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.     

Multiplication of Infectious Hematopoietic Necrosis Virus in Rainbow Trout following Immersion Infection: Organ Assay and Electron Microscopy

Abstract

Sequential spread of infectious hematopoietic necrosis virus (IHNV) to tissues of rainbow trout Oncorhynchus mykiss was examined following immersion infection with two different isolates of IHNV, a pathogenic strain and a nonpathogenic strain from rainbow trout. Virus strain 193–110 was highly pathogenic to 1-month-old rainbow trout and caused 100% mortality within 13 d, whereas strain RB-76 was much less virulent, causing 50% mortality by the 19th day. Virus titers of 1-month-old fingerling fish dying soon after infection were significantly higher than titers of those dying later. Assays of dissected tissues showed that gills of infected 2-month-old fingerlings contained virus as early as 16 and 20 h postinfection, with definite replication occurring at 48 h. The early presence of the virus in the gills followed shortly by appearance of the virus in the kidneys and spleen indicated that the virus spreads rapidly to the target organs. Virus was detected in many other organs at lower levels on the third day and increased to higher levels during the following days. Heart tissue had high titers later in the infection. When 4-month-old rainbow trout were infected with strain 193–110, the mortality was reduced and delayed, whereas those infected with strain RB-76 produced no mortality. Assays on the day of death of these older fingerlings infected with strain 193–110 revealed that fish dying soon after infection also had higher titers than those dying later. Electron microscopic examination offish organs showed the presence of typical IHNV particles budding off from various tissue cells of affected organs, including gill tissue. The destructive effect of the virus was particularly noticeable in the disarrangement of heart muscle organelles.     

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.     

Genotyping of Infectious Pancreatic Necrosis Virus Isolates from Mexico State

Abstract

The infectious pancreatic necrosis virus (IPNV; genus Aquabirnavirus) affects salmon and trout, causing high mortality in first-feeding fry. The classification of this virus includes nine serotypes and seven genogroups. In Mexico, two different isolates were identified in 2000 and 2008, respectively. Both isolates were classified into genogroup I according to the RNA genome of this virus. As Mexico is importing rainbow trout Oncorhynchus mykiss eggs from different countries, the aim of this study was to genotype IPNV isolates obtained from four rainbow trout producer regions within the state of Mexico. We utilized a fragment of the VP2* (outer capsid protein) gene sequence of Mexican IPNV isolates as a molecular marker to determine the genogroup to which they belong. Although all Mexican IPNV isolates were grouped into genogroup I, we identified genetic diversity among these isolates, and 14 unique nucleotide sequence types were associated with the four producer regions in Mexico State.

Received December 21, 2010; accepted July 27, 2011     

White Sturgeon as a Potential Vector of Infectious Hematopoietic Necrosis Virus

Abstract

Cell lines from white sturgeon Acipenser transmontanus were derived from peripheral blood cells, heart, and spleen. Incubated with infectious hematopoietic necrosis virus (IHNV) for 8 d at l5°C, these cell lines produced 0.7–53.2 plaque-forming units (PFU)/cell. Waterborne exposure of larval white sturgeons (60 d posthatch) to 10⁶ PFU/mL of IHNV resulted in 10% mortality 5–6 d postinfection, with virus concentrations consistently greater than 10⁵ PFU/g. A replicate group of larval white sturgeons that were sampled at different times post-IHNV exposure had no detectable virus at 24 h, but 72% of the fish had IHNV concentrations of 10²-10⁶ PFU/g when they were examined 2–9 d postinfection. Juvenile white sturgeons (mean weight, 35 g) immersed in or injected with IHNV exhibited no mortality, and virus was only detected immediately postexposure in just 25% of the fish tested. Juvenile white sturgeons fed either virus-free rainbow trout Oncorhynchus mykiss or dead IHNV-infected rainbow trout had no viable virus in their feces. Juvenile white sturgeons fed or exposed to IHNV failed to transmit the virus to cohabiting rainbow trout fry. These results suggest that IHNV can replicate in larval white sturgeons but presumably not in juveniles or adults. Virus neutralization activity was detected in serum from adult white sturgeons (4–6 years old) cultured with rainbow trout exposed to IHNV but not in white sturgeons kept in a pathogen-free environment and fed a manufactured diet. White sturgeon serum with IHNV-neutralizing activity was used to passively immunize rainbow trout, and it provided significant (P < 0.01) protection against IHNV challenge.     

