Inactivated infectious haematopoietic necrosis virus (IHNV) vaccines

The inactivation dynamics of infectious haematopoietic necrosis virus (IHNV) by b-propiolactone (BPL), binary ethylenimine (BEI), formaldehyde or heat and the antigenic and immunogenic properties of the inactivated vaccines were evaluated. Chemical treatment of IHNV with 2.7 mm BPL, 1.5 mm BEI or 50 mm formaldehyde abolished virus infectivity within 48 h whereas heat treatment at 50 or 100 degrees C rendered the virus innocuous within 30 min. The inactivated IHNV vaccines were recognized by rainbow trout, Oncorhynchus mykiss, IHNV-specific antibodies and were differentially recognized by antigenic site I or antigenic site II IHNV glycoprotein-specific neutralizing monoclonal antibodies. The BPL inactivated whole virus vaccine was highly efficacious in vaccinated rainbow trout challenged by waterborne exposure to IHNV 7, 28, 42 or 56 days (15 degrees C) after immunization. The formaldehyde inactivated whole virus vaccine was efficacious 7 or 11 days after vaccination of rainbow trout but performed inconsistently when tested at later time points. The other vaccines tested were not efficacious.     

Infectious pancreatic necrosis virus of salmonids: biological and antigenic features of a pathogenic strain and of a non-pathogenic variant selected in RTG-2 cells

Abstract. A non-pathogenic cell culture adapted variant was obtained from a normally pathogenic strain of IPN virus (Sp type) after several passages in RTG-2 cells. This cell culture modified (CCA) strain was compared with the original wild (W) strain in various tests. Cross neutralization tests showed no obvious antigenic difference between the two. However the CCA strain was neutralized by a 1:5000 dilution of normal trout serum whereas the W strain was not. In RTG-2 cells, CCA strain gave both large and small plaques, whereas the W strain gave only small ones. Both virus strains were heat sensitive and labile to cyclic freezing and thawing. The CCA virus was more stable in storage at 4°C under different pH conditions and its growth in RTG-2 cells was more rapid. The rt (supraoptimal temperature at which viral yield is depressed by 90%) appeared to be 19-20°C for the CCA strain and 18-19°C for the W strain. Pre-treatment of RTG-2 cells with ultraviolet inactivated CCA virus could inhibit growth of W virus, but had no effect on replication of homologous virus.     

Immunization of rainbow trout, Salmo gairdneri Richardson, against bacterial kidney disease: preliminary efficacy evaluation

Abstract. A bacterin for immunization against bacterial kidney disease of salmonid fishes caused by Renibacterium salmoninarum is described. Cultures were grown in Evelyn's KDM2 medium containing 10% calf serum in a fermenter under the following conditions: pH 7.2, 15°C, 800ml/min air, 200 rev/min agitation and 5–15 days of incubation. Possible substitutes for calf serum were 10% horse serum 0.15% starch and leptospira medium. The bacterins were inactivated with 0.3% formalin and no adjuvants were used. Other tests evaluated pH-lysed bacterin, 50% concentrated bacterin and 50% concentrated pH-lysed bacterin. Juvenile rainbow trout, salmo gairdneri Richardson, were vaccinated either by intraperiotoneal (i.p.) injection, 2 min immersion or 2-step hyperosmotic infiltration. Fish were held from four to six weeks at 11°C, then challenged by i.p. injection with the homologous virulent bacterium. Fish died from days 19 to 40 after challenge. The best preparation was pH-lysed bacterin given by a single i.p. injection; hyperosmotic and immersion vaccination were not effective. Typically when 80% or more of unvaccinated controls were infected as detected by Gram stain, 10% or less of the vaccinated fish were infected.     

