Effects of Acholeplasma laidlawii and an unidentified mycoplasma on selected fish cell cultures and the replication of fish viruses

Abstract. An unidentified mycoplasma was isolated from cultures of Atlantic salmon (AS) cells and implicated as the cause of cytopathic effects (CPE). The agent did not visibly affect RTG-2 cells. Experimental infection of RTG-2 cells with Acholeplasma laidlawii resulted in increased cellular granularity and destruction. Scanning electron microscopic (SEM) examination of infected RTG-2 and AS cultures revealed typical mycoplasmas distributed on cell surfaces and a marked effect on the cell surface topography, characterized by a loss of microvilli and cellular processes. SEM also revealed mycoplasmal contamination that was not detected by culturing. Comparisons of contaminated and uncontaminated RTG-2 cells showed enhanced (2–100 fold) replication of infectious pancreatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV) in the presence of A, laidlawii .     

Studies on a host range variant from different isolates of infectious pancreatic necrosis virus (IPNV)

Abstract. Eight isolates of infectious pancreatic necrosis virus (IPNV) propagated solely in RTG-2 cells (RTG-IPNV) were examined for ability to replicate in FHM cells. Seven isolates replicated in FHM cells; however, the plaque titres were 10- to greater than 100 000-fold less than titres in RTG-2 cells. The ability of IPNV to replicate in FHM cells was shown to result from the presence of a host range variant (FHM-IPNV) that produced plaques in both cell types. Variant-free preparations of two isolates differed in ability to generate FHM-IPNV during a single replication cycle in RTG-2 cells. One isolate did not generate the variant and failed to replicate in FHM cells even upon blind passage.
IPNV propagated in AS cells (AS-IPNV) was similar to RTG-IPNV, producing equal numbers of plaques in RTG-2 and AS cells but failing to form plaques in FHM cells. However, IPNV propagated in BF-2 cells (BF-IPNV) was similar to FHM-IPNV in producing equal numbers of plaques in all three cell lines.
RTG-IPNV and FHM-IPNV were identical in size, morphology and density in CsCl but differed in plaque size distribution and neutralization by specific antiserum.
Restriction of the replication of RTG-IPNV in FHM cells was partially explained by a reduction of 75% in adsorption to FHM cells. However, the synthesis of RTG-IPNV antigens was reduced 90 to >99% in FHM cells.     

Detection of infectious pancreatic necrosis in a carrier population of rainbow trout, Oncorhynchus mykiss(Richardson), by flow cytometry

Abstract. A non-lethal study of the disease status of adult rainbow trout, Oncorhynchus mykiss (Walbaum), suspected of being carriers of infectious pancreatic necrosis virus (IPNV) was carried out using purified leucocytes from pooled blood samples. Leucocytes were stained by indirect immunofluorcscence to detect IPN viral antigen and analysed by flow cytometry. Leucocytes from an IPN free source were also used as controls. Three populations of leucocytes were analysed: (1) leucocytes examined immediately following purification from blood, which gave positive results with 30–58% of fluorescent cells: (2) purified leucocytes cultured for 7 days in medium at 15 °C. which gave a higher number of fluorescent cells, suggesting multiplication of IPNV; and (3) leucocytes co-cultured on CHSE-214 cell monolayers for 7 days at 15 °C, which amplified the number of infected leucocytes to more than 90% but delayed the result 7 days. Isolation and serological identification of the pathogen was carried out on CHSE-214 cells, which confirmed the positive results obtained by flow cytometry analysis. Further experiments are in progress to complete the applications of flow cytometry to salmonid virus studies.     

