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Showing papers in "Diseases of Aquatic Organisms in 2005"


Journal ArticleDOI
TL;DR: An overview of the morphology and life cycle of Batrachochytrium dendrobatidis, the cause of chytridiomycosis of amphibians, is presented and adaptations that suggest it has long been evolved to live within cells in the dynamic tissue of the stratified epidermis are suggested.
Abstract: An overview of the morphology and life cycle of Batrachochytrium dendrobatidis, the cause of chytridiomycosis of amphibians, is presented. We used a range of methods to examine stages of the life cycle in culture and in frog skin, and to assess ultrastructural pathology in the skin of 2 frogs. Methods included light microscopy, transmission electron microscopy with conventional methods as well as high pressure freezing and freeze substitution, and scanning electron microscopy with critical point drying as well as examination of bulk-frozen and freeze-fractured material. Although chytridiomycosis is an emerging disease, B. dendrobatidis has adaptations that suggest it has long been evolved to live within cells in the dynamic tissue of the stratified epidermis. Sporangia developed at a rate that coincided with the maturation of the cell, and fungal discharge tubes usually opened onto the distal surface of epidermal cells of the stratum corneum. A zone of condensed, fibrillar, host cytoplasm surrounded some sporangia. Hyperkeratosis may be due to (1) a hyperplastic response that leads to an increased turnover of epidermal cells, and (2) premature keratinization and death of infected cells.

309 citations


Journal ArticleDOI
TL;DR: Although mortality in 3 groups of 15 green tree frogs Litoria caerulea exposed to 3 isolates of Batrachochytrium dendrobatidis was 100%, time to death varied with isolate, highlighting the importance of strain and/or passage history in pathogenicity studies and possibly in the epidemiology of chytridiomycosis.
Abstract: Although mortality in 3 groups of 15 green tree frogs Litoria caerulea exposed to 3 isolates of Batrachochytrium dendrobatidis was 100%, time to death varied with isolate, highlighting the importance of strain and/or passage history in pathogenicity studies and possibly in the epidemiology of chytridiomycosis. A standard naming scheme for isolates of B. dendrobatidis is proposed.

214 citations


Journal ArticleDOI
TL;DR: The biology and ecology of Hematodinium spp.
Abstract: Parasitic dinoflagellates in the genus Hematodinium are important parasites of marine Crustacea. Outbreaks of these parasites have damaged commercial stocks of Norway lobster Nephrops norvegicus, snow crab Chionoecetes opilio, Tanner crab C. bairdi, American blue crab Callinectes sapidus, and velvet swimming crab Necora puber. Species of Hematodinium can reach high enough levels to regulate their host populations, but mortalities are also centred on the unfished juveniles and females, hosts not normally sampled by fisheries; hence impacts are often underre- ported. Seasonal prevalences of up to 85% occur annually in many host populations; in effect, these parasites form cryptic blooms in the water column with crabs and other crustaceans at risk of disease. We review the biology and ecology of Hematodinium spp. infections in crustaceans. Included is a comparison of the different infections, a synthesis of what is known, and an attempt to highlight fruitful areas for continued research.

198 citations


Journal ArticleDOI
TL;DR: In vitro studies show that potential means of translocation may be moist soil and bird feathers, and raise the possibilities that B. dendrobatidis may be translocated by movement of moist river sand and that birds may carry the amphibian chytrid between frog habitats.
Abstract: Amphibian chytridiomycosis caused by Batrachochytrium dendrobatidis has spread at an alarming rate over large distances throughout sensitive frog populations in eastern Australia, Central America and New Zealand. Infected amphibians and contaminated water are implicated in translocation, but other vectors are unknown. Through in vitro studies we show that potential means of translocation may be moist soil and bird feathers. B. dendrobatidis survived for up to 3 mo in sterile, moist river sand with no other nutrients added. B. dendrobatidis attached to and grew on sterile feathers and were able to be transported by feathers to establish new cultures in media, surviving between 1 and 3 h of drying between transfers. If these in vitro results are valid in the natural environment, the findings raise the possibilities that B. dendrobatidis may be translocated by movement of moist river sand and that birds may carry the amphibian chytrid between frog habitats. However, further studies using sand and feathers containing normal microflora are essential.

192 citations


Journal ArticleDOI
TL;DR: This paper lists the pathological findings and causes of mortality of 93 sea turtles stranded on the coasts of the Canary Islands between January 1998 and December 2001.
Abstract: This paper lists the pathological findings and causes of mortality of 93 sea turtles (88 Caretta caretta, 3 Chelonia mydas, and 2 Dermochelys coriacea) stranded on the coasts of the Canary Islands between January 1998 and December 2001. Of these, 25 (26.88%) had died of spontaneous diseases including different types of pneumonia, hepatitis, meningitis, septicemic processes and neoplasm. However, 65 turtles (69.89%) had died from lesions associated with human activities such as boat-strike injuries (23.66%), entanglement in derelict fishing nets (24.73%), ingestion of hooks and monofilament lines (19.35%), and crude oil ingestion (2.15%). Traumatic ulcerative skin lesions were the most common gross lesions, occurring in 39.78% of turtles examined, and being associated with Aeromonas hydrophila, Vibrio alginolyticus and Staphylococcus spp. infections. Pulmonary edema (15.05%), granulomatous pneumonia (12.90%) and exudative bronchopneumonia (7.53%) were the most frequently detected respiratory lesions. Different histological types of nephritis included chronic interstitial nephritis, granulomatous nephritis and perinephric abscesses, affecting 13 turtles (13.98%). Ulcerative and fibrinous esophagitis and traumatic esophageal perforation were the most frequently observed lesions in the esophagus, being associated in the majority of the cases with ingestion of fishing hooks. Larval nematodes of the Anisakidae family caused gastritis in 15 turtles (16.13%). Necrotizing and/or granulomatous hepatitis were the lesions most commonly observed in the liver (27.95%). Traumatic lesions included necrotizing myositis (10.75%) mainly caused by entanglement in fishing nets or boat-strikes, and amputation of 1 or 2 flippers (25.81%) by netting. Traumatic erosions and/or fractures of the carapace/plastron mainly caused by boat-strikes were also observed (26.88%). Eye lesions included heterophilic keratoconjunctivitis, ulcerative keratitis and heterophilic scleritis, affecting 7 turtles (7.53%).

