The pathology of Lates calcarifer herpesviral disease-Disseminated intravascular coagulation explains mortality spikes.

Lates calcarifer herpesvirus (LCHV) causes an emerging serious disease in aquaculture. Sudden drops in feed rates and mortality spikes exceeding 40%-50% often accompany LCHV infections in juvenile L. calcarifer, soon after transfer into sea cages. Affected fish have patchy white skin and fins, corneal opacity and frequently hang in surface water column like 'ghost' or 'zombie' fish. Fish have pale gills, fluid-filled intestines with yellowish casts, lipid depleted liver, enlarged spleen and kidney and reddened brain. Epithelial hyperplasia, apoptosis, marginated nuclear chromatin, amphophilic intranuclear inclusion bodies and the occasional multinucleated cells are observed in gills, skin, intestines, liver and kidney. These are often accompanied by lymphocytic-monocytic infiltration and extensive necrosis in gills, skin, kidney and intestines. Martius scarlet blue stains indicate presence of fibrin in vasculature in brain, gills, intestines, kidney and liver, or disseminated intravascular coagulation (DIC). DIC has been reported in human herpesviral infections. Multifocal lifting of intestinal epithelium with proteinaceous exudate and necrosis of several adjacent villi often progress to involve entire gut sections. Atrophied livers with accentuated lobules may progress to marked loss of hepatic acini. Multifocal dilated attenuated renal tubules are often accompanied by casts and marked protein loosing renopathy. This study on LCHV demonstrates that it can cause significant pathology and mortality.

First detection of Edwardsiella ictaluri (Proteobacteria: Enterobacteriaceae) in wild Australian catfish.

The bacterium Edwardsiella ictaluri is considered to be one of the most significant pathogens of farmed catfish in the United States of America and has also caused mortalities in farmed and wild fishes in many other parts of the world. E. ictaluri is not believed to be present in wild fish populations in Australia, although it has previously been detected in imported ornamental fishes held in quarantine facilities. In an attempt to confirm freedom from the bacterium in Australian native fishes, we undertook a risk-based survey of wild catfishes from 15 sites across northern Australia. E. ictaluri was detected by selective culturing, followed by DNA testing, in Wet Tropics tandan (Tandanus tropicanus) from the Tully River, at a prevalence of 0.40 (95% CI 0.21-0.61). The bacterium was not found in fishes sampled from any of the other 14 sites. This is the first report of E. ictaluri in wild fishes in Australia.

Renal mucous production in Australian freshwater plotosid and ariid catfishes.

This study reports the presence of large mucous cells lining the large renal collecting ducts and opisthonephric ducts of the Australian freshwater catfishes Tandanus tandanus and Tandanus tropicanus. Histologic examination of the renal tissue from four other species of Australian freshwater catfishes suggests that this feature is unique to the two species of Tandanus studied. The potential functions of renal mucous secretion in T. tandanus and T. tropicanus are discussed.

Case studies of spinal deformities in ornamental koi, Cyprinus carpio L.

This is a study of vertebral deformities in ornamental koi based on computed radiography and skeletons cleaned by dermestid beetles (Dermestes maculatus). All koi developed gradual onset of swimming abnormalities as adults. Extensive intervertebral osteophyte formation correlated with age of fish and was associated with hindquarter paresis in one koi. Vertebral compression and fusion were the most common spinal deformities occurring at multiple sites, similar to findings in other farmed fish. Site-specific spinal deformities were thought to develop due to differences in swimming behaviour and rates of vertebral growth. One koi had offspring with spinal deformities. Spinal deformities are significant problems in both European and Australian food fish hatcheries. The heritability of vertebral deformities in farmed fish is reportedly low unless there is concurrent poor husbandry or nutritional deficiencies. The specific aetiologies for vertebral deformities in koi in this study could not be ascertained. Current knowledge on spinal deformities in the better studied European food fish species suggests multifactorial aetiologies. Future research should include prospective longitudinal studies of larger numbers of koi from hatch and consideration of all potential risk factors such as husbandry, nutrition, temperature, photoperiod and genetics.