Egtved virus: temperature-dependent immune response of trout to infection with low-virulence virus

Abstract. The present report deals with experiments concerning the theoretical basis for development of a live, attenuated vaccine against viral haemorrhagic septicaemia (VHS) of rainbow trout, Salmo gairdneri Richardson, on the basis of a virus strain derived from the Egtved virus reference strain F, the pathogenicity of which has been strongly reduced by in vitro passage. It is shown that the low pathogenicity of the virus (designated the Reva strain) is a genetically stable feature and that the protection against VHS induced by infection with the virus is due to an immune response. Following immunological priming at 10°C partial protection developed at 5° 10° and 15° but not at 20°C, whereas neutralizing antibody was produced at all four temperatures by some of the fish. The duration of virus persistence in trout was found to be inversely proportional to the water temperature.     

A new viral epizootic of Anguilla japonica Temminck and Schlegel

Abstract. In 1969, a new kind of epizootic occurred among eels in Japan and virological investigation was initiated. A new virus designated eel virus european (EVE) was isolated and biological, cytological, serological tests and infectivity trials were carried out. In its CPE on RTG-2 cells and additionally in its biological properties and particle size, EVE was found to be similar to infectious pancreatic necrosis virus (IPNV). Serologically, EVE was similar to the ď Honnincthun, France, strain of IPNV. However, infectivity trials showed that EVE and IPNV differed; EVE killed Japanese eels Anguilla japonica but not rainbow trout fry Salmo gairdneri , while IPN virus killed rainbow trout fry but not eels. We consider EVE to be the primary agent causing the new epizootic and propose the name viral kidney disease for the resulting clinical condition.     

Effects of temperature, irradiance, salinity and inorganic nitrogen concentration on coral zooxanthellae in culture

SUMMARY: Effects of temperature, irradiation, salinity and inorganic nitrogen concentration on two cultured strains of zooxanthellae isolated from the corals Pocillopora damicornis (strain P) and Montipora verrucosa (strain M) were studied. Each strain showed different growth responses in terms of temperature and light intensity. A maximum growth rate of strain P, 1.2 per day, was observed at 32°C under all light intensities examined (5–40 μEm⁻²s⁻¹). However, its photosystem 2 activity (FRI) was higher at 28°C than at 32°C under most light intensities. In contrast, the growth rate of strain M was affected more by light intensity and was almost invariably affected at all temperatures examined (24–36°C). Both algal strains had a comparable growth rate and FRI at salinities from 20 to 35 PSU under moderate temperature and irradiant conditions. High temperature and low irradiation reduced the algal tolerance against low salinity. The gross photosynthesis per cell was not affected by the ammonium enrichment more than 5 μM per day although the cellular chlorophyll a content and cell density increased in proportion with the ammonium enrichment up to 20 μM per day. A potential response of zooxanthellae to the multiple environmental stresses was shown from these results.     

Growth promoting effects of carp serum components on goldfish culture cells

ABSTRACT:   The growth promoting effect of carp Cyprinus carpio serum was investigated in a medium for goldfish Carassius auratus cell culture. The cell growth rate was decreased in the L-15 medium containing carp serum free from low molecular weight (LMW) fraction of less than 10 000. The addition of this fraction to the medium recovered the original growth rate, whereas the fraction itself did not enhance cell growth. Similar experiments were carried out with carp serum lipoprotein and albumin-like protein fractions. The high density lipoprotein (HDL) fraction containing apolipoprotein (apo) A-I and apoA-II enhanced cell growth at 20°C and 35°C when added with the above-mentioned LMW fraction. A good proliferation was observed at both 20°C and 35°C with the HDL fraction at 0.58 mg/mL. Carp albumin-like protein fraction, mainly containing a 71 kDa-component, also enhanced goldfish cell growth with the LMW fraction at the two temperatures. These results suggest that HDL and albumin-like proteins play an important role in the goldfish cell growth cooperatively with LMW substances contained in carp serum.     