184 citations


Journal ArticleDOI
TL;DR: Hemocytosis, diapedesis and hemocyte degeneration characterized by nuclear pycnosis and fragmentation were observed in infected oysters, which is consistent with previous observations of OsHV infections in France.
Abstract: Pacific Crassostrea gigas and eastern C. virginica oysters were examined between June 2002 and April 2003 from 8 locations along the east, west and south USA coasts for oyster herpes virus (OsHV) infections using the A primer set in a previously developed PCR test. Only surviving Pacific oysters from a mortality event in Tomales Bay, California, USA, where annual losses of oysters have occurred each summer since 1993, were infected with a herpes-like virus in 2002. PCR examination using template amounts of both 50 and 500 ng were essential for OsHV detection. Sequence analysis indicated that the Tomales Bay OsHV was similar to that identified in France with the exception of a single base pair substitution in a 917 bp fragment of the viral genome. However, unlike the French OsHV-1, the Tomales Bay OsHV did not amplify with the primer pair of a second OsHV-1 PCR assay, suggesting that further characterization of these viruses is warranted. No evidence of Cowdry type A viral infections characteristic of herpes virus infections or other pathogens were observed in OsHV-infected oysters. Hemocytosis, diapedesis and hemocyte degeneration characterized by nuclear pycnosis and fragmentation were observed in infected oysters, which is consistent with previous observations of OsHV infections in France. Together these data suggest that OsHV may be associated with the annual summer Pacific oyster seed mortality observed in Tomales Bay, but establishment of a causal relationship warrants further investigation.

172 citations


Journal ArticleDOI
TL;DR: It is proposed that an altered mechanical load could have caused the transformation of the bone growth zones and the concomitant replacement of the intervertebral (notochord) tissue by cartilaginous tissues in the 'short tails' studied here.
Abstract: Anterior/posterior (a/p) compression of the vertebral column, referred to as 'short tails', is a recurring event in farmed Atlantic salmon. Like other skeletal deformities, the problem usually becomes evident in a late life phase, too late for preventive measures, making it difficult to understand the aetiology of the disease. We use structural, radiological, histological, and mineral analyses to study 'short tail' adult salmon and to demonstrate that the study of adult fish can provide important insights into earlier developmental processes. 'Short tails' display a/p compressed vertebrae throughout the spine, except for the first post-cranial vertebrae. The vertebral number is unaltered, but the intervertebral space is reduced and the vertebrae are shorter. Compressed vertebrae are characterized by an unchanged central part, altered vertebral end plates (straight instead of funnel-shaped), an atypical inward bending of the vertebral edges, and structural alterations in the intervertebral tissue. The spongiosa is unaffected. The growth zones of adjacent vertebrae fuse and blend towards the intervertebral space into chondrogenic tissue. This tissue produces different types of cartilage, replacing the notochord. The correspondence in location of intervertebral cartilage and deformed vertebral end plates, and the clearly delimited, unaltered, central vertebral parts suggest that the a/p compression of vertebral bodies is a late developmental disorder that may be related to a metaplastic shift of osteogenic tissue into chondrogenic tissue in the vertebral growth zone. Given the lack of evidence for infections, metabolic disorders and/or genetic disorders, we propose that an altered mechanical load could have caused the transformation of the bone growth zones and the concomitant replacement of the intervertebral (notochord) tissue by cartilaginous tissues in the 'short tails' studied here. This hypothesis is supported by the role that notochord cells are known to play in spine development and in maintaining the structure of the intervertebral disk.

