Annual variation in lymphocystis infection frequency in flounder, Platichthys flesus (L.)

The infection frequency of the virus induced lymphocystis disease has been registered in flounder from the Oslofjord, Norway, during a 4 year period. The disease has a clear regular seasonal variation, and also an annual variation. The percentage infection in the summer was 1–10%, and in the winter up to 57%. The possibility that the high pollution level of the inner Oslofjord could be the reason for the high frequency of lymphocystis disease, is discussed.     

Whitespotted puffer Arothron hispidus, a new host for lymphocystis in Qingdao Aquarium of China

In April 2004 white nodular lesions were found on the fins of whitespotted puffer Arothron hispidus (Linnaeus). Diagnostic studies were carried out to confirm the disease using light and electron microscopy, histochemical methods and PCR. The results revealed that the nodules were composed of giant cells up to 400 microm in diameter. These cells were surrounded by a periodic acid-Schiff (PAS)-positive hyaline capsule containing dot-shaped, Feulgen-positive inclusion bodies in the cytoplasm and an irregular nucleus. Numerous virus particles 200 nm in diameter and with hexagonal profiles were observed in the cytoplasm. These features were consistent with those of lymphocystis disease. Additionally, based on the gene sequences of major capsid protein (MCP) of lymphocystis disease virus (LCDV) from Japanese flounder Paralichthys olivaceus, 2 pair primers were designed; after a nested PCR was performed for detection of LCDV in A. hispidus, a positive amplified product was obtained showing the presence of LCDV. Therefore, the white nodules were the lymphocystis lesions caused by LCDV infection and A. hispidus was demonstrated to be a new host for LCDV.     

Iridovirus infections in farm-reared tropical ornamental fish

A systemic viral infection in both gourami Trichogaster spp. and swordtail Xiphophorus hellerii and an outbreak of lymphocystis in scalare Pterophyllum scalarae and gourami are reported to have occurred in fish reared in ornamental fish farms in Israel. The systemic infection developed in endothelial cells that became hypertrophic and their contents were modified. The presence of such cells in light-microscopically examined stained smears and sections provides an initial indication for this systemic viral infection. Infection in gourami caused hemorrhagic dropsy. Transmission electron microscopic (TEM) images of iridovirus-like particles recovered from gouramies showed them to be 138 to 201 nm from vertex to vertex (v-v); those from swordtails were 170 to 188 nm v-v. TEM images of lymphocystis virions from scalare were 312 to 342 nm v-v and from gourami 292 to 341 nm v-v. Lymphocystis cells from the gourami were joined by a solid hyaline plate, which was lacking in the infection in scalare where the intercellular spaces between the lymphocystis cells consisted of loose connective tissue.     

A histopathological report of some epidermal anomalies of Dover sole, Solea solea L., and other flatfish species in coastal waters off south-east England

The gross pathological and histological changes of typical examples of epidermal anomalies of Dover sole, Solea solea L., and other flatfish from the sewage dumping areas of the Thames Estuary and two 'control' areas are described. Five per cent of flatfish showed anomalies including trawl damage abrasions, fin-rot, ulcers, nodules and tumours, lymphocystis and skeletal anomalies. Many of the anomalies suggested a traumatic aetiology and were often chronic. There was no significant difference in disease incidence between the areas sampled, except more evidence of lymphocystis in one of the control areas.     

Population dynamics of lymphocystis disease in estuarine flounder, Platichthys flesus (L.)

The population dynamics of viral lymphocystis disease in an age-structured population of flounder, Plarichthys flesus (Linnaeus 1758), is studied with a mathematical model. For a single cohort the model illustrates the influence of host density and acquired immunity on length-prevalence profiles. This case corresponds to a host population at demographic equilibrium. When the model is extended to several cohorts, seasonal recruitment of susceptible hosts is shown to drive seasonality in disease prevalence. In both cases, there is good qualitative agreement between model predictions and field data from the Elbe estuary, Germany.     

