Infectious Pancreatic Necrosis Virus: Transmission from Infectious to Susceptible Rainbow Trout Fry

Abstract

Fry of rainbow trout Oncorhynchus mykiss were exposed to serotype VR-299 of infectious pancreatic necrosis virus (IPNV) by using a standardized immersion challenge. In concurrent experiments, fish were monitored for 11 d for excretion of IPNV or monitored for 9 d for excretion and transmission of IPNV to susceptible rainbow trout fry. Immersion-challenged fish began excreting virus within 2 d after challenge. The rate of IPNV excretion per fish increased steadily from about day 4 to day 8 and then decreased. Virus concentrations in tissues of immersion-challenged fish increased exponentially. Susceptible fish became infected with IPNV within 4 d after being introduced to immersion-challenged fish (e.g., 2 d after the challenged fish began excreting virus). By 9 d, 84% of the susceptible fish were infected with IPNV.     

Early Kinetics of Infectious Hematopoietic Necrosis Virus (IHNV) Infection in Rainbow Trout

Abstract

A series of experiments was carried out with infectious hematopoietic necrosis virus (IHNV; 193-110 isolate) in rainbow trout Oncorhynchus mykiss (weight, ～1.2 g) to determine the duration of the patent period and the timing of onset of the infectious periods. We first attempted to transmit IHNV to recipient fish from infected rainbow trout 2–3 d after they had been exposed. No infection transfer occurred despite high titers (10^4.79 to 10^4.91 plaque-forming units 5–8 d postexposure (dpe). To determine the number of secondary cases produced by one infectious individual, we exposed approximately 50 rainbow trout (weight, ～1.5 g) in each of seven replicate tanks to a donor fish that had been infected with virus by bath exposure 3 d earlier. The prevalence of infection in recipient fish rose from 0.84% at 2 dpe to 7.9% at 6 dpe. Maximum incidence (22 cases) occurred between 2 and 4 dpe. No disease-specific mortalities occurred in recipient fish during the experiment. The titer of virus in both recipient and donor fish increased from 2 to 4 dpe. There was a positive correlation between the level of infection among donors and prevalence values among recipient fish (r ² = 0.60). The level of challenge by one infectious fish under the conditions provided was enough for infection transfer from sick cohabitant to susceptible fish but was not enough for initiation of a full-scale epizootic among recipients.     

Serological Properties of Neutralizing Antibodies Induced by Vaccination of Rainbow Trout with Distinct Strains of Infectious Pancreatic Necrosis Virus

Abstract

Adult rainbow trout Oncorhynchus mykiss were immunized with formalin-inactivated, concentrated infectious pancreatic necrosis virus (IPNV). Although the immune response was variable among fish inoculated with a given virus type, sera were obtained that contained high titers of antibodies against known representatives of each of the three major serotypes and several unclassified field isolates of IPNV. Preparations of semipurified macroglobulins from the rainbow trout were subsequently used for comparative cross-neutralization testing of viruses. Cross-reactions were generally low between serotypes; however, diversity and heterogeneity existed among viral isolates from North American hatcheries (e.g., within serotype 1). For example, the Jasper subtype was clearly serologically distinguishable from other western Canadian isolates and from typical eastern Canadian isolates, which were similar to U.S. isolate VR 299. Specific salmonid immunoglobulin is suggested as a possible supplemental reagent, together with mammalian polyclonal and monoclonal antibody, for determining the epidemiology of IPNV in North America.     

Early Life Stage Survival and Susceptibility of Brook Trout,Coho Salmon,Rainbow Trout,and Their Reciprocal Hybrids to Infectious Hematopoietic Necrosis Virus

Abstract

Triploid (heat-shocked) and diploid groups of rainbow trout Oncorhynchus mykiss, brook trout Salvelinus fontinalis, coho salmon Oncorhynchus kisutch, and reciprocal hybrids were produced, monitored for early life stage survival, and evaluated for susceptibility to infectious hematopoietic necrosis virus (IHNV). The female rainbow trout × male brook trout triploid hybrids had significantly greater (P < 0.01) survival than the diploid hybrids of this cross. The heat-shocked hybrid group of the female rainbow trout × male coho salmon also exhibited significantly greater survival to the eyed egg stage of development than the untreated group of this hybrid. Studies of the susceptibility of treatment groups to a 1990 IHNV isolate from the Hagerman Valley were conducted by using a standardized immersion exposure procedure at one or two different mean body weights. The diploid brook trout and coho salmon and two triploid hybrids (female rainbow trout × male brook trout or male coho salmon) were significantly less (P < 0.05) susceptible to IHNV than the pure-species diploid and triploid rainbow trout groups.     

