Gross Signs Of Disease Research Articles

Experimental Evidence of Epizootic Epitheliotropic Disease Virus (Salmoid Herpesvirus-3, Alloherpesviridae) Transmission via Contaminated Fomites and Subsequent Prevention Using a Disinfectant.

Epizootic epitheliotropic disease virus (EEDV) has caused considerable mortality in hatchery-reared lake trout Salvelinus namaycush in the Great Lakes Basin, and yet the routes of transmission and efficacious means of prevention remain poorly understood. To determine whether EEDV can be transmitted via contaminated fomites and clarify whether such transmission could be prevented via fomite disinfection, juvenile lake trout (n = 20 per treatment) were handled in nets previously soaked in an EEDV suspension (7.29 × 104–2.25 × 105 virus copies/mL of water) that were further immersed in either 1% Virkon® Aquatic (“disinfected” treatment, in triplicate) or in sample diluent (“EEDV-contaminated” treatment). Negative control nets were soaked in sterile sample diluent only. Characteristic gross signs of EED developed in the “EEDV-contaminated” treatment group, which was followed by 80% mortality, whereas no gross signs of disease and 0–5% mortality occurred in the negative control and “disinfected” treatment groups, respectively. EEDV was detected via qPCR in 90% of the “EEDV-contaminated” treatment fish, however, it was not detected in any fish within the negative control or “disinfected” treatment groups. Study findings not only demonstrate that EEDV can be readily transmitted via contaminated fomites, but importantly suggest that Virkon® Aquatic is an efficacious option for preventing EEDV contagion via the disinfection of hatchery tools, thereby highlighting a promising tool for improving lake trout hatchery biosecurity and minimizing EEDV-linked losses.

Application of multiplex quantitative polymerase chain reaction methods to detect common bacterial fish pathogens in Nile tilapia, Oreochromis niloticus, hatcheries in Costa Rica

Edwardsiella spp., Streptococcus spp., and Francisella noatunensis subsp. orientalis are some of the most important fish pathogens affecting global tilapia, Oreochromis spp., aquaculture. In Costa Rica, the aquaculture industry is dominated by freshwater‐cultured Nile tilapia, Oreochromis niloticus, which are raised in all seven national provinces. At present, little is known regarding the diversity of pathogens present in these facilities, and definitive identification of agents associated with disease outbreaks are rare. To evaluate the prevalence of common bacterial pathogens in these systems, this study used multiplex quantitative polymerase chain reaction (qPCR) assays targeting Edwardsiella, Streptococcus, and Francisella species as a diagnostic and surveillance tool. In 2017, seven different tilapia hatcheries were visited, and 350 fingerlings were subjected to necropsy and molecular diagnostic evaluation. Fish exhibiting gross signs of disease were subjected to histological and microbiological analysis. For the first time, Edwardsiella anguillarum was recovered and molecularly confirmed from diseased tilapia in Costa Rica. In addition, F. noatunensis subsp. orientalis was identified in a region of Costa Rica where it had not been previously reported.

Feasibility of dsRNA treatment for post-clearing SPF shrimp stocks of newly discovered viral infections using Laem Singh virus (LSNV) as a model

Using post-larvae derived from specific pathogen free (SPF) stocks in penaeid shrimp farming has led to a dramatic increase in production. At the same time, new pathogens of farmed shrimp are continually being discovered. Sometimes these pathogens are carried by shrimp and other crustaceans as persistent infections without gross signs of disease. Thus it is that a 5-generation stock of Penaeus monodon SPF for several pathogens was found, post-stock-development, to be persistently-infected with newly-discovered Laem Singh virus (LSNV). In this situation, the stock developers were faced with destroying their existing stock (developed over a long period at considerable cost) and starting the whole stock development process anew in order to add LSNV to its SPF list. As an alternative, it was hypothesized that injection of complementary dsRNA into viral-infected broodstock prior to mating might inhibit replication of the target virus sufficiently to reduce or eliminate its transmission to their offspring. Subsequent selection of uninfected offspring would allow for post-clearing of LSNV from the existing stock and for conversion of the stock to LSNV-free status. Testing this hypothesis using the LSNV-infected stock described above, we found that transmission was substantially reduced in several treated broodstock compared to much higher transmission in buffer-injected broodstock. Based on these results, the model is proposed for post-clearing of SPF stocks using dsRNA treatment. The model may also be applicable to post-clearing of exceptional, individual performers from grow-out ponds for return to a nucleus breeding center.