Ichthyophthiriasis of Atlantic salmon, Salmo salar L., at the Montta Hatchery in northern Finland in 1978–1979

Abstract. The occurrence of the ciliated protozoan Ichthyophthirius multifiliis on Atlantic salmon yearlings at a hatchery in northern Finland is described. During August 1978 a severe epizootic occurred in two earth ponds resulting in mortality of over 50%. Water temperature at this time was about 15°C. Fish were treated with a 1:4000 formalin bath administered every 3 days. Parasites disappeared from the fish by October after which they were refractory to further infection. The parasite occurred in other stocks in 1979 but was successfully controlled with formalin. Some aspects of the ecology of I, multifiliis are discussed.     

Comparative stability of two salmonid viruses and poliovirus in fresh, estuarine and marine waters

Abstract. The inactivation rates in the aquatic environment of two fish pathogens, infectious pancieatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV), were compared with that of poliovirus type 1 as a representative of the human enterovirus group. The survival studies were performed using untreated fresh, estuarine and sea water samples held at 15 and 20°C. The results indicated that the salmonid viruses survived longer than poliovirus in the saline waters, whereas in fresh water poliovirus was the most stable of the three viruses. IPN virus proved to be most stable in estuarine water at 15°C, whereas the survival of IHN virus was favoured in fresh water. We also observed that at 20°C the inactivation rate for each virus was independent of salt concentration in estuarine and sea water. Although temperature exhibited a marked effect on virus stability in fresh water, the salmonid viruses presented similar survival patterns at both temperatures in sea water. In general the period of greatest viral inactivation correlated with higher bacterial numbers in the waters.     

Neutralizing antibody levels for protection against betanodavirus infection in sevenband grouper, Epinephelus septemfasciatus (Thunberg), immunized with an inactivated virus vaccine

An inactivated betanodavirus, red‐spotted grouper nervous necrosis virus (RGNNV), is a vaccine candidate for viral nervous necrosis (VNN). The present study was conducted to examine inoculation doses of the vaccine and neutralizing antibody titre levels to protect fish against VNN. Young sevenband grouper, Epinephelus septemfasciatus, averaging 25.4 g, were immunized at 25 °C water temperature by a single intraperitoneal injection of formalin‐inactivated RGNNV. Fish immunized at vaccine doses of 10^8.5, 10^8.0, 10^7.5, 10^7.0 and 10^6.5 TCID₅₀ per fish produced antibodies at mean titres of 1:907, 1:511, 1:259, 1:197 and 1:96, respectively, at 20 days post‐immunization (p.i.). Neutralizing antibodies were not detected in any control fish (titre <1:80). When fish were challenged with RGNNV (10^5.0 and 10^4.0 TCID₅₀/fish) at 20 days p.i., cumulative mortalities of the fish groups immunized with 10^8.5, 10^8.0, 10^7.5 and 10^7.0 TCID₅₀ per fish were significantly lower than those of the control group, and the relative percent survival values were higher than 60% in fish groups immunized with 10^7.5 TCID₅₀ per fish or higher doses. However, no significant differences were found in mortality between the group immunized with 10^6.5 TCID₅₀ per fish and the control group. From these results, it was deduced that the minimum effective inoculation dose of the vaccine is 10^7.0 TCID₅₀ per fish and the minimum mean neutralizing antibody titre giving significant protection is approximately 1:200. This antibody titre level is a possible measure of vaccine efficacy against VNN in sevenband grouper, instead of a virus challenge test.     

