g., Aeromonas spp. and viral hemorrhagic septicemia virus). Before the 1980s, most publications reported the use of agglutination tests, but our review indicates that enzyme-linked immunosorbent assay (ELISA) has more recently become the dominant serological test. The main application of serological tests has been in the assessment of vaccine efficacy, with few applications for surveillance or demonstration of freedom from disease, despite the advantages of serological tests over direct detection at the population level. Nonlethal sampling, low cost, and postinfection persistence of antibodies make serological assays the test of choice in surveillance, especially of valuable broodstock. However, their adoption has been constrained by poor characterization and validation. The number of publications in our review reporting diagnostic sensitivity and specificity of serological tests in finfish was small (n = 7). Foreseeing a wider use of serological tests in the future for diagnostic end purposes, we offer recommendations for mitigating deficiencies in the development and evaluation of serological tests, including optimization, control of nonspecific reactions, informed cutoff points, diagnostic accuracy, and serological baseline studies. Achieving these goals will facilitate greater international recognition of serological testing in programs supporting aquatic animal health. Received March 21, 2016; accepted September 24, 2016.

Saprolegniosis is a fungal infection that leads to huge economic losses in tilapia aquaculture. Saprolegnia spp. are usually implicated as the etiological agents, but their identification is sometimes troublesome and confusing. In this study, two Saprolegnia strains (ManS22 and ManS33) were isolated from Nile Tilapia Oreochromis niloticus suffering from saprolegniosis. Both isolates were characterized morphologically and from internal transcribed spacer (ITS) sequence data. Additionally, both strains were tested for pathogenicity, and they were highly pathogenic and caused cumulative mortalities of 88.9% and 95.6%, respectively. Initially, the two strains were identified, by morphology of sexual and asexual stages, as members of the genus Saprolegnia. For more definitive identification and characterization, the ITS region of the ribosomal RNA genes was amplified and sequenced, and sequences were compared with other known sequences in GenBank. A phylogenetic tree constructed using the neighbor-joining method revealed that the two strains fell into two clusters within the species Saprolegnia parasitica. Cluster 1 included the ManS33 strain and cluster 2 the ManS22 strain. Cluster 1 grouped the ManS33 strain with other S. parasitica stains and shared 97-99% sequence similarity. Cluster 2 contained only the ManS22 strain and shared 93-94% similarity to several reference sequences of S. parasitica strains. Therefore, our findings suggest that ManS22 represents a newly described strain of S. parasitica. Received April 19, 2016; accepted October 27, 2016.

An aquarium-maintained female Red Irish Lord Hemilepidotus hemilepidotus presented with severe coelomic distension. The fish was anesthetized for ultrasonographic examination, which highlighted multiple cyst-like lesions in the liver and a distended ovary that was filled with follicles and an inspissated egg mass. Multiple exploratory celiotomies were performed for egg mass removal, liver biopsy, ovariosalpingectomy, and body wall rupture repair. Fourteen weeks after original presentation, and subsequent to 2 weeks of anorexia, the fish died. At necropsy, the liver was severely enlarged and distorted by multiple, coalescing, cyst-like spaces with no grossly normal liver parenchyma. The spleen also contained a raised cyst-like structure. Microscopically, the liver had well-demarcated foci of hepatocyte loss with retained meshworks of interconnected, perisinusoidal stellate cells. The fluid-filled spaces surrounded by stellate cells were not lined by epithelium or endothelium. The spleen had similar fluid-filled spaces formed of stellate cells. The cyst-like lesions in the liver were consistent with spongiosis hepatis; however, the concurrent development of a morphologically comparable lesion in the spleen is not typical of spongiosis hepatis cases. This case may represent the first report of spontaneously occurring spongiosis hepatis in a fish maintained in a public aquarium, as well as the first report in a fish of spongiosis hepatis-like lesions in an organ other than the liver.