Gill X-cell lesions in American plaice (Hippoglossoides platessoides) concurrent with lymphocystis infection

This paper documents the first report of concurrent infections of lymphocystis lesions and X-cells and the varied associations between multiple, clustered or single lymphocystis lesions and X-cells in a NW Atlantic pleuronectid.     

Molecular evidence for association of chlamydiales bacteria with epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer)

Epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer), previously associated with chlamydial bacterial infection using ultrastructural analysis, was further investigated by using molecular and immunocytochemical methods. Morphologically, all three species showed epitheliocystis cysts in the gills, and barramundi also showed lymphocystis cysts in the skin. From gill cysts of all three species and from skin cysts of barramundi 16S rRNA gene fragments were amplified by PCR and sequenced, which clustered by phylogenetic analysis together with other chlamydia-like organisms in the order Chlamydiales in a lineage separate from the family Chlamydiaceae. By using in situ RNA hybridization, 16S rRNA Chlamydiales-specific sequences were detected in gill cysts of silver perch and in gill and skin cysts of barramundi. By applying immunocytochemistry, chlamydial antigens (lipopolysaccharide and/or membrane protein) were detected in gill cysts of leafy seadragon and in gill and skin cysts of barramundi, but not in gill cysts of silver perch. In conclusion, this is the first time epitheliocystis agents of leafy seadragon, silver perch and barramundi have been undoubtedly identified as belonging to bacteria of the order Chlamydiales by molecular methods. In addition, the results suggested that lymphocystis cysts, known to be caused by iridovirus infection, could be coinfected with the epitheliocystis agent.     

Infection and propagation of lymphocystis virus isolated from the cultured flounder Paralichthys olivaceus in grass carp cell lines

The causative agent of lymphocystis disease that frequently occurs in cultured flounder Paralichthys olivaceus in China is lymphocystis virus (LV). In this study, 13 fish cell lines were tested for their susceptibility to LV. Of these, 2 cell lines derived from the freshwater grass carp Ctenopharyngodon idellus proved susceptible to the LV, and 1 cell line, GCO (grass carp ovary), was therefore used to replicate and propagate the virus. An obvious cytopathic effect (CPE) was first observed in cell monolayers at 1 d post-inoculation, and at 3 d this had extended to about 75% of the cell monolayer. However, no further CPE extension was observed after 4 d. Cytopathic characteristics induced by the LV were detected by Giemsa staining and fluorescence microscopic observation with Hoechst 33258 staining. The propagated virus particles were also observed by electron microscopy. Ultrastructure analysis revealed several distinct cellular changes, such as chromatin compaction and margination, vesicle formation, cell-surface convolution, nuclear fragmentation and the occurrence of characteristic 'blebs' and cell fusion. This study provides a detailed report of LV infection and propagation in a freshwater fish cell line, and presents direct electron microscopy evidence for propagation of the virus in infected cells. A possible process by which the CPEs are controlled is suggested.     

Sexing river buffalo (Bubalus bubalis L.), sheep (Ovis aries L.), goat (Capra hircus L.), and cattle spermatozoa by double color FISH using bovine (Bos taurus L.) X- and Y-painting probes