大鲵虹彩病毒β-丙内酯灭活方法的研究

为探讨β-丙内酯(β-propiolactone,BPL)灭活大鲵虹彩病毒(Giant salamander iridovirus,GSIV)的最适条件,研究了BPL对GSIV的灭活方法。采用终浓度分别为0.025%、0.05%、0.1%、0.2%的BPL灭活细胞培养的GSIV,4℃条件下分别灭活24 h、48 h、72 h、96 h,通过细菌培养、细胞培养、病毒核酸PCR扩增以及鱼体感染试验进行灭活病毒的安全性检验,确定最适灭活条件。试验结果表明,GSIV经终浓度为0.1%的BPL 4℃灭活处理72 h后可完全灭活病毒,灭活病毒无细菌污染,接种对GSIV敏感的鲤上皮瘤细胞系(EPC)细胞无细胞病变效应(CPE)出现,病毒主衣壳蛋白(MCP)基因特异性引物PCR反应未扩增出靶基因产物,灭活病毒对健康大鲵的感染试验未出现疾病症状。灭活效果检测结果表明,与未灭活GSIV相比较,最适灭活条件下的GSIV结构蛋白与抗原性未发生显著变化。结论显示BPL可用来灭活GSIV,本研究确立了BPL灭活GSIV的最适条件,为大鲵虹彩病毒细胞培养灭活疫苗研究奠定了重要基础。     

Lymphocystis disease virus persists in the epidermal tissues of olive flounder, Paralichthys olivaceus (Temminch & Schlegel), at low temperatures

Olive flounder artificially infected with lymphocystis disease virus (LCDV) were reared at 10, 20 and 30 °C for 60 days, to compare LCD-incidence. In the fish reared at 20 °C, lymphocystis cells appeared on the skin and fins at 35 days post-challenge, and the cumulative LCD-incidence was 80% at 60 days. High levels of LCDV, with a mean polymerase chain reaction (PCR) titre of 10⁶ PCR-U mg⁻¹ tissue, were detected in the fins and skin of LCD-affected fish at 20 °C, but were not detected in the spleen, kidney, brain and intestinal tissues of these fish. No LCD clinical signs were observed in the fish reared at 10 °C and 30 °C; however, a low level of LCDV (10³ PCR-U mg⁻¹ tissue) was detected in the fins and skin of these fish. By increasing the rearing temperature from 10 to 20 °C, lymphocystis clusters appeared on the skin and fins of the fish with no previous LCD clinical signs within 33 days after the temperature change. It was shown that permissive cells for LCDV infection exist in the epidermis of olive flounder. At low temperatures, small amounts of LCDV were able to persist over a period extended for a further 45 days in the fish epidermis, even though the fish showed no LCD clinical signs. The optimum growth temperature of LCDV is near 20 °C.     

Effects of temperature, density and early weaning on the survival and growth of Atlantic ditch shrimp Palaemonetes varians larvae

This investigation examined the effects of temperature, density and early weaning on the survival and growth of Palaemonetes varians larvae. Survival of larvae raised at 17.5 °C was not significantly different (average + standard deviation) (94 ± 5%) from the survival of those raised at 19.5 °C (95 ± 5%) and at 21.5 °C (94 ± 4%). However, the duration of the larval stage was significantly longer for shrimp reared at 17.5 °C (17.3 ± 0.8 days) compared with shrimp reared at 19.5 °C (14.3 ± 0.7 days) and at 21.5 °C (11.3 ± 0.6 days). No significant differences ( P >0.05) were found in the survival rate, final weight and length of larvae reared at the densities of 5, 10, 20 and 50 larvae L⁻¹. The survival of P. varians larvae fed solely on Artemia was significantly higher ( P <0.05) than larvae weaned with an artificial practical diet from Zoea II stage (94 ± 4% and 82 ± 1%, respectively, for Artemia and artificial diet-fed larvae), but no significant differences ( P >0.05) were observed in the final larval weight or length between these two treatments. The survival and growth of the larvae fed with the practical diet tested is a promising step ahead in the development of the culture of this species as it eliminates both the need for Artemia throughout all larval stages, and the need for more expensive artificial diets.     