Flavobacterium columnare, the causative agent of columnaris disease in fish, affects many economically important freshwater fish species. A colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein (LAMP-calcein) was developed and used to detect the presence of F. columnare in farmed tilapia (Nile Tilapia Oreochromis niloticus and red tilapia [Nile Tilapia × Mozambique Tilapia O. mossambicus]) and rearing water. The detection method, based on a change in color from orange to green, could be performed within 45 min at 63°C. The method was highly specific, as it had no cross-detections with 14 other bacterial species, including other fish pathogens and two Flavobacterium species. The method has a minimum detection limit of 2.2 × 10(2) F. columnare CFU; thus, it is about 10 times more sensitive than conventional PCR. With this method, F. columnare was detected in gonad, gill, and blood samples from apparently healthy tilapia broodstock as well as in samples of fertilized eggs, newly hatched fry, and rearing water. The bacteria isolated from the blood were further characterized biochemically and found to be phenotypically identical to F. columnare. The amplified products from the LAMP-calcein method had 97% homology with the DNA sequence of F. columnare.

Indices of test accuracy, such as diagnostic sensitivity and specificity, are important considerations in test selection for a defined purpose (e.g., screening or confirmation) and affect the interpretation of test results. Many biomedical journals recommend that authors clearly and transparently report test accuracy studies following the Standards for Reporting of Diagnostic Accuracy (STARD) guidelines ( www.stard-statement.org ). This allows readers to evaluate overall study validity and assess potential bias in diagnostic sensitivity and specificity estimates. The purpose of the present study was to evaluate the reporting quality of studies evaluating test accuracy for finfish diseases using the 25 items in the STARD checklist. Based on a database search, 11 studies that included estimates of diagnostic accuracy were identified for independent evaluation by three reviewers. For each study, STARD checklist items were scored as "yes," "no," or "not applicable." Only 10 of the 25 items were consistently reported in most (≥80%) papers, and reporting of the other items was highly variable (mostly between 30% and 60%). Three items ("number, training, and expertise of readers and testers"; "time interval between index tests and reference standard"; and "handling of indeterminate results, missing data, and outliers of the index tests") were reported in less than 10% of papers. Two items ("time interval between index tests and reference standard" and "adverse effects from testing") were considered minimally relevant to fish health because test samples usually are collected postmortem. Modification of STARD to fit finfish studies should increase use by authors and thereby improve the overall reporting quality regardless of how the study was designed. Furthermore, the use of STARD may lead to the improved design of future studies.

Nonlethal methods are needed to assess the health of wild fish and quantify the robustness of the broader population. Results could be used to indicate exposure to various stressors, such as contaminants, infectious disease, external parasite loads, and fishing pressure, to monitor changes in fish population health over time. The wild Red Drum Sciaenops ocellatus population in the Kennedy Space Center Reserve of Merritt Island National Wildlife Refuge was used to develop a protocol to define the health of free-ranging fish using nonlethal techniques. This health index incorporated morphometric measurements, weight, an evaluation for external parasite fauna, notation of physical deformities, and the presence of lesions. A total of 126 adult Red Drum were collected using hook-and-line angling during prespawning (May), spawning (September and October), and postspawning (December) periods. All fish were released alive back into their environment. The nonlethal health assessment scored fish in the "healthy" range of the health index during the prespawning and spawning periods. Fish caught during the postspawning period scored slightly below this range. Parasite load contributed to the depressed score during the postspawning period. Fish collected in all sampling periods were rated on average as "excellent" for condition factor, which suggests that the sampled population in the reserve were thriving.

The potential of fish to transfer salmonellae from heterogeneous aquatic biofilms into feces was assessed in controlled aquarium studies with Suckermouth Catfish Hypostomus plecostomus and with biofilms inoculated with salmonellae. Neither the presence of catfish nor inoculation with salmonellae had detectable effects on the abundance of the microbial community. Densities of the microbial community were about 10(5) cells/mL in the water during a 1-week period, whereas densities of the microbial community increased 10-fold (10(6) to 10(7) cells/mg) in catfish feces during the same period. Salmonellae were detected by both quantitative polymerase chain reaction (qPCR) and situ hybridization in water samples immediately after inoculation, in numbers of about 10(4) cells/mL, representing up to 20% of the cells of the microbial community. Numbers decreased by three orders of magnitude within the first 3 d of the study, which represented only 0.01% of the community, and became undetectable after day 5. In catfish feces, numbers of Salmonella initially increased to up to 6% of the cells of the community but then declined. These results suggest that Salmonella are not biomagnified during gut passage, and thus, fish only provide a means for the translocation of this pathogen.