River buffalo, sheep, and goat spermatozoa were cross-hybridized using double color fluorescence in situ hybridization (FISH) with bovine Xcen- and Y-chromosome painting probes, prepared by DOP-PCR of laser-microdissected-catapulted chromosomes, to investigate the possibility of using bovine probes for sexing sperm of other members of the family Bovidae. Before sperm analysis, the probes were hybridized on metaphase chromosomes of each species, as control. Frozen-thawed spermatozoa of cattle, river buffalo, sheep, and goat were decondensed in suspension with 5 mM DTT. Sperm samples obtained from three individuals of each species were investigated, more than 1,000 spermatozoa were scored in each animal. FISH analysis of more than 12,000 sperm revealed high level of sperm with X- or Y-signals in all of the species investigated, indicating FISH efficiency over 99%. Significant interspecific differences were detected in the frequency of aberrant spermatozoa (aneuploid and diploid) between goat (0.393%) and sheep (0.033%) (P < 0.01), goat and cattle (0.096%) (P < 0.5), as well as between river buffalo (0.224%) and sheep (P < 0.5). There was no significant difference between river buffalo and cattle. The present study demonstrated that it is possible to use bovine X-Y painting probes for sexing and analyzing sperm of other species of the family, thus facilitating future studies on the incidence of chromosome abnormalities in sperm as well as on sex predetermination of embryos for the livestock industry. Mol. Reprod. Dev. 67: 108-115, 2004.     

Molecular Evidence for Association of Chlamydiales Bacteria with Epitheliocystis in Leafy Seadragon (Phycodurus eques), Silver Perch (Bidyanus bidyanus), and Barramundi (Lates calcarifer)

Epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer), previously associated with chlamydial bacterial infection using ultrastructural analysis, was further investigated by using molecular and immunocytochemical methods. Morphologically, all three species showed epitheliocystis cysts in the gills, and barramundi also showed lymphocystis cysts in the skin. From gill cysts of all three species and from skin cysts of barramundi 16S rRNA gene fragments were amplified by PCR and sequenced, which clustered by phylogenetic analysis together with other chlamydia-like organisms in the order Chlamydiales in a lineage separate from the family Chlamydiaceae. By using in situ RNA hybridization, 16S rRNA Chlamydiales-specific sequences were detected in gill cysts of silver perch and in gill and skin cysts of barramundi. By applying immunocytochemistry, chlamydial antigens (lipopolysaccharide and/or membrane protein) were detected in gill cysts of leafy seadragon and in gill and skin cysts of barramundi, but not in gill cysts of silver perch. In conclusion, this is the first time epitheliocystis agents of leafy seadragon, silver perch and barramundi have been undoubtedly identified as belonging to bacteria of the order Chlamydiales by molecular methods. In addition, the results suggested that lymphocystis cysts, known to be caused by iridovirus infection, could be coinfected with the epitheliocystis agent.     

Seasonal prevalence of skin tumors from walleye (Stizostedion vitreum) from Oneida Lake, New York

A seasonal survey of skin tumor prevalence in walleyes (Stizostedion vitreum) was conducted during the ice-free period on Oneida Lake, New York in 1986. During the survey, 1,028 walleyes were collected and examined for the presence of lymphocystis disease, dermal sarcoma, discrete epidermal hyperplasia and diffuse epidermal hyperplasia. Skin growths were high in prevalence in early spring, low in prevalence during the summer, and again high in prevalence in the fall. Lymphocystis disease and dermal sarcoma were more frequently observed than either discrete or diffuse epidermal hyperplasia. Histologically, a moderate to severe inflammatory response was associated with dermal sarcoma in the early spring and late spring but not in the fall. Regardless of the time of year, varying degrees of inflammatory response were seen associated with lymphocystis disease. Samples were inadequate to assess seasonal trends in incidence of discrete and diffuse epidermal hyperplasia.     

Lymphocystis disease in the winter flounder, Pseudopleuronectes americanus.

Nodular lesions on the fins of winter flounder, Pseudopleuronemphocystis disease. Hypertrophied, encapsulated connective tissue cells contained cytoplasmic inclusions composed of icosahedral virus particles. The winter flounder is a new host for lymphocystis disease and is the second flatfish in the western North Atlantic to have the disease.     