Effect of temperature and feeding on conditioning of Ostrea chilensis Philippi, 1845 reproductors

Abstract. Adult oysters of Ostrea chilensis Philippi 1845, collected from the Quempilén River estuary, Ancud, Chile, were subjected to different temperature (14,17 and 20°C) and feeding treatments (daily rations of dry weight algae equivalent to 0.75% and 1.5% mean dry flesh wt) to measure their influence on gonad maturation. Males spawned under all these experimental conditions, after 25-27 conditioning days, while females spawned only at the same time at the higher temperatures (17 and 20°C). At high food ration (1.5%), the incubation period lasted for 29 days at 20°C, and 44 days at 17°C. At half food ration (0.75%), however, the incubation period was 24 days at 20°C and lasted for more than 54 days at 17°C, longer than under the natural estuary conditions. Larger larvae and better settlement were obtained from oysters kept at 17°C and high food ration (1.5%).     

Effect of chemical and physical treatments on the inactivation of Macrobrachium rosenbergii nodavirus and extra small virus

White tail disease (WTD) is found to cause immense economic losses in hatcheries, with mortalities often reaching 100% within 4 or 5 days. The pathogenic agents have been identified as Macrobrachium rosenbergii nodavirus (MrNV) associated with extra small virus (XSV), which are 27 and 15 nm in diameter respectively. The effects of some chemical disinfectants hydrogen ions (pH), heat and ultraviolet (UV) irradiation on the inactivation of MrNV and XSV were investigated. The viral inoculum exposed to UV irradiation for a period of 5 min and more was totally inactivated and failed to cause mortality in postlarvae of prawn. The viruses were totally inactivated by this high pH (8.5, 9 and 10). The viral suspension treated with sodium hypochloride, formalin, Benzalkonium chloride and Benzethonium chloride at the concentration of 200 ppm caused 100% mortality in postlarvae of prawn. Iodine was found to be effective to inactivate MrNV and XSV at the concentration of 100 ppm or more, whereas the viral suspension treated with iodine at the concentration of 50 ppm or less caused mortality in postlarvae. The infected postlarvae in treated and positive control groups showed positive by RT‐PCR for these viruses.     

Anaesthetic efficacy and physiological responses to clove oil-anaesthetized kelp grouper Epinephelus bruneus

The efficacy of clove oil as an anaesthetic and at producing a physiological response (plasma cortisol and glucose) was evaluated in the kelp grouper, Epinephelus bruneus . To acquire complete anaesthesia in less than 3 min and recovery in <10 min, three doses of clove oil were tested at 18, 22 and 26 °C. Although higher anaesthetic doses resulted in shorter induction times and longer recovery times, and a lower temperature resulted in longer anaesthesia induction and slower recovery, we found the optimal dose and administering temperature of clove oil to be 250–300 mg L⁻¹ at water temperature of 18 °C, 150–200 mg L⁻¹ at water temperature of 22 °C and 50–100 mg L⁻¹ at water temperature of 26 °C respectively. Following the administration of 150 mg L⁻¹ of clove oil at 22 °C, the plasma cortisol level was highest (4.24 ± 1.571 μg dL⁻¹) after 12 h and the plasma glucose was highest (92.7 ± 9.61 mg dL⁻¹) after 2 h. These results should be useful to the aquaculture industry, where anaesthesia is necessary for a range of activities.     

The influence of fish age and water temperature on mortalities of rainbow trout, Salmo gairdneri Richardson, caused by a European strain of infectious pancreatic necrosis virus

Abstract. Rainbow trout fry were infected by a pathogenic Sp type of infectious pancreatic necrosis virus isolated in France. Fry held at 10°C and infected at different ages showed a decrease of sensitivity with increasing age and ceased to be susceptible to the disease when 20 weeks old. Mortality was delayed at 6°C and lowered or suppressed at 16°C. When fry were moved from 10 to 16°C before infection mortality was also lowered but not when they were moved from 16 to 10°C. Late infection of siblings kept at different temperatures before infection suggested a relation between the number of degrees x days and the final mortality recorded.     

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.     