143 citations


Journal ArticleDOI
K. Hodneland1, A. Bratland, K E Christie, Curt Endresen, Are Nylund1 
TL;DR: C cultured a Norwegian salmonid alphavirus from moribund fishes diagnosed with cardiac myopathy syndrome (CMS) and fishes diagnosedWith PD, the virus has also been found in salmon suffering from haemorrhagic smolt syndrome in the fresh water phase.
Abstract: In Europe, 2 closely related alphaviruses (Togaviridae) are regarded as the causative agents of sleeping disease (SD) and salmon pancreas disease (SPD): SD virus (SDV) has been isolated from rainbow trout Oncorhynchus mykiss in France and the UK, while SPD virus (SPDV) has been isolated from salmon Salmo salar in Ireland and the UK. Farmed salmonids in western Norway also suffer from a disease called pancreas disease (PD), and this disease is also believed to be caused by an alphavirus. However, this virus has not yet been characterised at the molecular level. We have cultured a Norwegian salmonid alphavirus from moribund fishes diagnosed with cardiac myopathy syndrome (CMS) and fishes diagnosed with PD. The virus has also been found in salmon suffering from haemorrhagic smolt syndrome in the fresh water phase. The genomic organisation of the Norwegian salmonid alphavirus is identical to that in SPDV and SDV, and the nucleotide sequence similarity to the other 2 alphaviruses is 91.6 and 92.9%, respectively. Based on the pathological changes, host species and the nucleotide sequence, we suggest naming this virus Norwegian salmonid alphavirus (NSAV). Together with SPDV and SDV it constitutes a third subtype of salmonid alphavirus (SAV) species within the genus Alphavirus, family Togaviridae.

141 citations


Journal ArticleDOI
TL;DR: The number of sporangia was highly variable and this appeared to be related to the stage in the cycle of sloughing, and other pathological changes such as hyperkeratosis and congestion occurred much more frequently on ventral surfaces.
Abstract: Although histopathology is used routinely for diagnosis of chytridiomycosis in live and dead amphibians, there are no quantitative data on the distribution of the causative fungus, Batrachochytrium dendrobatidis, in the skin. We performed quantitative histological examinations on 6 sites on the body and 4 toes of 10 free-ranging adult green tree frogs Litoria caerulea found recently dead or dying from chytridiomycosis. Large numbers of sporangia occurred in all areas of ventral skin and toes; on average there were 94.3 sporangia mm(-1) of superficial epidermis. The number of sporangia was highly variable and this appeared to be related to the stage in the cycle of sloughing. The stratum corneum tends to build up with high intensities of infection and then sheds entirely rather than being shed continuously. Very few or no sporangia occurred on dorsal skin. This distribution could be explained by the dryness of the dorsal skin or possibly by the greater number of serous glands, which produce antifungal peptides, on the dorsum. In some frogs, ulceration and erosions occurred on skin on the back in the absence of sporangia. Other pathological changes such as hyperkeratosis and congestion occurred much more frequently on ventral surfaces.

135 citations


Journal ArticleDOI
TL;DR: It appears that use of a cross-genogroup challenge virus strain may lead to reduced efficacy of the DNA vaccine, and vaccinated fish responded to viral challenges with higher antibody titers than mock-vaccinated control fish.
Abstract: The level of protective immunity was determined for Chinook Oncorhynchus tshawytscha and sockeye/kokanee salmon (anadromous and landlocked) O. nerka following intramuscular vaccination with a DNA vaccine against the aquatic rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A DNA vaccine containing the glycoprotein gene of IHNV protected Chinook and sockeye/kokanee salmon against waterborne or injection challenge with IHNV, and relative percent survival (RPS) values of 23 to 86% were obtained under a variety of lethal challenge conditions. Although this is significant protection, it is less than RPS values obtained in previous studies with rainbow trout (O. mykiss). In addition to the variability in the severity of the challenge and inherent host susceptibility differences, it appears that use of a cross-genogroup challenge virus strain may lead to reduced efficacy of the DNA vaccine. Neutralizing antibody titers were detected in both Chinook and sockeye that had been vaccinated with 1.0 and 0.1 pg doses of the DNA vaccine, and vaccinated fish responded to viral challenges with higher antibody titers than mock-vaccinated control fish.

130 citations


Journal ArticleDOI
TL;DR: Two co-habitation studies with common carp were conducted to determine whether latent infections of koi herpesvirus (KHV) exist and reaction of the virus appeared to be temperature dependent.
Abstract: Two co-habitation studies with common carp were conducted to determine whether latent infections of koi herpesvirus (KHV) exist. Fish were exposed to KHV using 2 different temperature profiles, which induced low and high initial mortality. Subsequently, certain groups of fish were co-habited with naive fish while others were not. Koi herpesvirus was reactivated in fish from 3 of the 5 experimental tanks. Reactivation of the virus occurred regardless of the initial mortality associated with the virus or whether fish were co-habited with naive fish. The reactivation of the virus in our experiments occurred several months after the initial exposure to KHV and appeared to be temperature dependent.