Assessment of some innate immune responses in dab (Limanda limanda L.) from the North Sea as part of an integrated biological effects monitoring

The marine flatfish dab (Limanda limanda), which lives in direct contact with contaminated sediments, is frequently used as a sentinel species in international monitoring programmes on the biological effects of contaminants. In this study, immune responses were recorded as indicators of sublethal chronic effects of contaminants, in addition to measurement of the induction of mono-oxygenase ethoxyresorufin O-deethylase (EROD) in liver cells, the inhibition of acetylcholin esterase (AChE) in muscle and a quantification of grossly visible diseases and parasites. In total, 336 dab were analysed from five sampling areas in the North Sea, including the German Bight, the Dogger Bank, the Firth of Forth, and two locations close to oil and gas platforms (Ekofisk and Danfield). When considering plasma lysozyme levels, pinocytosis and respiratory burst activity of head kidney leucocytes, a clear gradient could be observed with decreased levels in individuals collected from the Firth of Forth and locations near the oil or gas platforms compared with dab from the Dogger Bank or the German Bight. Individuals with induced EROD activity displayed reduced lysozyme and respiratory burst activities. Lysozyme levels were also reduced in dab with lymphocystis or with nematodes. The data obtained indicate that the assessment of innate immune parameters in a monitoring programme provides supplementary information about immunomodulatory effects associated with the exposure of fish to contaminants. In particular, concentrations of plasma lysozyme, which can be analysed in an easy and inexpensive assay, are considered to be an appropriate parameter for use in a battery of other bioindicators. Communicated by H. von Westernhagen and A. Diamant     

The diet and growth of 0-group dace, Leuciscus leuciscus (L.), and roach, Rutilus rutilus (L.), in a lowland river

The diets of 0-group dace and roach in the Lower Welsh Dee were studied by stomach contents analysis of seasonal and diel samples. During development, dace preyed on a sequence of food types, initially being mainly carnivorous then becoming nocturnally carnivorous and diurnally omnivorous. Roach showed a similar pattern, although after the first two months of life they were more herbivorous/detritivorous. From the variations in the diets, it is hypothesised that both species are limited by food. This receives circumstantial support from their poor observed growth. The food supply routes by which flow regulation of the River Dee could affect juvenile fish production are noted.     

Predation by Nile perch Lates niloticus (L.) on Oreochromis niloticus (L.), Cyprinus carpio (L.), Mugil sp. and its role in controlling tilapia recruitment in Egypt

Vulnerability of three fish groups (tilapia, common carp, mullet) to predation by Nile perch Lates niloticus was evaluated. The study was conducted in aquaria and in cages placed in concrete ponds. Common carp Cyprinus curpio was the more vulnerable to predation by Nile perch followed in order by mullet ( Mugil cephulus and M. cupito ) and Nile tilapia Oreochromis niloricus . Medium-size Nile perch (30–32 cm t.l. ) were able to consume tilapia up to 14 cm t.l. , while tilapia of 15 cm total length were not eaten. Ratios of 1 : 10 to 1 : 15 between Nile perch and ready-to-spawn females of O. niloticus were found to be adequate to control tilapia reproduction.     

Electrophoretic identification of roach (Rutilus rutilus L.), rudd (Scardinim erythrophthalmus L.), bream (Abramis brama L.) and their natural hybrids

Roach, rudd, bream and their natural hybrids of 2 cm standard length or larger can be definitively identified by their enzyme electrophoretic patterns. Zymograms of lactate dehydrogenase and esterases as produced by vertical starch gel electrophoretic analysis of whole fry or adult eye extracts are the most useful in this respect. The lactate dehydrogenase isozymes, containing B sub-units, migrate more anodally in rudd and bream than in roach. Due to the tetrameric structure of lactate dehydrogenase, in hybrid rudd x roach and roach x bream, eleven isozymes can be observed as compared with six in the parental patterns. Esterases show unique patterns for all species and hybrids. With the exception of one fraction in rudd x bream, the esterase patterns of hybrids show summations of the parental phenotypes.     