In Vitro Infection of Salmonid Epidermal Tissues by Infectious Hematopoietic Necrosis Virus and Viral Hemorrhagic Septicemia Virus

Abstract

The ability of two rhabdoviruses, infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV), to infect fish skin was investigated by in vitro infection of excised tissues. Virus replication was determined by plaque assay of homogenized tissue extracts, and the virus antigen was detected by immunohistology of tissue sections. Gill, fin, and ventral abdominal skin tissues of rainbow trout Oncorhynchus mykiss that had been infected in vitro with a virulent strain of IHNV (193–110) produced substantial increases in virus titer within 24 h. Titers continued to increase up until day 3 of incubation; by this time, virus had increased 1,000-fold or more. This increase in IHNV titer occurred in epidermal tissues of fingerlings and of older fish. In another experiment, IHNV replicated in excised rainbow trout tissues whether the fish had been subject to prior infection with a virulent strain of IHNV (Western Regional Aquaculture Consortium isolate) or whether the fish had been infected previously with an attenuated strain of the virus (Nan Scott Lake, with 100 passes in culture). A virulent strain of VHSV (23/75) replicated effectively in excised gill tissues and epidermal tissues of rainbow trout and chinook salmon O. tshawytscha; however, the avirulent North American strain of VHSV (Makah) replicated poorly or not at all.     

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.     

Effects of Elevated Water Temperature and Immunosuppressant Treatment on Prevalence and Titer of Infectious Pancreatic Necrosis Virus in Naturally Infected Brook Trout

Abstract

Using fingerlings of brook trout Salvelinus fontinalis naturally infected with infectious pancreatic necrosis virus (IPNV), we demonstrated that elevated water temperature and treatment with the immunosuppressant triamcinolone acetonide (generic Kenalog®) significantly increases the titer of IPNV and probably also the prevalence of the infection. Stress-promoting treatments could potentially enhance the capability to detect various fish viral agents.     

Susceptibility of Four New Salmonid Cell Lines to Infectious Hematopoietic Necrosis Virus

Abstract

Four salmonid cell lines, CoE 45, CoE 115, CoE 345, and RBTE 45, were established from embryonic tissues of coho salmon Oncorhynchus kisutch and rainbow trout O. mykiss. In vitro challenges of the new lines were conducted with four isolates of infectious hematopoietic necrosis virus (IHNV). Two of the IHNV isolates used for the challenges were derived from infected tissues of rainbow trout, one was derived from chinook salmon O. tshawytscha, and the other isolate was derived from coho salmon. To standardize the virus challenges of the new cell lines, several established piscine cell lines (EPC, CHSE 214, CSE-119, RTH-149, RTG, and RTS) were challenged in the same way as the new lines. Each of the lines was challenged with virus at a single low multiplicity of infection (0.01 plaque-forming unit per cell). Virus yields were quantitated by plaque assay on epithelioma papulosum cyprini (EPC) cells on day 3. Results of the challenge experiments revealed different levels of production of virus for each isolate on the various cell lines. Overall, the new cell line derived from rainbow trout, RBTE 45, was quite susceptible to all viruses tested. The three cell lines newly derived from coho salmon embryo were not as resistant to the replication of IHNV as was the established coho salmon cell line, CSE-119. An established cell line, EPC, derived from an epithelial tumor of common carp Cyprinus carpio, remained the most susceptible to all four IHNV isolates tested.     

虹鳟鱼传染性胰脏坏死病病毒的分离与鉴定

目的探讨传染性胰脏坏死病毒（IPNV）在虹鳟鱼中的存在与传播,为我国北方地区IPNV疫情动态监测和防控提供依据。方法从辽宁省某渔场中患传染性胰脏坏死病的虹鳟鱼中分离出1株优势毒株,对新分离出来的菌株进行培养增殖、感染试验及理化性质的鉴定。结果新分离的IPNV在CHSE细胞上增殖良好,且可引起良好的病变,病毒滴度达10^-805／0．1mL。分离毒对酸、碱和热均稳定,对乙醚不敏感,病毒的复制不被FUDR抑制。根据IPNV的保守基因N基因序列,设计特异性引物,结果扩增出224bp的片段。对该片段进行测序分析,发现与IPNV的参考序列有较高的相似性,表明该毒株为传染性胰脏坏死病毒。结论IPNV已在北方虹鳟鱼中存在,可为我国北方IPNV疫情动态监测和防控提供依据。     

Assessment of the Risk of White Sturgeon to become Infected and Potential Carriers of Infectious Pancreatic Necrosis Virus