Isolation and Characterization ofVibrio parahaemolyticusandVibrio rotiferianusAssociated with Mass Mortality of Chinese Shrimp (Fenneropenaeus chinensis)

ABSTRACT Outbreaks of mass mortality among cultured juvenile Chinese shrimp Fenneropenaeus chinensis occurred during summer 2010 in Jiangsu Province, PR China. Gross signs of disease included lethargy, swimming near the water surface and close to pond edges, pale red shell and appendages, breaking of antenna, and reduction in food consumption. Green and yellow bacterial colonies were recovered on thiosulfate citrate bile salt sucrose agar plates from the diseased shrimp. The predominant observed colonies were purified (VP1 and VR1) and their virulence toward postlarvae of Fenneropenaeus chinensis confirmed by bath experimental challenges. The phenotypic characteristics of the isolates, including morphological, physiological, and biochemical traits, were determined. Three housekeeping genes (16S rRNA, RNA polymerase a-chain (rpoA), and DNA gyrase B subunit (gyrB)) were sequenced, and phylogenetic trees based on rpoA and gyrB sequences were constructed. Phenotypic and genetic results confirmed the diseased ...

A haplosporidian parasite associated with high mortality and slow growth in Penaeus (Litopenaeus) vannamei cultured in Indonesia

High mortality of juvenile Penaeus (Litopenaeus) vannamei less than one month old in Indonesia has been observed in shrimp farms and hatcheries since 2007. The gross signs of disease included shrinkage of the hepatopancreas, flaccid bodies, cuticular melanization and retarded growth. Overall loss in the severely-infected farms was 60 to 90% due to progressive mortality. The loss since 2007 is estimated to be more than 5millionUSdollars. PCR testing of shrimp samples for major viral pathogens gave negative results, but histopathological analysis of hepatopancreatic (HP) tissue showed evidence of a haplosporidian parasite indicated by cytoplasmic multinucleate plasmodia and trophonts in tubule epithelial cells. Plasmodia caused disruption of HP tubule epithelial cells when they discharged trophonts into the tubular lumen. To confirm the presence of haplosporidia, PCR was performed to amplify a small subunit rRNA gene (SSU rDNA) fragment and a cloned 1.5kb amplicon showed 96% sequence identity to that of a haplosporidian previously described in P. vannamei from Belize, Central America suggesting that the Indonesian haplosporidian was either conspecific or very closely related. In situ hybridization assays using a probe based on this amplicon gave positive results specifically with the putative haplosporidian lesions. Prevalence of infections in broodstock and ponds from 2004 to 2010 suggested that the haplosporidian disease outbreaks had resulted from stocking of infected post-larvae.

Molecular and phenotypic characterization of Vibrio aestuarianus, a pathogen of the cultured tongue sole, Cynoglossus semilaevis Günther