Edwardsiella tarda has become one of the most severe pathogens in aquaculture industries throughout the world; therefore, a specific and rapid identification method for this bacterium is urgently needed. In the present study, a novel loop-mediated isothermal amplification (LAMP) was developed by targeting the upstream region of the hlyb gene of E. tarda, which was then named as UH-LAMP. The Mg(2+) concentrations, the reaction temperature, and the reaction time of UH-LAMP were optimized to 10 mM, 65°C, and 45 min, respectively. The detection limit of the UH-LAMP was 100-times higher than that of conventional polymerase chain reaction (10 versus 1000 CFU/test). Furthermore, the new UH-LAMP assay showed no cross-reactivity to the E. ictaluri belonging to the other species in the genus Edwardsiella. The high specificity of the assay was also confirmed by testing the nine strains of E. tarda collected from different geographical locations and the other 20 bacteria species. The assay can be performed in a simple water bath or a heat block and the detection result can be visualized by adding a fluorescent reagent to the reaction mixture. Taken together, our preliminary results indicate that this UH-LAMP assay provided a rapid, sensitive, and species-specific diagnostic tool for E. tarda and can easily be applied for the diagnosis under clinical or onsite conditions.

Nucleospora salmonis is an intranuclear microsporidian that primarily infects lymphoblast cells and contributes to chronic lymphoblastosis and a leukemia-like condition in a range of salmonid species. The primary goal of this study was to evaluate the prevalence of N. salmonis in out-migrating juvenile hatchery and wild Chinook salmon Oncorhynchus tshawytscha and steelhead O. mykiss from the Snake River in the U.S. Pacific Northwest. To achieve this goal, we first addressed the following concerns about current molecular diagnostic tests for N. salmonis: (1) nonspecific amplification patterns by the published nested polymerase chain reaction (nPCR) test, (2) incomplete validation of the published quantitative PCR (qPCR) test, and (3) whether N. salmonis can be detected reliably from nonlethal samples. Here, we present an optimized nPCR protocol that eliminates nonspecific amplification. During validation of the published qPCR test, our laboratory developed a second qPCR test that targeted a different gene sequence and used different probe chemistry for comparison purposes. We simultaneously evaluated the two different qPCR tests for N. salmonis and foundthat both assays were highly specific, sensitive, and repeatable. The nPCR and qPCR tests had good overall concordance when DNA samples derived from both apparently healthy and clinically diseased hatchery rainbow trout were tested. Finally, we demonstrated that gill snips were a suitable tissue for nonlethal detection of N. salmonis DNA in juvenile salmonids. Monitoring of juvenile salmonid fish in the Snake River over a 3-year period revealed low prevalence of N. salmonis in hatchery and wild Chinook salmon and wild steelhead but significantly higher prevalence in hatchery-derived steelhead. Routine monitoring of N. salmonis is not performed for all hatchery steelhead populations. At present, the possible contribution of this pathogen to delayed mortality of steelhead has not been determined.

The infectious pancreatic necrosis virus (IPNV) affects several species of freshwater and marine fish. In Mexico, IPNV has an important impact on farming of rainbow trout Oncorhynchus mykiss; however, IPNV distribution in Mexico is unclear. The diagnosis of IPNV is laborious; usually it is based on isolation tests in cell culture followed by immunological identification using techniques of serum neutralization, immunofluorescence, or enzyme-linked immunosorbent assay. It has recently been demonstrated that reverse transcriptase polymerase chain reaction (RT-PCR) is an adequate method for the detection of aquatic birnaviruses. However, its diagnostic use is still limited because very low titers of viable virus cannot be easily detected. In this study, a combination of short-time cell culture and multiplex RT-PCR was established for the diagnosis of IPNV in rainbow trout obtained from farms in the state of Mexico. Three primer sets were used in a single reaction in the multiplex RT-PCR to increase the probability of identifying all serotypes of IPNV serogroup A as well as to help prevent a false-negative result. This approach was able to identify samples with an IPNV concentration of just 0.01 tissue culture infective dose with 50% endpoint (TCID50)/mL, and it identified more infected fish than RT-PCR alone or first-passage cell culture alone. Moreover, this technique made the same identifications as second-passage cell culture but in approximately 30% of the time needed for second-passage cell culture. Consequently, the time and cost efficiency of IPNV diagnosis were greatly reduced.