Abstract

Little scientific information is available to assess whether White Sturgeon Acipenser transmontanus can become infected and potential carriers of infectious pancreatic necrosis virus (IPNV). To assess this risk, monitoring results of adult and progeny White Sturgeon were examined from waters historically stocked with salmonid fish known to be IPNV carriers. From 1999 through 2004 White Sturgeon from a total of 30 separate families whose parentage came from waters historically stocked with IPNV carrier fish were tested. Duplicate groups of 25 juvenile Snake River White Sturgeon were waterborne exposed to 1.0×10⁴ 50% tissue culture infective dose (TCID50)/mL of water for 1 h and an additional group was injected intraperitoneally with 1.0×10⁵ TCID50/fish. A negative control group was handled similarly but was not exposed to the virus. No morbidity was detected in any of the treatment groups or the negative control. At 34, 40, 47, and 54 d postexposure to IPNV, virus reisolation was attempted on five fish from each group, and an additional five fish from each group were examined for histological changes consistent with an IPNV infection. At 34 and 40 d postinjection with IPNV, 20% (one of five) of the fish tested positive for the virus per sample interval; however, fish from groups that were waterborne-exposed to IPNV were all negative. At 47 and 54 d after exposure or injection with IPNV an additional five fish from each group were tested at each sample interval and all results were negative. Histological analysis of target tissue obtained from five fish per group at 34 and 54 d postinfection also failed to detect any consistent change associated with an IPNV infection. These results suggest that the risk of White Sturgeon to become infected and develop into potential carriers of IPNV is negligible.

Received May 21, 2013; accepted July 8, 2013     

Journal of Aquatic Animal Health: Guide for Authors 2012

Abstract

A virus adsorption-elution (viradel) method for use with positively charged microporous filters was previously developed in our laboratory to concentrate waterborne infectious pancreatic necrosis virus (IPNV). In the present study, this method was evaluated for the detection of IPNV in water of laboratory aquaria containing IPNV-infected rainbow trout Onchorhynchus mykiss. Waterborne IPNV samples concentrated by the viradel method were compared with samples of sediment, fish tissue homogenate, and untreated water to determine how much the viradel method could improve rapidity and efficiency of virus detection. Results of three separate experiments indicated a significant, positive correlation between the virus titers in fish tissue and in the viradel method samples and between the sediment and untreated water samples.     

Susceptibility of Selected Inland Salmonids to Experimentally Induced Infections with Myxobolus cerebralis,the Causative Agent of Whirling Disease

Abstract

Laboratory exposures to the infectious stages (triactinomyxons) of Myxobolus cerebralis demonstrated a range of susceptibility to whirling disease among four species of inland salmonids. Replicate groups of each species were exposed to two concentrations of triactinomyxons, a low dose (100–200 per fish) and a high dose (1,000–2,000 per fish). Exposed fish were evaluated for clinical signs, for severity of microscopic lesions at 35 d, 2 and 5 months, and for spore concentrations in the head cartilage at 5 months. A standard strain of rainbow trout Oncorhynchus mykiss matched for age served as a susceptible species control. Rainbow trout, westslope cutthroat trout O. clarki lewisi, Yellowstone cutthroat trout O. clarki bouvieri, and bull trout Salvelinus confluentus were susceptible to M. cerebralis infections. Clinical signs, including radical swimming (“whirling”) and black tails, were observed at 7 weeks postexposure among rainbow and cutthroat trout challenged at 3 weeks of age. Clinical signs were rare among bull trout exposed at an age of 4 weeks and absent among rainbow and cutthroat trout exposed at 3 months posthatch. Most rainbow, cutthroat, and bull trout were found to be infected when examined at 5 months postexposure. The most severe microscopic lesions among infected fish at 5 months postexposure were found among rainbow trout. Cutthroat trout had less severe lesions, bull trout had mild infections, and no evidence of infection was found among Arctic grayling Thymallus arcticus. Mean spore concentrations among infected fish correlated with the severity of microscopic lesion scores. Rainbow trout had mean concentrations of spores in head cartilage reaching 10⁶, whereas more resistant species such as bull trout had 10⁴ spores; no spores were found among Arctic grayling at 5 months postexposure.     

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.     