Establishment of a novel fin cell line from Brown-marbled grouper, Epinephelus fuscoguttatus (Forsskål), and evaluation of its viral susceptibility

To lay a solid foundation of in vitro investigations of fish viral diseases, cytotechnology and cytotoxicology, a novel fin cell line from brown-marbled grouper, Epinephelus fuscoguttatus , was established and its viral susceptibility was evaluated. The fin tissues, digested with hyaluronidase and collagenase II, were used to initiate primary culture at 24 °C by using 20% foetal bovine serum-Dulbecco's modified Eagle medium/F12 medium, which was further supplemented with carboxymethyl–chitooligosaccharide, basic fibroblast growth factor and insulin-like growth factor-I. The fibroblastic fin cells grew at a steady rate during subsequent subculture and had a population doubling time of 50.6 h at passage 60. The modal diploid chromosome number was 48. A brown-marbled grouper fin cell line (bmGF-1) has been established and subcultured to passage 75 by now. Viral susceptibilities revealed that typical cytopathic effects of bmGF-1 cells emerged after being infected by turbot reddish-body iridovirus (TRBIV) or lymphocystis disease virus (LCDV). However, a large number of TRBIV and LCDV particles were also found in infected bmGF-1 cells. All these indicate that the bmGF-1 cell line has good susceptibility to TRBIV and LCDV, which may serve as a valuable tool for studies of cell–virus interactions and have potential applications in fish virus propagation and vaccine development.     

Effect of temperature on resting egg formation of the tropical SS-type rotifer Brachionus rotundiformis Tschugunoff

ABSTRACT:   Tropical minute rotifer strains (SS-type) induce mixis at 30–35°C but sexual reproduction and resting egg formation do not proceed well due to rapid environmental change. The present study examined the effect of temperature regulation on rotifer Brachionus rotundiformis (Langkawi strain, SS-type) resting egg formation in small (500 mL in culture volume)- and large-scale (500 L in culture volume) experiments. Rotifers were cultured at 30°C in 15–17 p.p.t. seawater with an initial density of 1 individual (ind.)/mL. After 4 days, when cultures were in exponential growth stage with active mixis induction, the culture temperature of the experimental rotifers was changed to 25°C. Control rotifers were cultured at 30°C throughout the experiment. Fresh or frozen Nannochloropsis oculata and condensed freshwater Chlorella vulgaris were used as the rotifer diets in the small- and large-scale experiments, respectively. Significantly higher resting egg production was observed with the experimental rotifers (30→ 25°C) versus the control rotifers. In the large-scale trial, experimental rotifers produced 2.6 × 10⁶ resting eggs during a 9-day experiment, which was 1.6-fold more than the control rotifers. Moreover, the efficiency of resting egg formation was found to increase by a factor of 1.8. The present study indicates that decreasing culture temperature from 30 to 25°C after active mixis increased resting egg formation in B. rotundiformis (SS-type).     

Effect of temperature and cell density on ethanol fermentation by a thermotolerant aquatic yeast strain isolated from a hot spring environment

ABSTRACT: A thermotolerant, fermentative yeast strain named RND13 from a hot spring drainage was evaluated for its ethanol-producing ability at elevated temperatures at a high substrate concentration [15% (w/v) glucose] close to the level reflecting industrial practice. The RND13 was capable of utilizing glucose almost completely at 40°C with increasing inoculum size, producing ethanol up to 6.6% (w/v), which is comparable to levels (7.0–7.2%) at 30°C. The maximum rate of ethanol production by the RND13 was found to be 9.0 g/L per h at 40°C in an inoculum sized 5% (w/v). At 43°C, however, the RND13 could not utilize glucose to completion and showed a slight drop in the extent of produced ethanol [6.0% (w/v)]. Thus, the culture at 40°C with a 5% cell inoculum was considered to be the optimal condition for ethanol production at higher temperatures in terms of batch fermentation. In the phylogenetic analysis based on the small-subunit rDNA sequence, the strain was grouped together with both Candida glabrata and Kluyveromyces delphensis , which are relatively close to Saccharomyces cerevisiae .     