Cyprinid herpesvirus 2 (CyHV2) has been associated with epidemic mortality among cultured populations of goldfish Carassius auratus. As the principal target tissues are hematopoietic cells in the kidney and spleen, the disease is designated herpesviral hematopoietic necrosis (HVHN). Originally described from Japan, the virus is present in at least five other countries and probably has a global distribution in goldfish. Preventing the further spread of the virus via control programs that exploit sensitive viral detection methods is critical. We developed a conventional polymerase chain reaction (PCR) test based on unique sequences found in the putative helicase gene of CyHV2 and completed initial steps toward the validation of this test. The helicase CyHV2 PCR has an analytic sensitivity of at least 78 copies of the target sequence per reaction in serially diluted plasmid and 84 copies/microg of DNA from the kidney and spleen of goldfish experimentally infected with CyHV2. The analytic specificity of the helicase CyHV2 PCR was demonstrated by the lack of amplification of genomic DNA from cyprinid herpesvirus 1, cyprinid herpesvirus 3, and ictalurid herpesvirus 1 (IcHV1). The helicase CyHV2 PCR effectively detected DNA from CyHV2 from goldfish over a broad geographic range, including Japan, California, Ohio, and Pennsylvania. The performance of the helicase CyHV2 PCR was compared with that of the previously described real-time TaqMan PCR for CyHV2 on a set of 37 samples of DNA from goldfish after experimental or natural exposure to CyHV2. The two tests had very strong agreement (kappa coefficient = 0.907) in classifying fish as positive or negative for CyHV2. The helicase CyHV2 PCR therefore complements the real-time PCR test as a conventional diagnostic method for preventing the further spread of CyHV2.

There is strong evidence that Flavobacterium psychrophilum, the etiologic agent of coldwater disease, is transmitted vertically; it has been hypothesized that disease management at hatchery facilities can be improved through broodstock screening and implementation of culling programs. This paper describes the development of two assays used to screen broodstock tissues (kidney and ovarian fluid) for the presence of F. psychrophilum. Four monoclonal antibodies (MAbs) were generated against outer membrane preparations of F. psychrophilum strain CSF (Clear Springs Foods) 259-93. Of these, MAb FL43 was selected for assay development; this MAb reacted with 67 isolates of F. psychrophilum but exhibited no reaction with two strains of F. columnare or single strains of F. pectinovorum, F. aquatile, F. branchiophilum, and F. saccharophilum. An enzyme-linked immunosorbent assay (ELISA) was developed using MAb FL43 as the capture antibody and MAb FL43 conjugated to horseradish peroxidase (enzyme number 1.11.1.7; IUBMB 1992) as the secondary detection antibody. The ELISA had a lower F. psychrophilum detection boundary of approximately 1.6 X 10(3) colony-forming units (CFU)/mL in kidney tissue homogenates spiked with known bacterial concentrations. Asymptomatic broodstock of coho salmon Oncorhynchus kisutch (n = 50 fish) were sampled, and 100% tested positive for infection by ELISA analysis of kidney tissue; bacterial load was estimated at 2.0 x 10(3) to 9.4 x 10(3) CFU/mL. Ovarian fluid was also collected from these same coho salmon as well as from broodstock of rainbow trout O. mykiss; however, the ELISA proved to be unsuitable for use with ovarian fluid. A filtration-based fluorescent antibody test (FAT) was subsequently developed by conjugating MAb FL43 to Alexa Fluor 488. This FAT was able to detect F. psychrophilum in 74% of ovarian fluid samples collected from coho salmon and 42% of ovarian fluid samples from rainbow trout. Interestingly, yellow-pigmented bacteria were isolated on culture plates from 100% of kidney and ovarian fluid samples. All yellow-pigmented colonies were tested by polymerase chain reaction, and 100% of the coho salmon and rainbow trout were confirmed positive for infection with F. psychrophilum.