1株传染性造血器官坏死病病毒(IHNV)的分离与鉴定

将虹鳟鱼苗研磨过滤除菌后接种到胖头鱼肌肉细胞系(FHM)后出现了特征性病变(CPE),电镜下观察到IHNV病毒粒子。用传染性造血器官坏死病病毒(IHNV)和传染性胰腺坏死病病毒(IPNV)等水生动物病毒特异性引物对该毒株进行RTPCR试验,结果显示,用IHNV的引物能扩增出阳性片段。将分离株暂时命名为CJ-13。应用DNAStar和MEGA5.2软件,将CJ13的N基因与多株GenBank中已发表的IHNV毒株相应基因进行同源性比对和构建系统树。核苷酸同源性显示CJ-13株与HV7601株的N基因同源性高达97.1%,推导出的氨基酸序列同源性为95.2%。系统进化树表明,CJ-13分离株与HV7601株遗传进化关系较近,属于同一分支。     

Serological Evidence of Infectious Hematopoietic Necrosis in Rainbow Trout from a French Outbreak of Disease

Abstract

Following the detection of infectious hematopoietic necrosis virus (IHNV) in France in April 1987, a serological survey was conducted of the rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) from an infected cultured stock previously known to be contaminated with viral hemorrhagic septicemia virus (VHSV) for 3 years. The work lasted from April to December 1987, at which time all the remaining fish were slaughtered. Serum samples were assayed by a plaque-reduction test and a simplified neutralization test that is more suitable for processing large numbers of serum samples. Such investigations revealed that IHNV neutralization by trout antibodies depended on trout complement, as did neutralization of VHSV. Incubation for 16 h at 4°C increased the sensitivity of the test compared to incubation for 1 h at 20°C. During the course of clinical IHN from April to June, young fish did not display any neutralizing activity, but in September, 29 of 50 of them exhibited significant anti-IHN neutralizing antibody titers ranging from 21 to over 160, and 18 of 46 of these same fingerlings did so in December. Similarly, fish that had undergone VHS infection in August began to develop anti-VHSV antibodies in December (5 of 50), demonstrating that one fish can harbor neutralizing antibodies to both IHNV and VHSV, and that these antibodies had required 14 weeks to appear under fish culture conditions at 10°C. As could be expected from seroneutralization tests, neutralizing antibodies to IHNV did not result in protection against VHS. Sera from 13 of 20 adult fish sampled in mid-June revealed neutralizing antibodies to IHNV, suggesting that they harbored the virus prior to the clinical infection that affected their progeny. Only two of the fish showed low anti-VHSV antibody titers. Similarly, neutralizing antibodies to IHNV were detected in 53 of 73 other adult fish sampled in late October, 10 months after they had spawned and 7 months after mortality had occurred among their progeny. Given the prevalence, level, and persistence of neutralizing antibody titers, the seroneutralization test would be worth investigating more thoroughly to define the conditions that could make it a reliable tool for checking the virus status of trout carriers.     

Prevalence of Infectious Pancreatic Necrosis Virus on Salmonid Fish Farms in Spain

Abstract

In Spain, salmonid fish farming was commercially developed in the 1960s, and now there are 140 private farms that depend heavily on imported embryonate eggs. Infectious pancreatic necrosis was first clinically diagnosed in Spain in 1970, but the virus (IPNV) was not isolated and identified until 1980. Since that time, researchers have isolated IPNV from other samples in Spain. A diagnostic survey was conducted to determine how prevalent IPNV is on fish farms in Spain and whether the virus has been responsible for some of the major financial losses occurring every year on these farms. In total, 236 samplings of rainbow trout Oncorhynchus mykiss from 31 farms in eight hydrographic areas were done over a 3-year period. Infectious pancreatic necrosis virus was isolated in 94 cases, and serotyping of the viral strains revealed that 81% of these isolates were strain Sp and 19% were strain Ab. Neither IPNV strain VR-299 nor rhabdovirus (as infectious hematopoietic necrosis virus or viral hemorrhagic septicemia virus) was detected in any of the samples.     