Population studies ofBrevicoryne brassicae (L.), its parasites and hyperparasites in England

Studies on populations of Brevicoryne brassicae (L.), its parasites and hyperparasites were carried out by actual counting in the sprouts field and by sticky and water traps. B. brassicae was found to be attacked by one primary parasite, Diaretiella rapae (McIntosh ), which in turn is parasitized by Alloxysta brassicae (Ashm. ),Asaphes vulgarisWalker ,A. suspensus (Nees ),Pachyneuron minutissimum (Förster ) and Dendrocerus carpenterii (Curtis ). The aphid population in the field was started by immigrant alates which were found flying too early to be synchronized with the sprouts plants. Similarly D. rapae was not synchronized with the aphids although many individuals could have been carried into a plot through parasitized immigrant alates, of which less than 30% were found parasitized. Because of high hyperparasitism (especially by A. brassicae)D. rapae was not able to maintain a high rate of parasitism to curb the aphid population growth. The maximum percentage mummies being 27.8%, while the maximum, percentage parasitism being 56.6% recorded only during the early 1974 season (mean=12.9%). The decline of aphid population from September onwards was largely due to the cold weather, Syrphid predation and occasionally fungal attack. The high rate of hyperparasitism by A. brassicae is attributed to its better synchronization with D. rapae. The mean percentage of parasite that emerged from mummies collected during 1973–74 were D. rapae 31.3%,A. brassicae 64.3%,A. vulgaris and A. suspensus 4.3%,D. carpenterii 0.2% and P. minutissimum 0.1%.     

Linkage Mapping of Toll-Like Receptors (TLRs) in Japanese Flounder, <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

Toll-like receptors (TLRs) are responsible for the recognition of specific pathogen-associated molecular patterns and consequently activate signal pathways leading to inflammatory and interferon responses. The region surrounding several TLRs was previously found to be associated with resistance to specific disease. Hence, we determined the location of 11 TLRs in Japanese flounder (Paralichthys olivaceus) using polymorphic microsatellite markers. TLR1 and TLR3 were located on linkage group (LG) 21 and 7, respectively. Membrane TLR5 and soluble TLR5 were mapped to LG22. TLR7 and TLR8 were mapped to LG3. TLR9 was found on LG1 and TLR14 and TLR21 were located on the same linkage group, LG10. TLR22 was found on LG8. Interestingly, TLR2 was mapped with the previously reported Poli9-8TUF microsatellite marker which is tightly associated with lymphocystis virus disease resistance. Therefore, TLR2 is a candidate gene for resistance to lymphocystis disease. These results imply that the location of a TLR associated with a particular disease may be valuable for the research on the relationship between host immune response and disease resistance.     

The frequency, distribution, and pathology of three diseases of demersal fishes in the Bering Sea

During September and October, 1975, bottom-dwelling fish from the Bering Sea were examined for diseases. The three most commonly observed diseases and their frequency of occurrence were epidermal papillomas of rock sole ( Lepidopsetta bilineata Ayres), 1-0%; lymphocystis of yellowfln sole ( Limanda aspera Pallas), 2-1 %; and epidermal tumours of the pseudobranch of adult Pacific cod ( Gadus macrocephalus Tilesius), 7-4%. Both the appearance and the histological characteristics of epidermal papillomas on rock sole resembled those found on several species of flatfish in Puget Sound. The frequency of tumour-bearing rock sole ranged from 0-23%, with the highest occurrence at sampling stations around the periphery of the Bering Sea in water depths of 20-30 fathoms (36-54 m). The virus-caused lymphocystis lesions were usually on the 'blind' side fins and skin surfaces of yellowfin sole, the largest lesions being found on the operculum, and the most common site of infection was the pectoral fin. The frequency of lymphocystis was 0-15 %, with a gradual increase of prevalence from the northern to southern sampling stations. The Pacific cod tumours were always bilateral, and contained remnants of normal-appearing pseudobranchial tissue on the surface or deep inside the tumours. Portions of some tumours were necrotic and liquefied. Tumour-bearing cod were most commonly found in the south and south-eastern Bering Sea and the frequency of affected cod ranged from 0-100%.