Studies were conducted to determine the cause of high mortalities of cultured half-smooth tongue sole Cynoglossus semilaevis. Gross signs of disease included loss of appetite, erratic swimming, and haemorrhages on the head, opercula and base of fins, dorsal fin rot, swollen abdomen filled with ascitic fluid and herniation of the intestine. Histological examination of the liver showed focal areas of necrosis and extensive haemorrhages. Virtually pure, dense bacterial cultures were obtained from liver, kidney and spleen tissues, and high pathogenicity of the isolates to tongue sole was confirmed. The phenotypic characteristics of the isolates including morphological, physiological and biochemical characteristics were determined. The 16S rRNA and gyrB genes of the isolates were sequenced, and the phylogenetic trees representing genetic relatedness between the isolate and publicly available 16S rRNA and gyrB gene sequences from GenBank were constructed. The results confirmed that the diseased tongue soles were infected with Vibrio aestuarianus. The sequenced 16S rRNA gene of strain TS1 (GenBank Accession No. GQ372983) was 1446bp, the gyrB gene of strain TS1 (GenBank Accession No. GQ372984) was 1200bp and the two genes exhibited high similarity (98~99%) to those of V. aestuarianus from GenBank. In addition, the activities of extracellular enzymes and haemolysin were also studied; the results showed that the isolates produced beta haemolysis on rabbit blood agar, lecithinase, proteinase, DNase and lipase, but gelatinase was not produced.

Clearance of white spot syndrome virus (WSSV) and immunological changes in experimentally WSSV-injected Macrobrachium rosenbergii

A time course experimental challenge of WSSV was carried out to examine the clearance of WSSV in Macrobrachium rosenbergii and the consequent immunological changes. The experimental animals were injected with WSSV and the samples of gills, pleopods, head soft tissue and hemolymph were collected at different intervals of 1, 3, 5, 10, 25, 50, 75 and 100 days post infection (p.i.). WSSV infection and clearing were confirmed by single step PCR, nested PCR and bioassay. At 3 days p.i., M. rosenbergii became lethargic and stopped feeding in contrast to the control prawns that behaved and fed normally. However, the WSSV-injected prawns suffered no mortality during the experimental period and recovered without any further gross signs of disease or any mortality over a period of 100 days p.i. The single step PCR analysis showed positive at 1, 3 and 5 days p.i. in gills, head soft tissue, pleopods and hemolymph, and all the organs showed negative at 10 days p.i. onwards. The nested PCR results showed that all organs were positive for WSSV from 3 days p.i. and extended up to 25 days p.i. At 50 days p.i, head soft tissue sample alone showed WSSV-positive while all other organs were negative by nested PCR. All the organs at 75 and 100 days p.i. showed nested PCR negative for WSSV as observed in the control prawn. The hemolymph collected from experimentally infected M. rosenbergii at 1, 3 and 5 days p.i. caused 100% mortality at 40 h p.i., 55 h p.i. and 72 h p.i, respectively in Penaeus monodon whereas hemolymph collected at 10, 25, 50, 75 and 100 days p.i. failed to cause mortality in shrimp. The moribund shrimp showed WSSV-positive and surviving shrimp showed negative by PCR. Immunological parameters such as proPO, O 2 − and clotting time in WSSV-injected M. rosenbergii were found to be significantly higher than those of the control groups, whereas THC and superoxide dismutase were significantly lower when compared to control groups.

Preferential suppression of yellow head virus (YHV) envelope protein gp116 in shrimp that survive challenge with YHV

The DNA sequence that encodes the first 406 amino acid residues at the N-terminus of yellow head virus (YHV) protein gp116, namely N/2 gp116deltaTM, and the DNA sequence that encodes the next 392 amino acid residues at the C-terminus of gp116 (without the transmembrane region), namely C/2 gp116deltaTM, were cloned into pGEX-6P-1 plasmid and expressed in E. coli. Both recombinant proteins were expressed, purified by SDS-PAGE and used to immunize mice. The mouse anti-recombinant N/2 gp116 and C/2 gp116 antisera bound specifically to both the recombinant proteins and to natural gp116 protein in YHV-infected haemolymph as shown by Western blotting and in tissues as shown by immunohistochemistry. Immunohistochemical localization of YHV using anti-gp116 antiserum or monoclonal antibodies specific to gp116 (V3-2B), gp64 (Y18) and p20 (Y19) revealed similar immunoreactivity patterns for all these reagents in muscle and mandibular tissue in shrimp showing gross signs of yellow head disease. However, in gill, hepatopancreas, lymphoid organ and thoracic ganglion tissues from experimental YHV-infected shrimp (Penaeus vannamei and Palaemon serrifer) that did not show signs of disease, immunoreactivity to gp116 was reduced or absent while that for gp64 and p20 remained intense. Thus, some shrimp species were able to selectively inhibit the synthesis of gp116 in a manner that was associated with absence of gross signs of disease.