Comparative sensitivity of five fish cell lines to wild type infectious haematopoietic necrosis virus from two Oregon sources

Abstract. Five fish cell lines (CHSE-214, STE-137, RTG-2, EPC and FHM) were compared for sensitivity to infectious haematopoietic necrosis virus (IHNV) from samples obtained from naturally-infected fish. Infectious ovarian fluids were obtained from steelhead trout, Salmo gairdneri Richardson, at the Round Butte Hatchery in central Oregon and tissue homogenates were prepared from chinook salmon, Oncorhynchus tshawytscha (Walbaum), alevins during an IHN virus epizootic at the Elk River Hatchery in coastal Oregon. The only lines to show characteristic viral cytopathology by plaque or end-point dilution assay for the steelhead trout virus isolate were the EPC and FHM cell lines. The chinook salmon isolates produced CPE in CHSE-214, STE-137, FHM and EPC cells. The titre of the salmon virus isolate was 10-50-fold higher on FHM and EPC cells by both assay methods. Neither by end-point nor plaque assay did the Round Butte or Elk River isolates produce CPE on RTG-2 cells. With both virus isolants both cell lines showed that greater sensitivity was obtained with plaque assay than with end-point titration. Pre-treatment of the cells with the polycation, polybrene, did not increase the virus titre in either assay. However, a transient enhancement in virus titre was observed in polybrene-treated STE-137 and CHSE-214 cells.     

Incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids in phospholipid classes in cultured rainbow trout (Salmo gairdneri) cells

The incorporation and metabolism of various (n-3) and (n-6) polyunsaturated fatty acids supplemented to the culture medium was investigated in the rainbow trout cell line, RTG-2. The distribution, and the occurrence and relative extent of further desaturation and elongation of the incorporated acids was determined in individual phospholipid classes by analysis of the fatty acid compositions. RTG-2 cells exhibited Δ6 and Δ5 desaturase activities whereas Δ4 desaturase activity was almost totally absent. The percentage of precursor acids was greatest in the phosphatidic acid/cardiolipin fraction (PA/CL), suggesting a role for possibly PA in the initial incorporation of these acids into the phospholipid pool. The compositional data indicated that individual intermediates and products of the desaturation pathways were associated with specific phospholipid classes probably via mechanisms depending upon the specificities of the acylating enzymes. The composition of phosphatidylinositol (PI) and the tightly controlled mechanisms for generating/maintaining it are consistent with a role for this phospholipid in providing precursor fatty acid for eicosanoid synthesis.     

Establishment and characterization of a new fish cell line from head kidney of half-smooth tongue sole (Cynoglossus semilaevis)

A new cell line (TSHKC) derived from half-smooth tongue sole (Cynoglossus semilaevis) head kidney was developed. The cell line was subcultured for 40 passages over a period of 360?days. The cell line was optimally maintained in minimum essential medium supplemented with HEPES, antibiotics, fetal bovine serum, 2-Mercaptoethanol (2-Me), sodium pyruvate and basic fibroblast growth factor. The suitable growth temperature for TSHKC cells was 24?°C, and microscopically, TSHKC cells were composed of fibroblast-like cells. Chromosome analysis revealed that the TSHKC cell line had a normal diploid karyotype with 2n?=?42, contained the heterogametic W chromosome. The TSHKC cell line was found to be susceptible to lymphocystis disease virus. The fluorescent signals were observed in TSHKC when the cells were transfected with green fluorescent protein and red fluorescent protein reporter plasmids.     

Infectivity experiments with Cyprinus carpio iridovirus (CCIV), a virus unassociated with carp gill necrosis

Abstract. Experiments to determine Cyprinus carpio iridovirus (CC1V) virulence to fish of different ages and species were performed; viz. carp, Cyprinus carpio L., goldfish, Carassius auratus (L.), and rainbow trout, Oncorhynchus mykiss (Walbaum). Neither signs of disease nor mortality occurred in any of the fish species tested. Five passages of the virus in carp resulted in its loss and, not surprisingly, there was no enhancement of virulence. In carp inoculated intraperitoncally, the virus persisted for more than 6 weeks at 18-20°C, more than 12 weeks at a water temperature increasing from 10 to 20°C and more than 27 weeks at 6 5-8°C. The fish cleared itself of the virus after more than 6 weeks maintenance at 18-20°C. The viral infectivity via water is very low. It was impossible to infect the fish with the cell culture grown virus either by water immersion or by prolonged cohabitation with experimentally infected fish. From infectivity data, the authors conclude that the investigated iridovirus is not the aetiological agent of carp gill necrosis.     