Control of spawning activity in female Nile tilapia (Oreochromis niloticus) (L.) by temperature manipulation

The aim of this study was to maximize the spawning of Oreochromis niloticus females in a specific time period. Females were divided randomly into control and treatment groups. In the treatment groups, females were kept for one week at 28±0.5 °C, after which they were exposed to a reduced water temperature of 22±0.5 °C for 7, 14 and 28 days. Thereafter, the temperature was restored to 28 °C. Females in the control groups were kept continuously at a water temperature of 28 °C. All females were checked daily for signs of spawning for the duration of the experiments and were manually stripped if ready to spawn. The following parameters were calculated for period of 3 and 7 days following a 28 °C temperature restoration: spawning rate, number of eggs per female, weight of female, relative fecundity (eggs g⁻¹ body weight) and the percentage of hatched and swim-up fry. The highest spawning rate of 39.5% was obtained in the 14-day trial over a period of 7 days, while the corresponding value in the control was 12.5%. The percentages of hatched and swim-up fry in the 14- and 28-day trials, however, were significantly higher in the controls than in the corresponding treatment groups.     

Normal and aberrant tissue distribution of Loma salmonae (Microspora) within rainbow trout, Oncorhynchus mykiss (Walbaum), following experimental infection at water temperatures within and outside of the xenoma-expression temperature boundaries

Temperatures above 20 °C or below 9 °C interrupt the life cycle of the gill intracellular microsporidian parasite Loma salmonae (Microspora) prior to sporogony, inhibiting the production of xenomas. This study intended to characterize this life-cycle failure. Juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), were experimentally infected with L. salmonae spores, and the effect of water temperature on the progress of infection, as determined by polymerase chain reaction, was compared for fish held at water temperatures of 5, 15 and 21 °C. At 15 °C, parasite DNA was first detected in the heart (3 days post-exposure [PE]), and then in the gills and spleen (2 weeks PE). Branchial xenomas developed by week 4 PE. In contrast, at 5 °C, the arrival of the parasite in the heart was delayed until 7 days PE. However, even though parasite DNA was detected in the gills at 7 days PE, xenomas failed to form in the gill, and by week 4 PE, parasite DNA was no longer detected. In fish held at 21 °C, parasite DNA was detected in the heart, gills and spleen by 3 days post-infection, and similar results were observed at 7 days PE. Xenomas also failed to form in these fish and parasite DNA was no longer detected by week 2 PE. Within the range of temperatures tested in this study, spore germination and delivery of their DNA into the host through the intestinal wall was not blocked by temperature. At 5 or 21 °C, migration to the heart and gills occurred, but at aberrant periods of time. The normal life cycle of L. salmonae may depend on the completion of relatively lengthy, but yet unknown, stages of development within the heart, prior to reaching the gill. This development may be adversely affected by temperature, and explain the temperature limits of this parasite.     

Effects of developmental stage, seawater concentration and rearing temperature on cryopreservation of Pacific oyster Crassostrea gigas larvae

ABSTRACT: For the development of a stepwise cryopreservation technique for larvae of the Pacific oyster Crassostrea gigas , various conditions were examined. Larvae at 9, 12, 15, 18 and 21 h after insemination were cooled at a rate of −1°C/min (seeding at −8°C for 15 min) and then plunged into liquid nitrogen at −35 or −40°C using 1.5 M dimethyl sulfoxide (DMSO) and 250 mM trehalose as cryoprotectants. Among these larvae, 15 h after insemination (the trochophore stage before formation of the shell gland) showed the highest motility and the best external appearance after thawing. Trochophore larvae were cryopreserved in preservation media containing different dilutions (1/4, 1/6, 1/8, 1/10 and 1/30) of seawater. Larvae preserved in the 1/4 seawater medium showed the highest appearance of shelled larvae 4 days after thawing. Trochophore larvae reared in seawater at 21, 25 or 29°C were cryopreserved for 8 months and then reared at 26°C after thawing. Larvae reared at 25°C showed the highest survival rate and normal larval ratio at day 6 after thawing, although larvae reared at 21°C showed the highest rates until day 4. One larva developed at 25°C succeeded to settle.     