Lactococcus lactis subspecies lactis also causes white muscle disease in farmed giant freshwater prawns Macrobrachium rosenbergii

From May to August 2001 in Taiwan, 27 farms for the giant freshwater prawn Macrobrachium rosenbergii experienced white tail disease outbreaks in animals approximately 3 to 5 mo old, with total lengths from 6 to 8 cm. Examination of the infected prawns revealed not only previously reported Lactococcus garvieae (16 farms) but also the novel L. lactis subsp. lactis (10 farms). One farm had shrimp infected with both bacteria. In the farms with L. lactis infections, the cumulative mortality was approximately 25 to 60%. Gross signs of disease were opaque and whitish muscles, while histopathology included marked edema and necrotic lesions, with inflammation in the muscles and hepatopancreas. Bacteria isolated using brain/heart infusion medium or tryptic soy agar were Gram-positive and ovoid. Eleven isolates from different farms were identified as L. lactis subsp. lactis using API 20 Strep and Rapid ID32 Strep tests and using PCR assays specific for the L. lactis subsp. lactis 16S rDNA gene (650 bp amplicon) and for the 16S to 23S rDNA interspacer region (380 bp amplicon). In addition, sequencing of the full 16S rDNA genes of 2 of the isolates (MR17 and MR26; GenBank Accession Numbers AF493058 and AF493057, respectively) revealed 99.9% identity between the isolates and 98.7% identity to several complete 16S rRNA sequences of L. lactis subsp. lactis at GenBank. Experimental infections with our isolates gave gross signs and histopathological changes similar to those seen in naturally infected prawns. The mean lethal dose of 4 isolates and the reference strain L. lactis subsp. lactis BCRC 10791 ranged from 4.2 x 10(6) to 2.5 x 10(7) colony-forming units prawn(-1), indicating virulence similar to that previously reported for L. garvieae. This is the first report confirming L. lactis subsp. lactis as a pathogen in juvenile and adult prawns from aquaculture.

Metschnikowia bicuspidata dominates in Taiwanese cold-weather yeast infections of Macrobrachium rosenbergii

At water temperatures below 17 degrees C, yeast infections often occurred in 6 to 11 mo old giant freshwater prawn Macrobrachium rosenbergii (8 to 12 cm) in Taiwan from May 2001 to December 2003, with a cumulative mortality of 20 to 95%. Gross signs of disease included milky hemolymph, a yellow exoskeleton, opaque muscles, and a swollen hepatopancreas (HP). Histopathology included marked edema and extensive necrotic lesions associated with large numbers of yeast aggregates and inflammation within the muscles, HP, and other internal organs such as the heart, ovary, and intestine. Yeast cell numbers isolated from various tissues ranged from 4.5 x 10(8) to 9.0 x 10(9) colony forming units (CFU) per 100 mg. From diseased prawns from 12 affected farms, the presence of Metschnikowia bicuspidata (98.4% prevalence), Saccharomyces cerevisiae (0.8% prevalence), and Candida albicans (0.8% prevalence) was confirmed by biochemical tests and sequencing of PCR products from the D1/D2 domain of 26S rDNA. Experimental infection with these isolates caused gross signs and histopathological changes similar to those observed in naturally infected prawns, and lethal doses (LD50) were 3.8 x 10(3), 2.0 x 10(3), and 4.3 x 10(3) CFU prawn-1, respectively. Although the results of this study revealed that M. bicuspidata may be the major cause of yeast infections in the giant freshwater prawns in Taiwan, this is the first time that S. cerevisiae and C. albicans are also reported as pathogens.