A monoclonal antibody cross-reactive with three salmonid herpesviruses

Abstract. A monoclonal antibody cross-reactive with strain H-83 Oncorhynchus masou virus (OMV) and Herpesvirus salmonis has been prepared. Western blot analysis revealed that the monoclonal antibody, designated clo. 7, recognized the 16 000 (16K)-polypeptide of these herpesviruses but not the polypeptides of infectious pancreatic necrosis virus or infectious haematopoietic necrosis virus. Clo. 7 showed neither neutralizing activity against strain H-83 nor cytotoxicity to strain H-83-infected RTG-2 cells in the presence of complement. The purified antigen, recognized by clo. 7, from strain H-83 was a polypeptide with a molecular weight of 16K, and sensitive to proteolytic enzymes and sodium periodate, but not to neuraminidase and ethylether. The antigen was heat stable and stable at pH 5·0 – 9·0.     

Establishment and characterization of a continuous cell line (GF-1) derived from grouper, Epinephelus coioides (Hamilton): a cell line susceptible to grouper nervous necrosis virus (GNNV)

A new continuous cell line (GF-1) was established and characterized. The GF-1 cell line, derived from the fin tissue of a grouper, Epinephelus coioides (Hamilton), was maintained in L15 medium containing 5% foetal bovine serum (FBS) at 28 °C, and has been subcultured more than 160 times since 1995. The majority of GF-1 cells are fibroblast-like, together with some epithelioid cells. Spontaneous transformation of GF-1 cells occurred during subculture 50 to subculture 80, and led to an increase of plating efficiency, less requirement of FBS and de novo susceptibility to grouper nervous necrosis virus (GNNV). Cytopathic effects (CPEs) could be observed in GF-1 cells 3–5 days post-infection with pancreatic necrosis virus (IPNV), hard clam reovirus (HCRV), eel herpes virus Formosa (EHVF) and GNNV. In addition, abundant GNNV particles were found in the cytoplasm of GNNV-infected GF-1 cells using electron microscopy and nucleic acids of GNNV virus were detected by polymerase chain reaction in the culture medium of GNNV-infected cells after CPE appeared. The experimental results indicated that GF-1 can effectively proliferate fish nodavirus and is a promising tool for studying fish nodavirus.     

鲤疱疹病毒Ⅱ型的理化及生物学特性和超微形态发生

为了查明鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2,Cy HV-2)的理化与生物学特性以及病毒在细胞内的超微形态发生过程,利用新建立的对Cy HV-2敏感的异育银鲫脑组织细胞系(Gi CB),对Cy HV-2的理化及生物学特性进行了详细研究,比较了不同来源鱼类细胞系对Cy HV-2感染的敏感性,并对体外培养细胞中Cy HV-2病毒粒子及其超微形态发生过程进行了电镜观察。结果显示,Cy HV-2对热、酸、碱、有机溶剂和冻融敏感;常用鱼类细胞系EPC、RTG-2、Koi-Fin、CIK、CCK、PF-Fin对Cy HV-2的感染不敏感,特异性巢式PCR检测盲传至第7代Cy HV-2细胞培养物,结果均为阴性;Cy HV-2在Gi CB细胞中的增殖动态研究结果表明:病毒感染细胞经过12 h的隐晦期,24 h开始进入对数生长期,96 h病毒滴度达到最高值(107.52±0.26 TCID50/m L),然后进入平台期;透射电子显微镜观察结果显示,Cy HV-2感染细胞可分为吸附与侵入、复制与装配、成熟与释放3个主要过程,病毒进入对数生长期后,被感染细胞内可见形态典型的疱疹病毒颗粒。