Use of the Delphi Panel Method to Assess Expert Perception of the Accuracy of Screening Test Systems for Infectious Pancreatic Necrosis Virus and Infectious Hematopoietic Necrosis Virus

Abstract

Fifteen people, considered to be experts on fish virology, participated in a Delphi panel exercise to solicit opinion concerning the importance of factors that influence the ability of cell culture to detect infectious pancreatic necrosis virus (IPNV) or infectious hematopoietic necrosis virus (IHNV) in asymptomatic infected salmonids. Panelists rated many factors as having a strong impact on the sensitivity of cell culture and particularly emphasized the importance of technical and laboratory-related factors. Participants also provided their perceived estimates of the sensitivity and specificity of test systems—consisting of cell culture followed by serum neutralization, specific gene probes, enzyme-linked immunosorbent assay (ELISA), or fluorescent-antibody microscopy—for IPNV and IHNV in asymptomatic salmonids. The sensitivities estimated by panelists for optimal conditions were less than 70% for both IPNV and IHNV. There was substantial panelist uncertainty about the estimates, as indicated by large variances among individual responses. The system using serum neutralization for virus identification was perceived to have the highest sensitivity. All panelists estimated specificity to be very high. The importance of these findings with respect to the design of surveillance, quality assurance and control programs, and the interpretation of screening data are discussed.     

Regulatory Effects of Water Temperature on Loma salmonae (Microspora) Development in Rainbow Trout

Abstract

Water temperature, a pivotal factor influencing interactions between teleosts and pathogens, was examined to determine its effects on the kinetics of xenoma formation and dissolution subsequent to experimental exposure of rainbow trout Oncorhynchus mykiss to the microsporidian gill pathogen Loma salmonae. The permissive water temperature range in which xenomas developed was between 9° and 20°C. Parasite development was arrested at temperatures outside this range, as indicated by the absence of visible xenomas among exposed fish. In addition, when these trout were subsequently moved to temperatures within the permissive range, xenomas failed to develop. Water temperature, within the permissive range, had no significant effect on either the number of xenomas that formed or the proportion of fish that developed xenomas following gastric intubation with a standard dose of spores. The relationship between water temperature and xenoma onset-time was best described (R ² = 88.3%) by polynomial regression analysis: onset = 320 ? 33.4T + 0.9547T ², where T is temperature (°C). Xenoma onset rate was also described through a modified degree-days model, yielding a predictive equation appropriate for use under conditions of fluctuating temperature. The thermal units, expressed as days × (°C above 7°C) necessary for xenoma onset were 298.6 on average. Xenoma dissolution rates, from the time of onset, also appeared to have a trend; more rapid dissolution occurred as temperatures increased. However, this trend correlated minimally with regression models.     

壳寡糖对虹鳟生长性能、血清生化指标及非特异性免疫功能的影响

本文旨在研究壳寡糖对虹鳟生长性能、血清生化指标及非特异性免疫功能的影响.选用体重约为60 g的虹鳟480尾,随机分为4组(每组4个重复,每个重复30尾),分别饲喂在基础饲料中添加0、100、200和400 mg/kg壳寡糖的试验饲料.试验期为49 d.结果表明:1)与对照组相比,100、200和400 mg/kg壳寡糖组的特定生长率分别提高了5.60％ (P ＜0.05)、15.89％ (P ＜0.05)和18.69％(P＜0.05),增重率分别提高了7.53％ (P ＜0.05)、21.21％ (P＜0.05)和26.02％ (P ＜0.05),蛋白质效率分别提高了0.60％ (P ＞0.05)、7.02％ (P ＞0.05)和9.79％ (P ＜0.05),饲料系数分别降低了0.60％(P＞0.05)、6.55％ (P ＞0.05)和8.93％(P＜0.05);2)饲料中添加壳寡糖提高了虹鳟血清总蛋白、白蛋白、球蛋白含量(P＞0.05),降低了血清甘油三酯、胆固醇、葡萄糖含量以及谷丙转氨酶和谷草转氨酶活性(P＞0.05);3)饲料中添加200和400 mg/kg壳寡糖可显著或极显著提高虹鳟的白细胞吞噬百分率(P＜0.05)、吞噬指数(P＜0.05),血清杀菌百分率(P＜0.01)以及血清、肝脏和鳃中溶菌酶活性(P＜0.05);4)虹鳟全鱼水分、粗蛋白质、粗脂肪、灰分含量在各组间差异不显著(P＞0.05).由此得出,在本试验条件下,饲料中添加壳寡糖可提高虹鳟的生长性能和非特异性免疫功能,建议添加量为200 mg/kg.     