Cloning of eIF5A from shrimp Penaeus monodon, a highly expressed protein involved in the survival of WSSV-infected shrimp

Shrimp respond to viral infection with up- and down-regulation of many critical genes. In our previous work, we showed that white spot syndrome virus ( WSSV) infection of Penaeus monodon ( Pm) caused cellular syntenin levels to increase. In order to further explore the signal transduction pathway of syntenin, we constructed the cDNA library of WSSV-infected shrimp and performed a yeast two-hybrid screening of the library using syntenin as bait. Here we report that syntenin specifically binds eukaryotic translation initiation factor 5A (eIF5A), a 157 amino acid polypeptide that is implicated in cell proliferation and survival. GST-pull-down assays showed the presence of specific interaction between Pm-syntenin and eIF5A. Message analyses revealed that syntenin expression remained normal when shrimp were infected with WSSV but did not show any gross signs of disease. Syntenin expression, however, increased 1.4-fold when shrimp became dead. In contrast, eIF5A expression increased markedly during the stage of WSSV infection when there were no gross signs of disease, i.e., prior to the moribund stage. At the moribund stage, eIF5A expression was down-regulated. This data suggest that syntenin and eIF5A physically interact with each other in the setting of WSSV infection and that these two molecules exhibit dynamic changes in their expression patterns as infection progresses from grossly normal, infected shrimp to full-blown phase.

Hepatopancreatic nuclease of black tiger shrimp Penaeus monodon unlikely to be involved in viral triggered apoptosis

Nucleases are phosphodiesterases that hydrolyze DNA and/or RNA. In a search for shrimp nucleases involved in apoptosis, we discovered a nuclease from hepatopancreatic cDNA of the black tiger shrimp Penaeus monodon. The full-length nuclease gene was amplified and revealed to contain 1668 bp corresponding to 381 deduced amino acid residues in the mature enzyme. Sequence analysis indicated 83% nucleic acid identity and 89% amino acid identity to a nuclease from the Kuruma shrimp Penaeus japonicus (also called Marsupenaeus japonicus). Comparative analysis of sequences, conserved motifs and phylogenetic trees indicated that P. monodon nuclease (PMN) belonged to the family of DNA/RNA non-specific endonucleases (DRNSN). RT–PCR analysis using primers specific for PMN mRNA with seven different shrimp tissues revealed that expression in normal shrimp was restricted to the hepatopancreas. Semiquantitative RT–PCR analysis of PMN using hepatopancreatic mRNA from normal shrimp and from shrimp challenged with white spot syndrome virus (WSSV) indicated significant up-regulation of PMN in the hepatopancreas ( P < 0.05) at the early stage of viral infection but a return to baseline levels as gross signs of disease developed. At the same time, expression was always confined to the hepatopancreas and never seen in other tissues, including those reported to be prime targets for WSSV and subject to increased levels of apoptosis after infection. The results suggested that PMN is probably a digestive enzyme that is unlikely to be involved in hallmark DNA digestion associated with apoptosis.