Wild striped bass Morone saxatilis in Chesapeake Bay are experiencing a high prevalence of mycobacteriosis, which produces granulomatous lesions of the skin and visceral organs. Culture-based studies have indicated that the newly described species Mycobacterium shottsii and M. pseudoshottsii are the dominant isolates from diseased fish. The classical fish pathogen M. marinum is also found, albeit at much lower frequencies. Both M. shottsii and M. pseudoshottsii are extremely slow-growing on standard selective media, and up to 12 months may be required for isolation and characterization. Epidemiological studies of mycobacteriosis in Chesapeake Bay would therefore benefit from rapid molecular assays with which to detect these species in fish. In this paper, we describe the development of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays capable of detecting M. shottsii, M. pseudoshottsii, and, in most instances, coinfections thereof in striped bass tissues. In addition, PCR-RFLP assays were designed to detect M. marinum and other as-yet-undescribed Mycobacterium spp. present in Chesapeake Bay striped bass. Comparison of these molecular assays with culture-based techniques using splenic tissue from wild striped bass yielded generally concordant results and demonstrated the applicability of these techniques to the study of wild fish.

Fluorescein has been used for rapid and sensitive detection of fish skin and corneal ulceration. Effective use of the fluorescein test requires knowledge of conditions that might cause misleading interpretations or otherwise interfere with test reliability. Examination of fish health and the clinical workup often require tricaine as one of the most commonly used anesthetics. However, tricaine may interfere with correct interpretation of the fluorescein test and might also cause significant fish injury. The effects of tricaine exposure sequence on the fidelity of the fluorescein test was studied in Pacific halibut Hippoglossus stenolepis, walleye pollock Theragra chalcogramma, and northern rock soles Lepidopsetta polyxystra by examining the fluorescence of experimentally induced epidermal wounding. Tricaine can quench fluorescence that is emitted by fluorescein retained in skin ulcers, causing a false-negative reaction. Thus, for the fluorescein test to work properly, it is important to avoid the exposure of fluorescein-treated and rinsed ulcers to tricaine. The effects of exposure to buffered versus unbuffered tricaine on epidermal and corneal integrity were studied in Nile tilapia Oreochromis niloticus and channel catfish Ictalurus punctatus subjected to the fluorescein test and histological examination. Fluorescein could detect not only ulcers but also areas with only a partial loss of epithelium (i.e., erosion). The use of unbuffered tricaine to anesthetize these fish caused serious epidermal and corneal damage. If fish are euthanized with unbuffered tricaine for clinical workup, this severe epidermal or corneal damage could be misinterpreted as an antemortem lesion, leading to misdiagnosis. Even in water with alkalinity exceeding 50 mg/L as CaCO3, it would seem prudent to always buffer tricaine with sodium bicarbonate to prevent a pH change that might lead to iatrogenic effects from unbuffered tricaine. Thus, current general recommendations suggesting that tricaine does not need to be buffered in waters with alkalinity greater than 50 mg/L might need to be modified.

Infectious hematopoietic necrosis virus (IHNV) has been found to cause disease in cultured salmon of the Pacific Northwest region of North America. Diagnosis of IHNV by virus isolation (VI) can take over 2 weeks. Recently, a rapid reverse transcriptase (RT) polymerase chain reaction (PCR) test on fish tissues has been used for diagnosis. Test performances of the VI and RT PCR assays were compared using samples collected in the field. The effect of different storage conditions (tissue frozen with or without RNAlater [Ambion, Inc., Austin, Texas] versus fresh tissue) on the diagnostic tests was also evaluated. Based on the limited number of samples tested, the operating characteristics of RT PCR were very similar to those of VI; therefore, this method is likely suitable for testing field samples for IHNV. The ability of the tests to identify a positive fish ranged from 74% to 89%. Freezing samples at -80 degrees C before testing did not negatively affect the performance of RT PCR or VI. However, due to reduced test performance, RNAlater frozen storage is not recommended without further investigation.

The use of quantitative polymerase chain reaction (QPCR) to test for largemouth bass virus (LMBV) was evaluated during a challenge experiment in which largemouth bass Micropterus salmoides were immersed in the type strain of LMBV. The real-time PCR and cell culture methods were both used to measure LMBV present in the inoculum. Additional samples tested by QPCR included gill, gonad, kidney, liver, mucus, spleen, and swim bladder. A plasmid clone containing a 248-base pair (bp) fragment of the major capsid protein gene (MCP*) was serially diluted and used as a standard to quantify the number of LMBV DNA copies present in the samples tested. A 62-bp fragment of DNA located in MCP* was amplified in the real-time PCR assay. This work has demonstrated the value of the QPCR assay in LMBV surveys.