Additional novel Cryptosporidium genotypes in ornamental fishes.

Current knowledge on the prevalence and genotypes of Cryptosporidium in fishes is still limited. This study investigated the prevalence of Cryptosporidium species in 171 ornamental fishes, belonging to 33 species, collected from 8 commercial aquariums around Perth, Western Australia. All samples were screened by nested PCR targeting the 18S rRNA locus. A total of 6 positives were identified by PCR at the 18S locus from 4 different species of fishes (red eye tetra, Moenkhausia sanctaefilomenae; gold gourami, Trichogaster trichopterus; neon tetra, Paracheirodon innesi; goldfish, Carassius auratus auratus), giving an overall prevalence of 3.5% (6/171). Four different genotypes were identified, only one of which has been previously reported in fish; piscine genotype 4 in a neon tetra isolate, a rat genotype III-like isolate in a goldfish, a novel genotype in three isolates from red eye (piscine genotype 7) which exhibited a 3.5% genetic distance from piscine genotype 1 and a piscine genotype 6-like from a gold gourami (1% genetic distance). Further biological and genetic characterisation is required to determine the relationship of these genotypes to established species and strains of Cryptosporidium.

The pathology of 'scale drop syndrome' in Asian seabass, Lates calcarifer Bloch, a first description.

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.

The molecular characterization of an Eimeria and Cryptosporidium detected in Asian seabass (Lates calcarifer) cultured in Vietnam.

An intestinal Eimeria was previously reported as a significant pathogen of Asian seabass (Lates calcarifer) in nurseries in Vietnam. In the present study, both Eimeria and Cryptosporidium were detected by sequence analyses of fragments of the 18S rRNA gene amplified from these Vietnamese L. calcarifer tissues. Based on these analyses, the Eimeria from the Vietnamese L. calcarifer formed clades with the Eimeria detected in L. calcarifer tissues from Australia, but clustered separately from other known Eimeria and Goussia species. The Cryptosporidium detected in L. calcarifer from Vietnam clustered closest with C. parvum and C. hominis. In situ hybridization using DIG-labeled DNA probes generated from 18S PCR products on the Vietnamese L. calcarifer wax block tissues showed that this method could not be used to distinguish between Eimeria and Cryptosporidium, due to the conserved nature of the 18S locus. A previously published study on the morphology of parasite developmental stages and oocysts in the Vietnamese L. calcarifer tissues showed only an intestinal Eimeria infection. The Cryptosporidium could be present at very low levels undetectable by microscopy in intestines, or being ubiquitous, was a possible contaminant from feed or water. While molecular analysis is a very useful tool in the study of disease and identification of aetiological agents, this study reiterates the importance of demonstrating organisms in situ in tissues.

An intestinal Eimeria infection in juvenile Asian seabass (Lates calcarifer) cultured in Vietnam--a first report.

This is the first report of an intestinal Eimeria infection in Asian seabass (Lates calcarifer) at the histopathological and ultrastructural levels. The Eimeria infection was often associated with severe pathology and significant mortality in the absence of other pathogens. This showed that it is an important disease of juvenile L. calcarifer in small scale nurseries in Vietnam. Heavy infection and high prevalence levels of the Eimeria infection are suspected to be linked to the low daily water exchange rates practised in these nurseries. Although systemic iridovirus infection was concurrently observed in some of the fish examined, it was not as consistently present in diseased fish as the Eimeria infection.

Reduced postsynaptic GABAA receptor number and enhanced gaboxadol induced change in holding currents in Purkinje cells of the dystrophin-deficient mdx mouse.