Presence of multiple viruses in non-diseased, cultivated shrimp at harvest

Histological examinations were carried out with 400 cultivated black tiger shrimp ( Penaeus monodon) from 12 commercial rearing ponds from three different areas in Thailand over a period of 3 years. The shrimp were collected at or near harvest time as two arbitrary size groups of 10–20 each from each pond. Aside from size difference, they showed no gross signs of disease and were normally active. Pathognomonic histopathological lesions were found only for hepatopancreatic parvovirus (HPV) or monodon baculovirus (MBV). Although these were relatively frequent, no unusual shrimp mortality had occurred in any of the ponds examined. Severity of these infections was negatively correlated with shrimp size. When grouped together, HPV-infected shrimp gave mean lengths of approximately 6.5 cm that were significantly different from uninfected shrimp at 9 cm length, early in the cultivation cycle while MBV-infected groups of approximately 9 cm length were not readily distinguishable until uninfected shrimp were 10 cm or more, later in cultivation. Thus, HPV infection was correlated with more severe stunting than MBV. In addition to histopathological examination, polymerase chain reaction (PCR) assays for HPV, white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) were carried out on one large sample of 240 shrimp from 6 ponds where visible lesions were apparent for MBV only. Surprisingly, 94% of the specimens gave a positive test for at least one of the four viruses. HPV and IHHNV alone or in combination were detected at high prevalence (approximately 60%) despite the absence of visible histological lesions and were confirmed by southern blot hybridization. Although the prevalence of the four viral pathogens was very high, it would normally have gone unnoticed, since normal shrimp are rarely examined for viruses.

Pathology of cultured paua Haliotis iris infected with a novel haplosporidian parasite, with some observations on the course of disease.

Mortalities among juvenile paua Haliotis iris Martyn 1784 in a commercial culture facility were reported in April 2000. Histology of moribund paua showed heavy systemic infections of a uni- to multi-nucleate stage of a novel organism later confirmed by transmission electron microscopy (TEM) and molecular studies to be a haplosporidian. Multinucleate plasmodia up to 25 microm diameter with up to 17 nuclei were detectable in wet preparations of hemolymph from heavily infected paua. The presence of the haplosporidian in the affected facility was associated with mortalities of slow growing 'runt' paua during the summer months. Total mortalities in affected raceways 6 mo after mortalities began were between 82.5 and 90%. Heavily infected paua exhibited behavioural abnormalities including lethargy, loss of righting reflex, and were easily detached from surfaces. Some heavily infected paua exhibited oedema and pale lesions in the foot and mantle, but no reliable gross signs of disease were noted. Light infections of the haplosporidian were also found in apparently healthy paua from the facility. Histology indicated that the early stages of infection were characterised by small numbers of plasmodia in the connective tissue surrounding the gut, amongst glial cells adjacent to nerves in the mantle and foot and within gill lamellae. In heavy infections, large numbers of small plasmodia (mean size 5.5 x 7 microm in histological sections) were present in the hemolymph, gills, heart, kidneys, mantle, foot, epipodium and connective tissue of the digestive gland. Infections were not transferred horizontally at 14 and 19 degrees C after cohabiting heavily infected paua with uninfected paua for 3 mo in aquaria, or 3 mo after injecting healthy paua with hemolymph containing haplosporidian plasmodia. This may indicate that the prepatent period for disease is longer than 3 mo, that disease is not expressed below 20 degrees C, or that an intermediate host is required for transmission. Spore formation was not observed in juvenile paua but sporocyst-like bodies containing putative spores were observed amongst haplosporidian plasmodia in the right kidney of poorly performing adult paua collected from the wild.

Virulence differences between two field isolates of feline immunodeficiency virus (FIV-A petaluma and FIV-C pgammar) in young adult specific pathogen free cats