Effects of Chito-Oligosaccharides on Growth Performance,Serum Biochemical Indices and Non-Specific Immunity Function of Rainbow Trout (Oncorhynchus mykiss)

本文旨在研究壳寡糖对虹鳟生长性能、血清生化指标及非特异性免疫功能的影响.选用体重约为60 g的虹鳟480尾,随机分为4组(每组4个重复,每个重复30尾),分别饲喂在基础饲料中添加0、100、200和400 mg/kg壳寡糖的试验饲料.试验期为49 d.结果表明:1)与对照组相比,100、200和400 mg/kg壳寡糖组的特定生长率分别提高了5.60％ (P ＜0.05)、15.89％ (P ＜0.05)和18.69％(P＜0.05),增重率分别提高了7.53％ (P ＜0.05)、21.21％ (P＜0.05)和26.02％ (P ＜0.05),蛋白质效率分别提高了0.60％ (P ＞0.05)、7.02％ (P ＞0.05)和9.79％ (P ＜0.05),饲料系数分别降低了0.60％(P＞0.05)、6.55％ (P ＞0.05)和8.93％(P＜0.05);2)饲料中添加壳寡糖提高了虹鳟血清总蛋白、白蛋白、球蛋白含量(P＞0.05),降低了血清甘油三酯、胆固醇、葡萄糖含量以及谷丙转氨酶和谷草转氨酶活性(P＞0.05);3)饲料中添加200和400 mg/kg壳寡糖可显著或极显著提高虹鳟的白细胞吞噬百分率(P＜0.05)、吞噬指数(P＜0.05),血清杀菌百分率(P＜0.01)以及血清、肝脏和鳃中溶菌酶活性(P＜0.05);4)虹鳟全鱼水分、粗蛋白质、粗脂肪、灰分含量在各组间差异不显著(P＞0.05).由此得出,在本试验条件下,饲料中添加壳寡糖可提高虹鳟的生长性能和非特异性免疫功能,建议添加量为200 mg/kg.     

Relation of Water Temperature to Bacterial Cold-Water Disease in Coho Salmon,Chinook Salmon,and Rainbow Trout

Abstract

The effect of water temperature on the progress of experimentally induced Cytophaga psychrophila infection was investigated in juveniles of coho salmon Oncorhynchus kisutch, chinook salmon O. tshawytscha, and rainbow trout O. mykiss (formerly Salmo gairdneri). A virulent strain of C. psychrophila was administered to fish by subcutaneous injection. Infected fish were held in tanks containing pathogen-free well water at temperatures ranging from 3 to 23°C. Mean times from infection to death of the fish were shortest at 12–15°C, which were the temperatures associated with the shortest time for doubling the population of this bacterium in vitro. Juvenile steelhead (anadromous rainbow trout) injected with viable C. psychrophila cells and held in 22°C water did not become diseased.     