Journal ArticleDOI
TL;DR: If the AHL molecules regulate the expression of the virulence phenotype in these bacteria, as shown to occur in some bacterial pathogens, novel disease control measures may be developed by blocking AHL-mediated communication and suppressing virulence.
Abstract: The aim of the present study was to investigate the production of quorum sensing signals (specifically acylated homoserine lactones, AHLs) among a selection of strains of Gram-negative fish bacterial pathogens. These signals are involved in the regulation of virulence factors in some human and plant-pathogenic bacteria. A total of 59 strains, representing 9 different fish pathogenic species, were tested against 2 AHL monitor bacteria (Agrobacterium tumefaciens NT1 [pZLR4] and Chromobacterium violaceum CV026) in a well diffusion assay and by thin-layer chromatography (TLC). Representative samples were further characterized by high performance liquid chromatography-high resolution mass spectrometry (HPLC-HR-MS). AHLs were produced by all strains of Aeromonas salmonicida, Aeromonas hydrophila, Yersinia ruckeri, Vibrio salmonicida, and Vibrio vulnificus. Some strains of atypical Aeromonas salmonicida and Vibrio splendidus were also positive. Aeromonas species produced N-butanoyl homoserine lactone (BHL) and N-hexanoyl homoserine lactone (HHL) and 1 additional product, whereas N-3-oxo-hexanoyl homoserine lactone (OHHL) and HHL were detected in Vibrio salmonicida. N-3-oxo-octanoyl homoserine lactone (OOHL) and N-3-octanoyl homoserine lactone (OHL) were detected in Y. ruckeri. AHLs were not detected from strains of Photobacterium damselae, Flavobacterium psychrophilum or Moritella viscosa. AHLs were extracted from fish infected with Y. ruckeri but not from fish infected with A. salmonicida. In conclusion, the production of quorum sensing signals, AHLs, is common among the strains that we examined. If the AHL molecules regulate the expression of the virulence phenotype in these bacteria, as shown to occur in some bacterial pathogens, novel disease control measures may be developed by blocking AHL-mediated communication and suppressing virulence.

Journal ArticleDOI
TL;DR: Temperature strongly influenced percent mortality and time to death of salamanders exposed to the Ambystoma tigrinum virus (iridovirus) (ATV) and Polymerase chain reaction (PCR) confirmed the presence of virus in ATV-exposed salamander but was found to be less sensitive than cell culture in detecting ATV at low concentrations.
Abstract: Temperature strongly influenced percent mortality and time to death of salamanders exposed to the Ambystoma tigrinum virus (iridovirus) (ATV). Most salamanders survived when exposed at 26°C, whereas all died at 18°C and nearly all died at 10°C. Some asymptomatic salamanders that survived 60 d at 10 or 26°C were found to be carrying virus. Polymerase chain reaction (PCR) confirmed the presence of virus in ATV-exposed salamanders but was found to be less sensitive than cell culture in detecting ATV at low concentrations. PCR products were 100% identi- cal to ATV in the major capsid protein sequence. Virus titer was higher in salamanders held at 10°C than at 18°C but little virus, if any, was present in the small number of salamanders that died at 26°C. These results may help explain periodic viral epizootics in field populations of A. tigrinum where water temperatures fluctuate widely.

Journal ArticleDOI
TL;DR: A herpes-like virus is demonstrated for the first time to be associated with high mortality rates in maricultured abalone Haliotis diversicolor supertexta in Taiwan and historians indicated that the nerve system was the primary target tissue.
Abstract: A herpes-like virus is demonstrated for the first time to be associated with high mortality rates in maricultured abalone Haliotis diversicolor supertexta in Taiwan. Histopathology of moribund abalone indicated that the nerve system was the primary target tissue. The lesions were characterised by tissue necrosis accompanied with infiltration of haemocytes. Electron microscopic examination demonstrated viral particles within the degenerated cerebral ganglion cells. The viruses were hexagonal, approximately 100 nm in diameter and had a single coat. Some viral particles contained a dense nucleoid, while others were empty. The ultrastructure and morphogenesis of the virus particles were consistent with those of the herpesvirus described from the oyster Crassostrea virginica. Experimental infection using supernatant collected from minced visceral organs and muscle of moribund abalone induced 100 % mortality through both intramuscular injection and bath treatments.

Journal ArticleDOI
TL;DR: A specific and rapid PCR detection method for Flavobacterium columnare based on the 16S-23S rDNA intergenic spacer region of the ribosomal RNA operon has been developed and confirmed that F. columnare can be transmitted horizontally indirectly through the water column without fish-to-fish contact.
Abstract: A specific and rapid PCR detection method for Flavobacterium columnare based on the 16S-23S rDNA intergenic spacer region (ISR) of the ribosomal RNA operon has been developed. The ISR of 30 F. columnare strains and other Flavobacterium species was amplified using universal primers and sequenced. Once F. columnare specific sequences within the ISR were recognized, spe- cific PCR primers were designed against them (FCISRFL and FCISRR1). The primers were sensitive and able to detect as low as 7 colony forming units from pure culture by PCR. The new PCR detection method was applied to experimentally infected channel catfish. Two different experiments in which channel catfish fingerlings were infected by intramuscular injection or by immersion bath showed the advantage of the PCR method over standard culture techniques. F. columnare was detected by PCR in both tank water and catfish tissue samples with a higher frequency and in less time than standard microbiological methods. Furthermore, PCR detection confirmed that F. columnare can be transmit- ted horizontally indirectly through the water column without fish-to-fish contact. The newly devel- oped PCR detection method for F. columnare was more sensitive and rapid than standard culture on bacteriological media for detection of F. columnare in channel catfish tissues and in tank water.

Journal ArticleDOI
TL;DR: The results demonstrated that the probable cause of this disease was a ranavirus, and affected amphibians were found to exhibit necrosis within the hematopoietic cells.
Abstract: Using field, molecular and histological methods, an epizootic, systemic disease causing death within wood frog Rana sylvatica tadpoles and leopard frog Rana pipiens metamorphs at 3 different locations within Southern Ontario, Canada, has been investigated. Our results demonstrated that the probable cause of this disease was a ranavirus. Affected amphibians were found to exhibit necrosis within the hematopoietic cells. Liver tissue samples were found positive for the virus by PCR amplification of the ranavirus (Family: Iridoviridae) major capsid protein (MCP). Positive samples were confirmed by sequence analysis. Clinically normal, laboratory-raised wood frog egg broods were also found to test weakly positive for ranavirus. The population effects of disease on these amphibian communities have not yet been conclusively associated with population declines, but warrant more focused consideration.