Effects of formalin on water quality and parasitic monogeneans on silver perch (Bidyanus bidyanus Mitchell) in earthen ponds

Infestations of parasitic monogenean trematodes (Lepidotrema bidyana and Gyrodactylus sp.) on freshwater silver perch (Bidyanus bidyanus Mitchell) in earthen ponds were treated with formalin (37% formaldehyde). Concentrations of 30 and 40 mg L^?1 formalin were effective, but fish in ponds treated with 20 or 25 mg L^?1 remained infested. At temperatures of 24.1–26.9°C, concentrations of 30 or 40 mg L^?1 formalin caused dissolved oxygen (DO) to decline from 10.1–11.9 to 3.0–3.3 and 1.2–1.7 mg L^?1, respectively, within 36–42 h of treatment. In addition, pH declined from 7.2–8.4 to 6.3–6.7, within 36 h and turbidity decreased over 48 h. In the ponds where DO was 1.2–1.7 mg L^?1, silver perch showed signs of severe stress, but continuous aeration (10 hp ha^?1) for 3 days and inflow of well‐oxygenated water for 6–8 h prevented mortalities. At temperatures of 13.2–15.7°C, concentrations of 30 or 40 mg L^?1 formalin caused DO to decline from 9.0–10.0 to 6.0–8.1 mg L^?1 and pH from 7.0–7.3 to 5.9–6.6 within 72 h. Total ammonia‐nitrogen increased over 72 h in ponds treated with 30 or 40 mg L^?1 formalin. Fish became re‐infested with L. bidyana in all ponds within 30 days of treatment. A concentration of 30 mg L^?1 formalin is recommended as a treatment for monogeneans on silver perch in ponds, but aeration is necessary to maintain adequate water quality at higher temperatures.     

A method of experimental infection of kuruma shrimp larvae, Penaeus japonicus Bate, with baculoviral mid-gut gland necrosis (BMN) virus

Abstract. Infectivity experiments were undertaken by water-borne inoculation of kuruma shrimp, Penaeus japonicus Bate, larvae with BMN virus. Mysis stage larvae were inoculated with the virus by exposure for 2h in sea water containing a homogenized and filtered preparation (450nm) of naturally BMN virus-infected shrimp stored for about 7 weeks at −80°C. Inoculated shrimp stocked in rearing jars were examined to determine whether nuclear hypertrophy of the mid-gut gland epithelial cells characteristic for BMN virus infection could be observed in fresh squash preparations under dark field illumination equipped with a wet-type condenser. Four days post-inoculation at 25–30°C incubation temperature were considered to be satisfactory for the experimental trial.     

Effects of hormone treatments and temperature on sex-reversal of Nigorobuna Carassius carassius grandoculis

ABSTRACT: Functional sex reversal of all-female nigorobuna Carassius carassius grandoculis to phenotypic males was examined by immersion exposure of fry to 17-methyltestosterone (17-MT) and controlled water temperature during early development. Fry were reared in water containing different concentrations of 17-MT at 24 and 30°C for 80 days starting 20 days after hatching. Although the fish exposed to 0.1 and 1.0 μg/L 17-MT at 24°C were all male, treatment with 10.0 μg/L 17-MT resulted in 43% females. Twenty-two percent males appeared in the control treatment at 30°C but the control at 24°C was entirely female. The proportion of males in treatments exposed to 0.01– 1.0 μg/L 17-MT at 30°C was slightly lower than in the respective treatments at 24°C. These results indicate that the phenotypic expression of sex in nigorobuna is thermolabile and that sex determination is under the control of genetic factors and temperature. Also, control of temperature during early development has been shown to be important for the production of all-female offspring for use as breeding stock for pond culture of fish suitable for preparation as 'funazushi'.