The First Isolation in North America of Infectious,Hematopoietic Necrosis Virus (IHNV) and Viral Hemorrhagic Septicemia Virus (VHSV) in Coho Salmon from the Same Watershed

Abstract

In November 1989, infectious hematopoietic necrosis virus (IHNV) was found for the first time in the Soleduck River at the Washington Department of Fisheries Soleduck Hatchery. The virus was isolated from ovarian fluid and kidney-spleen tissue pools from chinook salmon Oncorhynchus tshawytscha and ovarian fluid pools from coho salmon O. kisutch returning to the Soleduck Hatchery. The virus was identified as IHNV by neutralization assays. In December 1989, the virus causing viral hemorrhagic septicemia (VHSV) was found in ovarian fluid and milt pools from wild coho salmon obtained from the Soleduck and Bogachiel rivers and held at the Soleduck Hatchery. The virus was identified as VHSV by neutralization and immunoblot assays. These findings and their implications for routine broodstock sampling are discussed.     

Studies on the Motility and Cryopreservation of Rainbow Trout (Salmo Gairdneri) Spermatozoa

The very short duration of vigorous movement (1 1/2 to 7 min) in fresh water and physiological solutions make trout spermatozoa difficult subjects for cryopreservation studies. Solutions consisting of 250 to 280 mmol sucrose and 5 to 12% dimethyl sulphoxide (DMSO) (4 parts) did not activate trout spermatozoa (1 part), but after dilution with fresh water vigorous motility could be fully restored. These sucrose-DMSO solutions were employed in cryopreservation studies. Using straws and a fast freezing — fast thawing procedure, post-thaw dilution with fresh water resulted in 25%-60% of spermatozoa becoming motile, all with vigorous forward progression. Some existing methods for the cryopreservation of other freshwater fish spermatozoa were repeated on trout without success.     

Survivability of Infectious Hematopoietic Necrosis Virus in Fertilized Eggs of Masu and Chum Salmon

Abstract

The possibility of vertical transmission of infectious hematopoietic necrosis virus (IHNV) was studied with the eggs of masu (cherry) salmon Oncorhynchus masou and chum salmon O. keta. The surfaces of eggs and sperm were contaminated with IHNV (10^3.8-10^4.8 50% tissue culture infective dose [TCID50]/egg) and then the eggs were fertilized. Eggs just after fertilization and embryonated eggs also were infected by injection with IHNV (10^3.8 TCID50/egg) directly into the yolk. During incubation, eggs were held in running water at 10°C. Mortality of the eggs or hatched progeny was determined and isolation of IHNV on the surface or inside of the eggs was determined during the incubation period. No mortality occurred and no virus was detected in fertile eggs from contaminated gametes. For injected eggs, IHNV was not detected on the surface of masu and chum salmon eggs after 1 d of incubation. Infectivity of IHNV inside the eggs decreased gradually and could not be detected after 1 month of incubation. This rate of IHNV reduction in the fertilized egg was similar to that found in a mixture of IHNV and homogenized yolk contents. Several individual yolk components also showed anti-IHNV activity. When eyed eggs were injected with IHNV, the embryos of both masu and chum salmon became infected, and the concentration of virus increased rapidly and reached more than 10^6.5 TCID50/fish. The cumulative mortality of eggs injected at the eyed stage for both masu and chum salmon was 90%. The susceptibilities of hatched-out larvae of masu and chum salmon to IHNV were different; cumulative mortality was more than 90% in masu salmon and 20–30% in chum salmon artificially infected with the virus. We concluded that vertical transmission of IHNV is doubtful because the virus is apparently unable to survive in eggs before the eyed stage.     

A Biopsy Procedure for the Nondestructive Determination of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) Infection in Penaeus vannamei

Abstract

The shrimp Penaeus vannamei is frequently an asymptomatic carrier of infectious hypodermal and hematopoietic necrosis virus (IHHNV). In addition, there is increasing evidence that P. vannamei may also suffer severe disease from infection by IHHNV under some culture conditions. Hence, a nondestructive technique has been developed that has the potential to replace the old method of destructive subsampling, at least with this penaeid species. The new procedure is simpler, faster, less labor intensive, and equally precise when compared with the old method. The procedure involves the excision of the first pereiopod, followed by standard histological examination of the appendage nerve cord for the presence of pathognomonic Cowdry type A intranuclear inclusions. The nondestructive nature of this new technique may be its most important asset; animals tested for IHHNV via this method need not be sacrificed and are thus still available for use as brood stock.     