Journal ArticleDOI
TL;DR: Based on the onset of clinical signs of infection and mortality, L. vannamei appears to be the most susceptible of these 3 species to IMNV infection, and skeletal muscle cells produced the strongest ISH reactions.
Abstract: Infectious myonecrosis virus (IMNV) was recently found to be the cause of necrosis in the skeletal muscle of farm-reared Litopenaeus vannamei from northeastern Brazil. Nucleic acid extracted from semi-purified IMN virions showed that this virus contains a 7.5 kb RNA genome. A cDNA library was constructed, and a clone, designated as IMNV-317, was labeled with digoxigenin-11-dUTP and used as a gene probe for in situ hybridization (ISH). This probe specifically detected IMNV in infected tissues. To determine the susceptibility of 3 species of penaeid shrimp (L. vannamei, L. stylirostris, Penaeus monodon) to IMNV infection, juveniles were injected with purified virions and observed for clinical signs of infection and mortality over a 4 wk period. All L. vannamei exhibited typical lesions after 6 d, and lesions were visible in all L. stylirostris by Day 13. The clinical signs of opaque muscle were not seen in P. monodon, due to their highly pigmented exoskeleton precluding visual detection of lesions. Moderate mortality (20%) occurred in infected L. vannamei. No mortalities were observed in either L. stylirostris or P. monodon. Histological examination and ISH indicated that all 3 species are susceptible to IMNV infection. Using ISH, IMNV was detected in tissues including the skeletal muscle, lymphoid organ, hindgut, and phagocytic cells within the hepatopancreas and heart. In all 3 species, skeletal muscle cells produced the strongest ISH reactions. Based on the onset of clinical signs of infection and mortality, L. vannamei appears to be the most susceptible of these 3 species to IMNV infection.

Journal ArticleDOI
TL;DR: A baseline survey of coral diseases in 2 regions in the Philippines shows that Porites is a major reef-builder in the Indo-Pacific comprising 30% of hard coral colonies on the authors' surveyed reefs, and is generally thought to be a hardy, long-lived genus.
Abstract: While it is generally assumed that Indo-Pacific reefs are not widely affected by diseases, limited data suggest a number of diseases and syndromes that appear to differ from those currently under study in the Caribbean. This report presents the results of a baseline survey of coral diseases in 2 regions in the Philippines: the Central Visayas and the Lingayen Gulf. Mean prevalence for all diseases observed was 8.3 +/- 1.2% (mean +/- SE; n = 8 reefs), with Central Visayas reefs showing higher disease prevalence (11.6 +/- 2.8%; n = 4 reefs) than those of Lingayen Gulf (5.1 +/- 1.4%; n = 4 reefs). Five diseases and syndromes were described; 3 of these-Porites ulcerative white spot disease (PUWS) (prevalence = 8.96 +/- 2.2%), tumors (prevalence = 1.0 +/- 0.5%) and pigmentation response (prevalence = 0.5 +/- 0.2%)--occurred frequently in both regions and targeted the genus Porites. Correlation between disease prevalence and number of Porites colonies was fairly strong (r2 = 43.4), though not significant, and no correlation was seen between prevalence and either the amount or diversity of hard coral. Porites is a major reef-builder in the Indo-Pacific comprising 30% of hard coral colonies on our surveyed reefs, and is generally thought to be a hardy, long-lived genus. Diseases targeting this robust group present an as yet unquantified risk to Philippine reefs and could result in major changes in reef structure.

Journal ArticleDOI
TL;DR: Though the present study indicates only a low level infectivity in wild polychaetes, laboratory experiments clearly indicated the possibility of WSSV transfer from the live feed to shrimp broodstock, suggesting that polychaete worms could play a role in the epizootiology of W SSV.
Abstract: The present work provides the first evidence of polychaete worms as passive vectors of white spot syndrome virus (WSSV) in the transmission of white spot disease to Penaeus monodon broodstocks The study was based on live polychaete worms, Marphysa spp, obtained from worm suppliers/worm fishers as well as samples collected from 8 stations on the northern coast of Tamilnadu (India) Tiger shrimp Penaeus monodon broodstock with undeveloped ovaries were experimentally infected with WSSV by feeding with polychaete worms exposed to WSSV Fifty percent of polychaete worms obtained from worm suppliers were found to be WSSV positive by 2-step PCR, indicating high prevalence of WSSV in the live polychaetes used as broodstock feed by hatcheries in this area Of 8 stations surveyed, 5 had WSSV positive worms with prevalence ranging from 167 to 75% Polychaetes collected from areas near shrimp farms showed a higher level of contamination Laboratory challenge experiments confirmed the field observations, and > 60% of worms exposed to WSSV inoculum were proved to be WSSV positive after a 7 d exposure It was also confirmed that P monodon broodstock could be infected with WSSV by feeding on WSSV contaminated polychaete worms Though the present study indicates only a low level infectivity in wild polychaetes, laboratory experiments clearly indicated the possibility of WSSV transfer from the live feed to shrimp broodstock, suggesting that polychaete worms could play a role in the epizootiology of WSSV