Empirical estimates of pathogen prevalence in samples of fish may underestimate true prevalence because available detection techniques are incapable of perfect detection. Trout of several species were collected from enzootic (Myxobolus cerebralis, causative agent in whirling disease) habitats, and individual fish were examined for presence of the parasite two or six times by one of four methods: pepsin-trypsin digest (brown trout Salmo trutta), plankton centrifuge (brown trout), polymerase chain reaction (rainbow trout Oncorhynchus mykiss), or histopathology (brook trout Salvelinus fontinalis). The presence-absence data were modeled for prevalence of infection (psi) and probability of detection (p) of the parasite via occupancy models that accounted for imperfect detection of the organism. Based on estimates from the most-supported model for comparison, two myxospore concentration methods underestimated prevalence by about 12% for whole-head results and 34% for the expected value of half-head analysis. Polymerase chain reaction and histopathology gave virtually the same prevalence estimates for whole-head results as the best models but underestimated prevalence by about 6% and 12%, respectively, for the expected value of half-head analysis. The probability of detecting the parasite in a single survey of a fish head, conditional on the parasite's presence, was 0.66 for myxospore concentration methods, 0.81 for histopathology, and 1.0 (left halves) or 0.89 (right halves) for polymerase chain reaction. The occupancy models used in this study may be extended to large-scale monitoring of M. cerebralis to estimate expansion or contraction of the parasite's range over time.

Two koi (Cyprinus carpio) from the same pond developed similar lesions of scoliosis. Radiographic examinations showed that their spines had become malaligned as a result of vertebral compression fractures involving T14 to T16. The vertebrae in both fish were stabilised with screws, k-wire and polymethylmethacrylate. They both appeared to improve after surgery, but they began to decline and died within three months. A postmortem examination revealed multi-organ inflammation that was not associated with the surgical implants.

Using the polymerase chain reaction (PCR) and two primers for conserved regions of the small subunit ribosomal RNA (SSU-rRNA) of Microsporidia, a DNA segment about 1,195 base pairs long was amplified from a DNA template prepared from purified spores of the microsporidian species Pleistophora anguillarum. These spores had been isolated from adult eels (Anguilla japonica) with "Beko Disease." A comparison of sequence data from other microsporidian species showed P. anguillarum SSU-rRNA to be most similar to Vavraia oncoperae. When juvenile eels were artificially infected with P. anguillarum, enzyme-linked immunosorbent assay could detect a positive infection only 12 days post-infection. However, when suitable PCR primers were used, a DNA fragment of about 0.8 kb was detected from these juvenile eels after only 3 days post infection. No PCR product was obtained with templates prepared from clinically healthy control animals.

Between February 1990 and July 1991, 18 harbour porpoises (Phocoena phocoena) and 248 common guillemots (Uria aalge), found dead along the Belgian and German coasts, were examined for their burden of helminths. A total of three species were found in the guillemots (one cestode, one nematode and one pentastomid), and six species in the porpoises (one trematode, one cestode and four nematodes). Among the guillemots the burden of helminths was not statistically different between juvenile and adult birds. The deaths of the birds were apparently not related to the parasite infections. In contrast, the adult porpoises were more heavily parasitised than the juveniles, except for one young porpoise stranded on the Belgian coast. In the porpoises, four species of parasites had a pathological effect and Torynurus convolutus was responsible for the death of one animal from the Belgian coast and three from the German coast.

Plasma lipase concentrations were determined in Atlantic salmon post smolts at weekly intervals on two farms from late June. On one farm there was a significant increase (P < 0.001) in lipase concentration which coincided with a suspicion of pancreas disease on clinical grounds. A definitive diagnosis was subsequently confirmed by histopathology. The exercise was repeated on 10 farms in the following year and the results provided additional evidence of the value of monitoring lipase concentration as an indication of pacreas disease at an earlier stage than it can be detected by clinical signs and histopathology alone.