The goal of this study was to identify a strain of feline immunodeficiency virus (FIV) that would be more virulent for adult cats than the prototype FIV-A petaluma and, thereby, enhance the FIV infection model for HIV-1 related research. Diehl et al. reported that one clade C strain of FIV, FIV-C pgammar, was more virulent than other known FIV isolates. Mortalities from 58 to 100% were reported for kittens 12 weeks of age and less following intravenous inoculation. A more variable and somewhat less virulent disease course was observed in neonatal to 8–10-week-old kittens infected orally, intravaginally or intrarectally with this same isolate ( Obert and Hoover, 2000). However, no studies have been done with FIV-C pgammar in adult cats. Therefore, the virulence of FIV-C pgammar for young adult cats was compared to that of FIV-A petalulma, the original FIV isolate. One group of five cats were inoculated intraperitoneally with 470 TCID 50 of FIV-C pgammar in the form of pooled plasma from acutely infected cats, while a second group was infected with plasma containing the 750 TCID50 of FIV-A petaluma. The cats were observed for 20 weeks for gross signs of disease, hematologic abnormalities, time of antibody appearance, and plasma and peripheral blood mononuclear cell (PBMC) associated virus levels. Viral RNA and proviral DNA were measured by a real-time PCR, sensitive to 50 copies per milliliter. The only outward sign of disease was lymphadenopathy, which occurred at a similar time and intensity in both groups of cats. Cats infected with FIV-C pgammar were more likely to be neutropenic and lymphopenic during the first 10–12 weeks of infection than cats infected with FIV-A petaluma. Both groups of cats showed similar overall declines in absolute mean CD4 cell counts and identical concomitant increases in CD8 cells. CD4/CD8 cell ratios were also similar. Antibody, as measured by an ELISA against recombinant FIV-TM antigen, appeared in all cats by 4 weeks post-infection. The most significant differences were in plasma viral RNA and PBMC proviral DNA levels. Cats infected with FIV-C pgammar had up to 100 times higher mean levels of viral RNA during the first few weeks of infection than cats infected with FIV-A petaluma. This difference was also mirrored in levels of proviral DNA in PBMC, which were significantly higher in the FIV-C pgammar infected cats. Plasma viral RNA and PBMC proviral DNA levels were virtually identical in both groups of cats at 20 weeks post-infection. However, proviral DNA in tissues such as thymus and popliteal lymph nodes was 10-fold or so higher in FIV-C pgammar infected cats at 20 weeks and histopathologic lesions were more severe. Based on these various parameters, we concluded that FIV-C pgammar was more virulent than FIV-A petaluma in young adult cats during the 20-week study period. However, we were not able to recreate the severe and rapidly progressive disease previously reported for kittens, suggesting an age-related resistance similar to that observed previously for FIV-A petaluma ( George et al., 1993).

A mycosis of juvenile spiny rock lobster, Jasus edwardsii (Hutton, 1875) caused by Haliphthoros sp., and possible methods of chemical control

Mortalities of puerulus and juvenile rock lobsters, Jasus edwardsii (Hutton, 1875), held in shore‐based ongrowing facilities at water temperatures between 10 and 18 °C were associated with infection by an invasive oomycete identified as Haliphthoros sp. Gross signs of disease included loss of appetite, lethargy and 1–3 brown/black focal necrotic lesions in the gills near insertion of the walking legs. Hyphae were observed in wet preparations of gill filaments excised from lesions. Histology of gill lesions showed hyphae inside the gill cuticle, invasion and lysis of the skeletal muscle and massive haemocyte infiltration and melanization at the base of walking legs adjacent to infected gill filaments. Lobsters over approximately 30 mm carapace length appeared resistant to infection. Death of affected lobsters usually occurred prior to or during the moult and in some cases may have been associated with secondary bacterial infection. Haliphthoros sp. was isolated in pure culture and marine agar 2216 was the medium that produced best growth in vitro. Two isolates were exposed in vitro to five chemicals to determine if an effective treatment could be found. Chemicals that interrupted the life cycle by killing zoospores or preventing sporulation included malachite green, trifuralin, formalin and copper sulphate. The appearance of the disease was associated with poor hygiene and its elimination may be achievable by improving husbandry practices.

Vibrio species associated with mortalities in hatchery-reared turbot ( Colistium nudipinnis) and brill ( C. guntheri) in New Zealand

Studies were conducted to determine the cause of the acute mortality of juvenile turbot Colistium nudipinnis and brill, C. guntheri in an experimental rearing facility. Gross signs of disease included loss of appetite, erratic swimming, distended abdomens caused by an accumulation of clear fluid in the stomach and intestines, and haemorrhagic lesions on the underside and bases of the fins. Histological examination of the liver and kidney showed focal areas of necrosis and extensive haemorrhaging. Other lesions included necrosis and sloughing of the mucosa of the stomach and intestine, and sparse vacuolation in the brain and spinal chord. Bacteria isolated from the liver, kidney, and spleen included Vibrio splendidus I, and V. campbellii-like variants. Examination of the liver, kidney, and brain by electron microscopy failed to detect the presence of viral particles and samples of brain were negative against anti-SJNNV rabbit serum. It appears that the mortalities were due to infection by opportunist bacteria in fish predisposed by a combination of adverse factors including an acute period of poor water quality and perhaps an inadequate diet.