茶多酚对虹鳟生长性能、生化指标和非特异性免疫指标的影响

本试验研究了不同水平茶多酚对虹鳟生长、肉质、血浆生化指标和免疫指标的影响.选取2日龄初始体重(152.23±4.90)g的虹鳟560尾,随机分为6组,分别饲喂添加0、25、50、100、500、1 000 mg/kg茶多酚饲料,每组3个重复,每个重复30尾鱼,试验进行8周.结果显示:与对照组相比,添加不同水平的茶多酚对虹鳟增重率、特定生长率和饵料系数未产生显著的影响(P>0.05),但显著提高肝体比和脏体比(P<0.05).除茶多酚25 mg/kg组外其它试验组鱼体水分含量显著降低(P<0.05),茶多酚1 000 mg/kg组显著降低鱼体粗脂肪含量(P<0.05),茶多酚25 mg/kg组显著升高鱼体粗蛋白质含量(P<0.05).随着茶多酚添加量的增加,谷草转氨酶(GOT)逐渐下降,除25 mg/kg组外各试验组均显著低于对照组(P<0.05);谷丙转氨酶(GPT)、总胆固醇(CHO)、低密度脂蛋白(LDL)和高密度脂蛋白(HDL)差异不显著(P>0.05).随着茶多酚从25 mg/kg提高到100 mg/kg,肝脏和肌肉中的SOD活性升高,茶多酚25 mg/kg组肌肉SOD和50 mg/kg组肝脏SOD显著升高(P<0.05);肌肉和血浆中MDA下降,25、100 mg/kg组肌肉和50、100 mg/kg组血浆MDA显著(P<0.05或P<0.01)下降;茶多酚50 mg/kg组肝脏、鳃中AKP、ACP活性与对照组相比显著升高(P<0.05),但500和1 000 mg/kg组却未表现出明显的作用.以上结果表明:添加50～100 mg/kg茶多酚可改善虹鳟非特异性免疫和肌肉品质;但茶多酚超过500 mg/kg对虹鳟未产生有益作用.     

Differences between Plasma and Serum Samples for the Evaluation of Blood Chemistry Values in Rainbow Trout,Channel Catfish,Hybrid Tilapias,and Hybrid Striped Bass

Abstract

Blood chemistry analytes are determined for fish from either serum or plasma samples. Serum and plasma are similar in that they both represent the fluid component of blood; however, plasma contains clotting factors that are not present in serum. This study was conducted to determine whether the type of sample—plasma or serum—had an effect on measured blood analytes in rainbow trout Oncorhynchus mykiss, channel catfish Ictalurus punctatus, hybrid tilapia Oreochromis spp., and hybrid striped bass (striped bass Morone saxatilis × white bass M. chrysops). Paired plasma and serum samples were analyzed for the following standard biochemical analytes: total protein, albumin, globulin, creatinine, total bilirubin, alkaline phosphatase, aspartate aminotransferase, sodium, potassium, chloride, calcium magnesium, phosphorus, glucose, and cholesterol. For all four taxa, values for potassium were lower in the serum and magnesium and phosphorus values were higher in the serum. Glucose values were lower in the serum from rainbow trout, hybrid striped bass, and channel catfish; whereas cholesterol values were higher in the serum of rainbow trout, channel catfish, and hybrid tilapias. The differences observed between serum and plasma were distinct from changes occurring with hemolysis and, therefore, do not represent release of erythrocyte constituents. The differences most likely represent metabolic utilization of blood constituents while the blood was clotting. This work indicates that plasma should be used preferentially to serum for biochemical analysis because analyte levels determined from serum may not accurately reflect those found in circulating blood.     

传染性法氏囊病病毒分子结构与功能研究进展

本文对传染性法氏囊病病毒的分子结构与功能的关系及致病的分子基础作了综述。ＩＢＤＶ的主要保护性抗原及抗原表位的变异位于结构蛋白ＶＰ２上。病毒多肽成熟需经二次切割,ＮＣＲ可影响病毒的转录和／或翻译,ＶＰ１－ＶＰ３复合体的形成是ＩＢＤＶ装配的重要环节。不同毒力型的ＩＢＤＶ的出现除与基因突变、重组、重排有关,还可能与ＶＰ５有关,也证实了ＮＣＲ与血清型的致病性无关。