Journal ArticleDOI
TL;DR: Results suggest that parasitized fish in polluted environments are in a poorer state of health than uninfected fish, and suggest that lipid peroxidation may be used as a biomarker of pathological effects caused by parasitism.
Abstract: Yellow perch Perca flavescens were collected from a contaminated site and a reference site in the St. Lawrence River, Quebec, Canada. Fish were assessed for oxidative stress (lipid perox- idation and reduced glutathione levels) and parasitism by the nematode Raphidascaris acus and metacercariae of the digenean Apophallus brevis. Lipid peroxidation is not only considered a mea- sure of oxidative stress, but of stress in general, and thus serves as an indicator of fish health. Fish from the contaminated site exhibited higher levels of lipid peroxidation than those from the reference site, independent of parasitic infections. However, fish infected with R. acus at the contaminated site tended to have higher levels of lipid peroxidation than uninfected fish at the same site, whereas no difference was observed between infected and uninfected fish at the reference site. Yellow perch infected with >10 metacercariae of A. brevis expressed higher levels of lipid peroxidation than those infected with <10 metacercariae at both the contaminated and the reference sites. No differences were found in levels of reduced glutathione in liver or muscle in relation to site or either parasite species. Results support the use of lipid peroxidation as a biomarker of water contamination. They further suggest that lipid peroxidation may be used as a biomarker of pathological effects caused by parasitism. Most importantly, results demonstrate that contaminants and parasites occurring together exacerbate oxidative stress in fish, suggesting that parasitized fish in polluted environments are in a poorer state of health than uninfected fish.

Journal ArticleDOI
TL;DR: Shrimp that survived challenges on the 5th and 10th days after cessation of IVP administration survived repeated challenges although they were sometimes positive for the presence of WSSV by a polymerase chain reaction (PCR) assay specific for W SSV.
Abstract: Fenneropenaeus indicus could be protected from white spot disease (WSD) caused by white spot syndrome virus (WSSV) using a formalin-inactivated viral preparation (IVP) derived from WSSV-infected shrimp tissue. The lowest test quantity of lyophilized IVP coated onto feed at 0.025 g(-1) (dry weight) and administered at a rate of 0.035 g feed g(-1) body weight d(-1) for 7 consecutive days was sufficient to provide protection from WSD for a short period (10 d after cessation of IVP administration). Shrimp that survived challenges on the 5th and 10th days after cessation of IVP administration survived repeated challenges although they were sometimes positive for the presence of WSSV by a polymerase chain reaction (PCR) assay specific for WSSV. These results suggest that F. indicus can be protected from WSD by simple oral administration of IVP.

Journal ArticleDOI
TL;DR: Temporal dynamics of the infection by Perkinsus olseni in a clam (Tapes decussatus) bed was studied over 5 yr and diagnosis techniques were compared to assess their suitability for epizootiological purposes.
Abstract: Temporal dynamics of the infection by Perkinsus olseni in a clam (Tapes decussatus) bed was studied over 5 yr (March 1996 to December 2000). Diagnostic techniques were compared to assess their suitability for epizootiological purposes. A technique based on incubation of 2 gill lamellae in Ray's fluid thioglycollate medium (RFTM) was more sensitive, quicker and cheaper than examination of histological sections. Incubation of the whole-clam soft tissues in RFTM allowed detection of very light infections that were not detected with incubation of only 2 gill lamellae. Nevertheless, the correlation between the infection intensity estimated by both RFTM incubations was high. Infection intensity was significantly and positively correlated with clam size/age. No infected clam smaller than 20 mm was found. There was an annual pattern of infection involving lower mean infection intensity and prevalence in winter and higher values for both variables from spring to autumn, with 2 main annual peaks in spring and late summer-early autumn. This temporal pattern was significantly associated with the seawater temperature. The annual spring peak of infection intensity occurred when seawater temperature was around 15 degrees C. Monthly mortality in the clam bed peaked in spring and summer--after peaks of P. olseni infection intensity and concurrently with high seawater temperature. A comparison of percentage mortality between clams from 2 sources (a perkinsosis-affected and a non-affected area) placed in the same clam bed revealed significantly higher mortality in the clams originating from the perkinsosis-affected area.

Journal ArticleDOI
TL;DR: One of the strains investigated was identified as Vibrio neptunius and is the first description of this species as a molluscan pathogen, which may constitute new species within this genus.
Abstract: Shellfish hatcheries are often affected by disease outbreaks. Three such episodes were investigated in different Galician hatcheries in order to establish the relationship between present microbiota and mortalities. Isolates were obtained from various parts of the hatcheries. Experimental tests for pathogenicity were carried out in microscale experiments using selected strains on Ostrea edulis larvae. The pathogenicity of 1 strain from each outbreak was demonstrated and shown to cause high mortalities (ranging from 98.5 to 100%) in 72 to 96 h after inoculation of larval cultures. All 3 strains belong to the genus Vibrio. One of the strains was identified as Vibrio neptunius and is the first description of this species as a molluscan pathogen. The other 2 strains showed low similarity with the Vibrio species analysed and may constitute new species within this genus.