Aeromonas hydrophila isolated from wild freshwater fish in Croatia.

Aeromonas hydrophila was recovered from fish living in lake Vrana on the Croatian island of Cres. The occurrence of the bacterium in the fish was assessed and related to gross signs of disease and findings at necropsy as a potential health hazard for fish. Isolated bacteria were subjected to morphological, physiological, biochemical and antibiotic susceptibility tests. A total of 26 A. hydrophila isolates were obtained. There was a clear seasonality, as no isolates were recovered in the summer months. Most of the isolates were sensitive to all the antimicrobials used in the study except novobiocin and penicillin G. Affected fish manifested haemorrhages over the skin, in the liver, kidney and swim bladder, spleen infarcts, fatty liver, ascitic fluid and swollen haemopoietic tissues. A. hydrophila does not appear to pose a major threat for the fish in the lake at present but under unfavourable and stressful conditions it could seriously compromise fish health.

Taura syndrome in Pacific white shrimp Penaeus vannamei cultured in Taiwan

Mass mortality of cultured white shrimp in southern Taiwan was diagnosed as an outbreak of Taura syndrome (TS). From late 1998 to early 1999, 90% of the shrimp ponds were abandoned at 30 to 45 d after stocking post larvae from Ecuador and elsewhere. The shrimp began to die within 2 to 3 d after they stopped feeding. There were no other gross signs, except that some affected shrimp had reddened tails. Histologically, multifocal necrosis of the cuticular epithelium was the main lesion. The necrotic foci contained pyknotic and karyorrhectic nuclei, and many lightly and densely stained intracytoplasmic and intercellular, spherical inclusions. By transmission electron microscopy, small, icosahedral, picornalike virus particles 30 nm in diameter were seen. Taken together, the observations resembled those for outbreaks caused by Taura syndrome virus (TSV). It is assumed that TSV was transferred to Taiwan via contaminated post larvae and spawners from epizootic areas. This is the first report of a TS outbreak in Taiwan. this newly introduced species. By early 1998, P. vannarnei became the most popular and highest-yield cultured shrimp in southern Taiwan. However, from late 1998 to early 1999, due to high mortality, shrimp production dropped sharply to as little as 10% of the production in early 1998. Based on the gross signs of disease, histopathological changes and evidence from transmission electron microscopy, we report in this paper that Taura Syndrome Virus (TSV) was responsible for the high shrimp mortality. Materials and methods. Moribund shrimp collected from cultivation ponds experiencing high mortality were fixed by Davison's fixative and processed as described in Bell & Lightner (1988). For electron microscopy, whole moribund shrimp were mixed with

Infectious Salmon Anemia Virus (ISAV) in Brown Trout

Abstract Infectious salmon anemia (ISA) is a viral disease of Atlantic salmon Salmo salar that have been exposed to seawater in fish farms or hatcheries. This disease was previously believed to be exclusively one of salmon. However, it has been shown that anadromous brown trout Salmo trutta may carry the ISA virus (ISAV). Propagation of the ISAV in brown trout without the trout's showing any gross clinical signs of disease could be a result of a longstanding host-pathogen relationship between the virus and brown trout. A brown trout population isolated from the sea during the last 5,000 years and expected to be naive to the virus was challenged. These fish did not develop any gross signs of disease, but a few ISAVs were present as late as 46 d postchallenge. It was also shown that the ISA virus was present in brown trout as late as 7 months after challenge.