UNLABELLED: Duchenne muscular dystrophy (DMD) is caused by the absence of a functional transcript of the protein dystrophin. DMD is associated with a range of cognitive deficits that are thought to result from a lack of the protein dystrophin in brain structures involved in cognitive functions. The CNS involvement extends to an impairment of cognitive abilities, with many DMD boys having significant reduction in IQ. In the cerebellum, dystrophin is normally localized at the postsynaptic membrane of GABAergic synapses on Purkinje cells. Here, we investigate the effect of an absence of dystrophin on the number of GABA(A) channels located at the synapse in cerebellar Purkinje cells of the dystrophin-deficient mdx mouse. Whole-cell patch-clamp recordings of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) were performed in cerebellar slices from mdx and littermate control mice. Our results showed that the number of receptors at GABAergic synapses in the cerebellar Purkinje cell was significantly reduced in mdx mice (38.38 ± 2.95) compared to littermate controls (53.03 ± 4.11). Furthermore, when gaboxadol was added to the bath, the change in holding current in mdx mice was significantly enhanced (65.01 ± 5.89pA) compared to littermate controls (37.36 ± 3.82pA). The single channel unitary conductance and the rise and decay time of mIPSCs were not significantly different in these two groups of mice, indicating that those GABA(A) channels located at the postsynaptic sites in the mdx mice function normally. CONCLUSION: There is a reduction in the number of functional receptors localized at GABAergic synapses in the cerebellar Purkinje cells of dystrophin-deficient mdx mice and an increase in a gaboxadol induced holding current, which is evidence for an increase in extrasynaptic GABA(A) receptors in mdx mice. We hypothesize that the absence of dystrophin, from mdx Purkinje cells, reduces the number of post-synaptic GABA(A) receptors and as a result there is an increase in extrasynaptic receptors. If similar changes occur in the CNS in boys with DMD, it will impact on the function of neural networks and may contribute to some of the motor, behavioral and cognitive impairment apparent in many boys with DMD.

Proposal for a fourth aquabirnavirus serogroup.

Four putative aquabirnaviruses, based on morphology, nucleic acid type and partial RNA-dependent RNA polymerase gene (VP1) sequence, isolated from three tropical freshwater fish species were not neutralised by antisera against type members of the Aquabirnavirus genus serogroups A, B or C. Antisera produced against two of the isolates neutralised the homologous and heterologous isolates, but not any type member of Aquabirnavirus serogroups A, B or C. The serological comparisons suggest that the four isolates should be regarded as members of a fourth Aquabirnavirus serogroup, D.

GABA(A) receptor expression and inhibitory post-synaptic currents in cerebellar Purkinje cells in dystrophin-deficient mdx mice.

1. Duchenne muscular dystrophy (DMD) is the second most common fatal genetic disease and arises as a consequence of an absence or disruption of the protein dystrophin. In addition to wasting of the skeletal musculature, boys with DMD have a significant degree of cognitive impairment. 2. We show here that there is no difference between littermate control and mdx mice (a murine model of DMD) in the overall expression of the GABA(A) receptor a1-subunit, supporting the suggestion that it is the clustering at the synapse that is affected and not the expression of the GABA(A) receptor protein. 3. We report a significant reduction in both the frequency and amplitude of spontaneous inhibitory post-synaptic currents in cerebellar Purkinje cells of mdx mice compared with littermate controls, consistent with the reported reduction in the number and size of GABA(A) receptor clusters immunoreactive for a1- and a2-subunits at the post-synaptic densities. 4. These results may explain some of the behavioural problems and cognitive impairment reported in DMD.

A systemic iridoviral disease in mullet, Mugil cephalus L., and tiger grouper, Epinephelus fuscoguttatus Forsskal: a first report and study.

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.

The pathology of systemic iridoviral disease in fish.

Iridoviruses have been associated with severe disease and economic loss in farmed food fish and ornamental fish, with mortality often reported to reach 50% or more. In the present study, three tropical marine food fish species and four tropical freshwater ornamental fish species with systemic iridovirus infections were examined histopathologically and ultrastructurally. Light microscopy consistently revealed pale to intensely basophilic hypertrophied virus-infected cells in spleen, kidney and intestine from all seven species. Ultrastructural examination showed changes in the vascular endothelium overlying hypertrophied virus-infected cells suggestive of pressure necrosis. Viral isolation was improved by the use of fibroblastic cell lines. This, together with the sub-endothelial location of infected cells in all infected species examined, suggests that systemic iridoviruses are mesotheliotropic.

Characterization of a novel ranavirus isolated from grouper Epinephelus tauvina.

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 approximately 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.