Journal ArticleDOI
TL;DR: Vital information about the importance of wild and man-made reservoirs for the emergence of ISA in salmon farming is still lacking and information can only be gained by further screening of possible reservoirs, combined with the development of a molecular tool for typing virulence and the geographical origin of the virus isolates.
Abstract: Studies of infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Ireland, Canada, the USA and Chile, suggest that natural reservoirs for this virus can be found on both sides of the North Atlantic. Based on existing information about ISAV it is believed to be maintained in wild populations of trout and salmon in Europe. It has further been suggested that ISAV is transmitted between wild hosts, mainly during their freshwater spawning phase in rivers, and that wild salmonids, mainly trout, are possible carriers of benign wild-type variants of ISAV. Change in virulence is probably a result of deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates after transmission to farmed salmon. Hence, it has been suggested that the frequency of new outbreaks of ISA in farmed salmon could partly reflect natural variation in the prevalence of ISAV in wild populations of salmonids. The aims of the present study were to screen for ISAV in wild salmonids during spawning in rivers and to determine the pathogenicity of resultant isolates from wild fish. Tissues from wild salmonids were screened by RT-PCR and real-time PCR. The prevalence of ISAV in wild trout Salmo trutta varied from 62 to 100% between tested rivers in 2001. The prevalence dropped in 2002, ranging from 13 to 36% in the same rivers and to only 6% in 2003. All ISAV were nonpathogenic when injected into disease-free Atlantic salmon, but were capable of propagation, as indicated by subsequent viral recovery. However, non-pathogenic ISAV has also been found in farmed salmon, where a prevalence as high as 60% has been registered, but with no mortalities occurring. Based on the results of the present and other studies, it must be concluded that vital information about the importance of wild and man-made reservoirs for the emergence of ISA in salmon farming is still lacking. This information can only be gained by further screening of possible reservoirs, combined with the development of a molecular tool for typing virulence and the geographical origin of the virus isolates.

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TL;DR: It is concluded that collection of hemolymph from the adductor sinus is safe for sampled E. complanata and should be explored as a relatively non-invasive, and potentially useful, approach to the evaluation of freshwater mussel health.
Abstract: Hemolymph, the circulatory fluid of bivalves, transports nutrients, respiratory gases, enzymes, metabolic wastes, and toxicants throughout the body. Hemolymph can provide information pertinent to health assessment of animals or populations, but is not commonly used in freshwater bivalves partly because of the lack of tested, practical techniques for its nonlethal collection. The objective of this study was to evaluate the effect of hemolymph collection on the growth and survival of Elliptio complanata, a freshwater bivalve (Unionidae). We describe a simple technique for the collection of hemolymph from the anterior adductor muscle sinus of E. complanata. To evaluate the effect of hemolymph sampling on mussel survival and growth, 30 mussels sampled using the technique and 30 unsampled controls were followed for 3 mo post collection. Nine animals were sampled 3 times over 7 mo to monitor effects of repeated sampling. No negative impacts on survival or growth were observed in either the singly or repeatedly sampled animals. We also compared the composition of hemolymph collected from the adductor muscle sinus with that collected from the ventricle of the heart. Calcium levels and cell count of hemolymph obtained from the adductor sinus and ventricle were significantly different. There was no significant difference between collection sites for magnesium, phosphorus, ammonia, protein, sodium, potassium, or chloride. We conclude that collection of hemolymph from the adductor sinus is safe for sampled E. complanata and should be explored as a relatively non-invasive, and potentially useful, approach to the evaluation of freshwater mussel health.

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TL;DR: The molecular epidemiology of IHNV outbreaks in Germany since its first introduction is investigated, finding IHN viruses in Europe seem to be of a monophyletic origin, again most closely related to North American isolates in the M genogroup.
Abstract: Infectious hematopoietic necrosis virus (IHNV) was first detected in Europe in 1987 in France and Italy, and later, in 1992, in Germany. The source of the virus and the route of introduction are unknown. The present study investigates the molecular epidemiology of IHNV outbreaks in Germany since its first introduction. The complete nucleotide sequences of the glycoprotein (G) and non-virion (NV) genes from 9 IHNV isolates from Germany have been determined, and this has allowed the identification of characteristic differences between these isolates. Phylogenetic analysis of partial G gene sequences (mid-G, 303 nucleotides) from North American IHNV isolates (Kurath et al. 2003) has revealed 3 major genogroups, designated U, M and L. Using this gene region with 2 different North American IHNV data sets, it was possible to group the European IHNV strains within the M genogroup, but not in any previously defined subgroup. Analysis of the full length G gene sequences indicated that an independent evolution of IHN viruses had occurred in Europe. IHN viruses in Europe seem to be of a monophyletic origin, again most closely related to North American isolates in the M genogroup. Analysis of the NV gene sequences also showed the European isolates to be monophyletic, but resolution of the 3 genogroups was poor with this gene region. As a result of comparative sequence analyses, several different genotypes have been identified circulating in Europe.