Experimental Infection of Brook Trout with Infectious Hematopoietic Necrosis Virus Types 1 and 2

Abstract

Fry of brook trout Salvelinus fontinalis became infected and diseased after immersion exposure to infectious hematopoietic necrosis virus (IHNV), but a long-lasting IHNV carrier state was not induced. Duplicate groups of 100 fish were immersed for 6 h in baths containing a type 1 (Round Butte, RB) or a type 2 (Rangen, RA) IHNV isolate at a high or low dose. Brook trout mortalities induced by immersion in a bath of the RB or RA IHNV isolate at 10² plaque-forming units (pfu) per milliliter were equivalent (1 and 0%), but fish were more susceptible to infection with RA IHNV. Only the single dead fish in the RB group was infected, but 24% of the RAexposed fish were infected 1 week after exposure. At a dose of 10⁶ pfu/mL, exposure to RB IHNV resulted in a higher mortality (35%) and prevalence of infection (89% of live fish sampled at 1 week postexposure), but no infectious virus was detectable by 5 weeks after exposure. In contrast, RA IHNV exposure at a dose of 10⁴ pfu/mL resulted in only 5% mortality, and live fish killed at 1 week postexposure had a 22% prevalence of infection, but infectious virus was not detectable by week 3. Although brook trout have been previously considered to be resistant to IHNV, this study has shown that brook trout become diseased and die after exposure to a high dose of one type I IHNV isolate and can be infected after immersion exposure to even a low dose of type 1 or type 2 IHNV.     

Infectious Salmon Anemia Virus (ISAV) in Brown Trout

Abstract

Infectious salmon anemia (ISA) is a viral disease of Atlantic salmon Salmo salar that have been exposed to seawater in fish farms or hatcheries. This disease was previously believed to be exclusively one of salmon. However, it has been shown that anadromous brown trout Salmo trutta may carry the ISA virus (ISAV). Propagation of the ISAV in brown trout without the trout's showing any gross clinical signs of disease could be a result of a longstanding host-pathogen relationship between the virus and brown trout. A brown trout population isolated from the sea during the last 5,000 years and expected to be naive to the virus was challenged. These fish did not develop any gross signs of disease, but a few ISAVs were present as late as 46 d postchallenge. It was also shown that the ISA virus was present in brown trout as late as 7 months after challenge.     

Occurrence of Aeromonas salmonicida,Renibacterium salmoninarum,and Infectious Pancreatic Necrosis Virus in Ontario Salmonid Populations

Abstract

The results of samples collected from private and government fish farms and wild and feral fish populations in Ontario from 1981 to 1995 were synthesized to obtain prevalence estimates in salmonids at both the fish and site levels for three pathogens. Renibacterium salmoninarum and Aeromonas salmonicida were both detected on at least one site for every year investigated. Ontario Ministry of Natural Resources (OMNR) culture stations had the highest percentages of sites with infected fish for R. salmoninarum. Natural water bodies had the highest percentages of sites with infected fish for A. salmonicida. Infectious pancreatic necrosis virus (IPNV) was only detected sporadically on some commercial farms and never in OMNR hatcheries or in wild or feral fish. Although screening for any virus that would yield cytopathological effect was carried out during all the years surveyed, no virus other than IPNV was isolated. The low prevalence and “source-specific” presence of IPNV in Ontario demonstrates the necessity of representative sampling for the detection of rare pathogens. It was estimated that, overall, less than 1% of all fish in the sampled populations were infected with each of the three pathogens for almost every year studied. The importance of summarizing pathogen-testing data and the possible implications on disease control policy planning and assessment are discussed.     

Detection of Infectious Pancreatic Necrosis Virus from the Leeches Hemiclepsis marginata and Hirudo medicinalis

Leeches have been reported to harbor several important fish pathogens, including spring viremia of carp virus, infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV), and also may contain blood protozoa. In the present study, leeches were collected from water bodies located in Kurdistan province, Iran. The specimens were tested for IHNV, VHSV, and infectious pancreatic necrosis virus (IPNV) using the PCR method. The results showed that two different species of leeches, Hemiclepsis marginata and Hirudo medicinalis, were infected by IPNV among the seven species studied. The infected leeches were found in areas that were polluted with untreated sewage coming from upstream fish farms culturing Rainbow Trout Oncorhynchus mykiss. In addition, the fish at fish farms in the vicinity had been infected with IPNV 9 months previously. Our results showed that the virus causing infectious pancreatic necrosis is present in the leeches H. marginata and H. medicinalis, suggesting that leeches are a potential source of IPNV in fish farms.

Received October 14, 2015; accepted June 1, 2016 Published online September 29, 2016     

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.