A large icosahedral virus was isolated from diseased grouper Epinephelus tauvina. The virus grew well in several cultured fish cell lines, with stable and high infectivity after serial passages in grouper cell line (GP). The virus was sensitive to both acid and heat treatments. Virus replication was inhibited by 5-iodo-2-deoxyuridine (IUDR), indicative of a DNA-containing genome. The virus infectivity was reduced with ether treatment, suggesting that the virus was lipid-enveloped. Electron micrographs showed abundant cytoplasmic icosahedral virons in the virus-infected GP cells. The size of the intracellular nucleocapsid was 154 nm between the opposite sides, or 176 nm between the opposite vertices with an inner electron-dense core of 93 nm. Virus particles were released through budding from plasma membranes with a size of 200 nm in diameter. SDS-PAGE of purified virus revealed 20 structural protein bands and a major capsid protein (MCP) of 49 kDa. A DNA fragment of ~500 nucleotides was successfully amplified by polymerase chain reaction (PCR) using the primers from conserved regions of the MCP gene of frog virus 3 (FV3), the type species of Ranavirus. Subsequent multiple alignment and phylogenetic analysis showed that the newly isolated grouper virus was closely related to largemouth bass virus (LMBV), FV3 and Regina ranavirus (RRV). Our data suggests that the virus isolate is a novel member of genus Ranavirus, family Iridoviridae. We tentatively name the virus as Singapore grouper iridovirus (SGIV). SGIV was able to cause serious systemic disease capable of killing 96% of grouper fry. KEY WORDS: Fish virus · Iridovirus · Ranavirus · Iridoviridae · Grouper · Epinephelus tauvina Resale or republication not permitted without written consent of the publisherDis Aquat Org 53: [1][2][3][4][5][6][7][8][9] 2003 significant economic losses in some Singapore marine net-cage farms. The pathogen was suggested as an iridovirus based on histopathological and morphological evidence. However, the virus was not isolated by cell culture, and no biochemical data are available to confirm the virus as a member of the family Iridoviridae (Chua et al. 1994). In 1998, an outbreak of the same disease occurred in fry and adult brown-spotted groupers. The grouper fry were imported from other SE Asian countries and cultured in fish farms in Singapore. The outbreak lasted several weeks and resulted in more than 90% mortality. The present work describes isolation of the viral pathogen in cell culture, investigation of virus infectivity and pathogenicity, and characterization of the virus based on biochemical, structural and molecular properties. MATERIALS AND METHODSCell lines and maintenance. Three local tropical marine-fish cell lines and 3 commercial fish cell lines were used. Grouper (GP) embryo cells from brownspotted grouper Epinephelus tauvina (Chew-Lim et al. 1994), Asian seabass fry (SF) cells from Lates calcarifer (Chang et al. 2001), and Asian seabass (SB) embryo cells (Chong et al. 1987) were cultured in Eagles' mi...

No abstract

This is the first pathological description of 'scale drop syndrome' (SDS) in Asian seabass, Lates calcarifer Bloch. Cumulative mortality was estimated at 40-50%. The vasculitis in all major organs including the skin and associated tissue necrosis was distinctive. The dermis overlying scale beds was often necrotic and associated with scale loss. Necrosis of splenic ellipsoids, renal glomeruli and choroid rete glands of eye were further hallmarks of a disease with systemic vascular involvement. The brain was not spared vascular damage, and the resulting multifocal encephalomalacia probably accounts for the spiral swimming behaviour in some affected fish. Other lesions included accentuated hepatic lobulation and gastric gland necrosis. Nuclear chromatin margination and karyolysis in hepatocytes, renal tubular epithelium and gastric and intestinal epithelium suggest specific targeting of cells. Basophilic cytoplasmic inclusions were present in spleen, kidney, liver, heart and choroid rete, but they were not prominent. Using transmission electron microscopy, two morphological forms of virions were observed: single- and double-enveloped hexagonal virions. Based on size and morphology, these virions resemble iridovirus or herpesvirus. The cause of SDS is unknown, but the pathological changes, especially the vasculitis, suggest an infectious aetiology, possibly viral.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The morphological, biological, and molecular characteristics of Cryptosporidium 28 piscine genotype 1 from the guppy (Poecilia reticulata) are described, and the species 29 name Cryptosporidium huwi n. sp. is proposed to reflect its genetic and biological 30 differences from gastric and intestinal Cryptosporidium species. Oocysts of C. huwi n. within the epithelium. However, phylogenetic analysis of 18S rRNA sequences 36 indicated that C. huwi n. sp. exhibited 8.5-9.2% and 3.5% genetic distance from C. 37 molnari isolates and piscine genotype 7 respectively. At the actin locus, the genetic 38 distance between C. huwi n. sp. and C. molnari was 16.6%. The genetic distance 39 between C. huwi n. sp. and other Cryptosporidium species at the 18S locus was 40 13.2%-17% and at the actin locus was 18.9%-26.3%. Therefore C. huwi n. sp. is 41 genetically distinct from previously described Cryptosporidium species. Cryptosporidium huwi

a b s t r a c tCryptosporidium is an enteric parasite that infects humans and a wide range of animals. Relatively little is known about the epidemiology and taxonomy of Cryptosporidium in fish. In the present study, a total of 775 fish, belonging to 46 species and comprising ornamental fish, marine fish and freshwater fish were screened for the prevalence of Cryptosporidium by PCR. The overall prevalence of Cryptosporidium in fish was 5.3% (41/775), with prevalences ranging from 1.5 to 100% within individual host species. Phylogenetic analysis of these Cryptosporidium isolates as well as 14 isolates from previous studies indicated extensive genetic diversity as well as evidence for mixed infections. At the 18S locus the following species were identified; Cryptosporidium molnari-like genotype (n = 14), Cryptosporidium huwi (n = 8), piscine genotype 2 (n = 4), piscine genotype 3-like (n = 1), piscine genotype 4 (n = 2), piscine genotype 5 (n = 13), piscine genotype 5-like (n = 1) and five novel genotypes (n = 5). At the actin locus, species identification agreed with the 18S locus for only 52.3% of isolates sequenced, indicating high levels of mixed infections. Future studies will need to employ both morphological characterization and deep sequencing amplicon-based technologies to better understand the epidemiological and phylogenetic relationships of piscine-derived Cryptosporidium species and genotypes, particularly when mixed infections are detected.

An intestinal Eimeria infection in juvenile Asian seabass (Lates calcarifer

A systemic iridoviral disease associated with high mortality was initially recognized in cultured mullet, Mugil cephalus L., and tiger grouper, Epinephelus fuscoguttatus Forsskal, by histopathology and transmission electron microscopy. Polymerase chain reaction was performed on tissues and viral isolates, using four published primer sets developed for the Red Sea bream iridovirus (RSIV). An indirect fluorescent antibody test was also performed on virus-infected ATCC gruntfin (GF) and seabass, Lates calcarifer Bloch, (SB) cells using a monoclonal antibody, RSIV M10. Our results suggested that the mullet and tiger grouper iridovirus bears genetic and antigenic similarities to RSIV.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 19A c c e p t e d M a n u s c r i p t