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TL;DR: To determine the origin of the TSV in Thailand, the viral coat protein gene VP1 was amplified by RT-PCR from several shrimp specimens found positive for TSV by RT, which revealed that the Thai, Burmese and Chinese TSV types formed aClade distinct from a clade of TSVtypes from the Americas.
Abstract: The cultivation of exotic Penaeus vannamei in Thailand began on a very limited scale in the late 1990s, but a Thai government ban on the cultivation of P. monodon in freshwater areas in 2000 led many Thai shrimp farmers to shift to cultivation of P. vannamei. Alarmed by the possibility of Taura syndrome virus (TSV) introduction, the Thai Department of Fisheries required that imported stocks of P. vannamei be certified free of TSV by RT-PCR (Reverse Trasciption Polymerase Chain Reaction) testing. During the interval of allowed importation, over 150,000 broodstock shrimp were imported, 67% of these from China and Taiwan. Despite the safeguards, TSV outbreaks occurred and we confirmed the first outbreak by RT-PCR in early 2003. This resulted in a governmental ban on all shrimp broodstock imports from February 2003, but TSV outbreaks have continued, possibly due to original introductions or to the continued illegal importation of stocks. To determine the origin of the TSV in Thailand, the viral coat protein gene VP1 was amplified by RT-PCR from several shrimp specimens found positive for TSV by RT-PCR from January to November 2003. These included 7 samples from P. vannamei disease outbreaks in Thailand, 3 other non-diseased shrimp samples from Thailand and Burma and 6 samples including P. vannamei and P. japonicus from China. Comparison revealed that the Thai, Burmese and Chinese TSV types formed a clade distinct from a clade of TSV types from the Americas.

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TL;DR: The clinical manifestations, pathology and electron microscopy observations supported by the RT-PCR analysis suggest that the nerve necrosis was due to nodavirus infection in the larvae of the Asian sea bass Lates calcarifer, the first report of piscine nodav virus infection from the Indian sub-continent.
Abstract: Larvae (15 to 21 d post hatch, dph) of the Asian sea bass Lates calcarifer (Bloch) suffered heavy mortalities (60 to 90%) during the hatchery-rearing phase Darkened and moribund larvae showed no evidence of bacterial or parasitic infections Tissue sections of brain and spinal cord showed clear necrotic vacuolation Electron microscopy revealed membrane-bound viral particles in the cytoplasm of the nerve cells The viral particles measured 28 to 30 nm in diameter Primer sets, designed for the amplification of the RNA2 segment of the piscine nodavirus coat protein gene, were used in the RT-PCR analysis of moribund larvae of 20 and 21 dph which produced the amplified product of 430 bp The clinical manifestations, pathology and electron microscopy observations supported by the RT-PCR analysis suggest that the nerve necrosis was due to nodavirus infection in the larvae This is the first report of piscine nodavirus infection from the Indian sub-continent

Journal ArticleDOI
TL;DR: In this article, the authors determined the infectious dose 50% endpoint (SID50 ml(-1)) of a Thai isolate of WSSV by intramuscular inoculation (i.m.) in 60 and 135 d old specific pathogen-free (SPF) Litopenaeus vannamei using indirect immunofluorescence and 1-step polymerase chain reaction (PCR).
Abstract: White spot syndrome virus (WSSV) is a devastating pathogen in shrimp aquaculture. Standardized challenge procedures using a known amount of infectious virus would assist in evaluating strategies to reduce its impact. In this study, the shrimp infectious dose 50% endpoint (SID50 ml(-1)) of a Thai isolate of WSSV was determined by intramuscular inoculation (i.m.) in 60 and 135 d old specific pathogen-free (SPF) Litopenaeus vannamei using indirect immunofluorescence (IIF) and 1-step polymerase chain reaction (PCR). Also, the lethal dose 50% endpoint (LD50 ml(-1)) was determined from the proportion of dead shrimp. The median virus infection titers in 60 and 135 d old juveniles were 10(6.8) and 10(6.5) SID50 ml(-1), respectively. These titers were not significantly different (p > or = 0.05). The titration of the WSSV stock by oral intubation in 80 d old juveniles resulted in approximately 10-fold reduction in virus titer compared to i.m. inoculation. This lower titer is probably the result of physical and chemical barriers in the digestive tract of shrimp that hinder WSSV infectivity. The titers determined by infection were identical to the titers determined by mortality in all experiments using both i.m. and oral routes at 120 h post inoculation (hpi), indicating that every infected shrimp died. The determination of WSSV titers for dilutions administered by i.m. and oral routes constitutes the first step towards the standardization of challenge procedures to evaluate strategies to reduce WSSV infection.

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TL;DR: In 2003, 13 isolates of iridovirus were obtained from cultured flounders Paralichthys olivaceus during epizootics in Korea and were grouped into an unknown genus, different from the 2 genera known to infect fish, Ranavirus and Lymphocystivirus.
Abstract: In 2003, 13 isolates of iridovirus were obtained from cultured flounders Paralichthys olivaceus during epizootics in Korea. The full open reading frames (ORFs) encoding the major capsid protein (MCP) (1362 bp) from the 13 flounder iridoviruses (FLIVs) were sequenced and the deduced amino acid sequences were phylogenetically analyzed. Phylogenetic analysis of the MCP revealed that all 13 FLIVs were the same species as rock bream iridovirus (RBIV), red sea bream iridovirus (RSIV), and infectious spleen and kidney necrosis virus (ISKNV), and were grouped into an unknown genus which was different from the 2 genera known to infect fish, Ranavirus and Lymphocystivirus. This is the first report on the isolation and phylogenetic analysis of the iridovirus of unknown genus from flounders during epizootics.